Oxidative Stress and Antioxidants - Their Role in Human Disease-Nova Biomedical Books (2009)

OXIDATIVE STRESS
AND ANTIOXIDANTS: THEIR ROLE

IN HUMAN DISEASE
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OXIDATIVE STRESS
AND ANTIOXIDANTS: THEIR ROLE
IN HUMAN DISEASE
RAMON RODRIGO
EDITOR
Nova Biomedical Books

New York
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Library of Congress Cataloging-in-Publication Data

Rodrigo, Ramon.
Oxidative stress and antioxidants : their role in human disease / Ramon Rodrigo.
p. cm.
Includes bibliographical references and index.
ISBN 9781607415541
1. Oxidative stress. 2. Antioxidants. I. Title.
RB170.R636 2009
616.07--dc22
2009013526
Published by Nova Science Publishers, Inc.  New York

Contents
Preface vii
Chapter I Oxidative Stress: Basic Overview 1
Joaquin Toro and Ramón Rodrigo
Chapter II Hypertension 25
Ramón Rodrigo
Chapter III Atherosclerosis 63
Víctor Molina and Ramón Rodrigo
Chapter IV Postoperative Atrial Fibrillation 91
José Vinay and Ramón Rodrigo
Chapter V Acute Renal Failure 111
Joaquín Toro, Víctor Molina and Ramón Rodrigo
Chapter VI Pre-Eclampsia 135
Mauro Parra
Chapter VII Metabolic Syndrome 159
Rodrigo Castillo
Chapter VIII Diabetes Mellitus 193
Rodrigo Castillo
Chapter IX Nonalcoholic Steatohepatitis 223
Juan Gormaz and Ramón Rodrigo
Chapter X Neurodegenerative Disorders 257
Rodrigo Pizarro
Chapter XI Glaucoma 297
Leonidas Traipe, Rodrigo Castillo and Ramón Rodrigo
Index 321
Preface
Oxidative stress is a relatively new concept that has been widely implicated in
biomedical sciences during the last 20 years. It significantly participates in the
pathophysiology of highly prevalent diseases such as diabetes, hypertension, preeclampsia,
atherosclerosis, acute renal failure, Alzheimer and Parkinson diseases, among others. The
metabolism of oxygen by cells generates potentially deleterious reactive oxygen species
(ROS). Under normal conditions the rate and magnitude of oxidant formation is balanced by
the rate of oxidant elimination However, an imbalance between pro-oxidants and antioxidants
results in oxidative stress. Increased ROS levels in the cell have a substantial impact either
leading to defective cellular function, aging, or disease. Therefore, a better understanding of
the roles of ROS-mediated signaling in normal cellular function as well as in disease is
necessary for developing therapeutic tools for oxidative stress-related pathologies. The
potential beneficial role of antioxidants is discussed in the light of experimental studies, as
well as clinical trials aimed to determine the outcome of patients. “Oxidative Stress and
Antioxidants: Their Role in Human Disease” is a practical guide for pathophysiology of
oxidative stress and the latest therapeutic advances to modulate the antioxidant defense. This
includes evidence from clinical trials, regarding the use of antioxidants and preconditioning,
to protect the organism against ROS.
Chapter I - Over the last decades, a new concept involving the biological effects of
highly reactive oxygen and nitrogen species in the mechanisms causing disease has filled the
scientific journals. These reactive species, mainly free radicals, are found in normal
physiological condition and can be beneficial when produced at low levels. However, they
are harmful at high concentrations when the endogenous antioxidant defense systems are
overwhelmed (oxidative stress), what has been implicated in disease states. This paradigm
has been widely documented for many settings, and a causal relationship has been suggested
for oxidative stress and some highly prevalent human pathologic alterations, such as
atherosclerosis, hypertension, preeclampsia, diabetes, among others.
This chapter provides a comprehensive review of the basis for the biochemical and
physiological mechanisms involving reactive oxygen species generation and depuration, as
well as their effects on biological molecules. In addition, the defensive response of the
antioxidant defense system in vivo against oxidative damage is also analyzed. Nevertheless,
before extensively examining the specific mechanisms hypothesized for the diverse
viii Joaquin Toro and Ramón Rodrigo
pathologies, it is included a basic review of the biochemistry accounting for the alterations
mediated by oxidative stress.
Chapter II - Reactive oxygen species (ROS) and reactive nitrogen species play a key role
in the modulation of the vasomotor system. Thus, ROS are recognized as mediators of the
vasoconstriction induced by angiotensin II, endothelin-1 or urotensin-II, among others; while
nitric oxide (NO) is a major vasodilator. In physiological conditions, low concentrations of
intracellular ROS play an important role in normal redox signaling involved in maintaining
vascular function and integrity. In addition, under pathophysiological conditions ROS
contribute to vascular dysfunction and remodeling through oxidative damage. The fact that
ROS play a key role in development of hypertension is supported by the findings of increased
production of superoxide anion and hydrogen peroxide, reduction of NO synthesis, and a
decrease in bioavailability of antioxidants in human hypertension. In both animal models
and humans, increased blood pressure has been associated with an excessive endothelial
production of ROS (oxidative stress) which may be both a cause and an effect of
hypertension.
Antioxidants, whether synthesized endogenously or exogenously administered, are
reducing agents that neutralize these oxidative compounds before they can cause damage to
biomolecules. In the management of hypertension and other cardiovascular diseases, the
primary interest was focused on the therapeutic possibilities of antioxidants to target ROS,
thus avoiding hypertensive end-organ damage. The use of antioxidant vitamins, such as
vitamin E and vitamin C, has gained considerable interest for their role as protecting agents
against vascular endothelial damage, in this way contributing to ameliorate chronic diseases,
beyond its essential function associated to body deficiencies. However, promising findings
from experimental investigations, the results of clinical trials aimed to demonstrate
antihypertensive effects of antioxidant supplementation are disappointing. Nevertheless, the
methodology used in some of these studies makes them a matter still to be debated. Some
studies reported a potential antihypertensive effect, particularly when using association of
two or more antioxidants. Even more, antioxidant diets low in fat, have found to be of most
significant benefit in hypertensive patients.
Taken together data are consistent with the view that while an antioxidant alone has not
yet demonstrated its efficacy as a therapeutic antihypertensive agent, the synergistic actions
among the various antioxidants appear to be effective to counteract the ROS effect on the
vascular wall. These effects could arise from their complex biological actions, from their
ability not solely to scavenge ROS, but also to prevent their formation through down
regulation of NADPH oxidase and up-regulation of endothelial NO synthase and antioxidant
enzymes.
Chapter III - Atherosclerosis is a major source of mortality, being the underlying cause
for most cases of cardiovascular diseases such as ischemic heart disease and cerebrovascular
disease. Reactive oxygen species (ROS) can regulate several cellular processes, having a key
role in the homeostasis of the vascular wall. There is compelling evidence pointing to ROS as
important factors for the development of atherosclerosis. Many of the proatherogenic actions
of ROS occur through the generation of oxidized LDL. Also, ROS can contribute to the
development of endothelial dysfunction through the consumption of nitric oxide and
generation of peroxynitrite. Endothelial dysfunction constitutes an early feature of
Preface ix
atherogenesis, preceding the alterations that later perpetuate the lesion formation.
Atherogenesis includes several processes, such as accumulation and oxidation of LDL in the
subendothelial space, expression of adhesion molecules and chemoattractant mediators,
adhesion of monocytes, generation of foam cells, production of inflammatory mediators and
proliferation of certain cell types. Since most of these processes can be modulated by ROS,
supplementation with antioxidants is expected to exert some degree of protection against
atherosclerosis. Several lines of evidence support a role of antioxidant supplementation in
attenuating some of the processes involved in atherogenesis. However, clinical trials have
failed to consistently prove a protective effect. The potential role of antioxidant
supplementation against atherosclerosis development or progression remains an open
question.
Chapter IV - Atrial fibrillation is an arrhythmia occurring frequently within the first few
days in 10% to 65% of patients after major cardiothoracic surgery (postoperative atrial
fibrillation, POAF). It is associated with increased morbidity and mortality and longer, more
expensive hospital stays. Despite the use of strategies to prevent POAF through the
prophylactic use of agents such as β-adrenergic blockers, amiodarone, or others, a
considerable percentage of the patients still presents the arrhythmia. The involvement of
oxidative stress in the mechanism of POAF is supported by an increasing body of evidence
indicating that the formation of reactive oxygen species (ROS) released following
extracorporeal circulation are involved in the structural and functional myocardial
impairment derived from the unavoidable ischemia–reperfusion cycle of this setting. ROS
behave as intracellular messengers mediating pathological processes, such as inflammation,
apoptosis and necrosis, thereby participating in the pathophysiology of POAF. Consequently,
myocardial electrical and structural remodeling associates with the appearance of functional
impairment consistent with alterations in electrical conduction. Therefore, it seems
reasonable to assume that the reinforcement of the antioxidant defense system should protect
the heart against functional alterations in the cardiac rhythm in this setting. Interestingly,
exposure to low to moderate doses of ROS could trigger a cellular defensive response
characterized by a prevailing effect of survival over apoptotic pathway, what should be
considered a therapeutic target. The present chapter examines the molecular basis accounting
for the contribution of oxidative stress to the development of POAF. In addition, it is
presented the clinical and experimental evidence to support a new paradigm based in the
prophylactic reinforcement of the antioxidant defense system toward reduction in the
susceptibility of cardiomyocytes to ROS-induced injury.
Chapter V - Acute renal failure (ARF) is a condition characterized by a rapid decrease in
renal function, leading to an imbalance in water and solutes metabolism. It constitutes a
major cause of morbidity and mortality in hospitalized patients worldwide, mainly in elderly
population. Despite the medical advances, over the past fifty years the mortality of ARF has
not diminished. This is often attributed to increased risk factors prevalence, mainly those
derived from changes in our lifestyle. However, it is also possible that the therapeutic
methods used until these days are not aiming on the right direction, probably due to lack of
knowledge about some of the mechanisms leading to the development and progression of
ARF.
x Joaquin Toro and Ramón Rodrigo
Over the last decades a large body of evidence has emerged supporting a role of
oxidative stress in the pathogenesis of a variety of diseases, including ARF. Indeed, both
reactive oxygen and nitrogen species are thought to enhance tubular damage caused from
either renal ischemia or direct toxic injury. Nevertheless, the role of oxidative stress in ARF
pathogenesis has not been fully established and some evidence is even contradictory. A better
understanding regarding the real contribution of oxidative stress to ARF development and
progression is required for the design of potentially preventive interventions, such as
antioxidant supplementation. Indeed, clinical trials on this matter have been carried out with
promising results.
This chapter presents an update of the current evidence supporting a role of oxidative
stress in ARF pathophysiology, and the potential role of antioxidants in the prevention and
treatment of this disease.
Chapter VI - Pre-eclampsia (PE) is the most important complication of human pregnancy
worldwide and a major contributor to maternal and fetal morbidity and mortality. It is a
disease of two stages. The first stage concerns the relative failure of early trophoblast
invasion and remodeling of the spiral arteries, leading to a poor blood supply to the feto-
placental unit, exposing it to oxidative stress. The second stage is characterized by maternal
endothelial dysfunction, leading to the clinically recognized symptoms of the syndrome,
which include hypertension, proteinuria, thrombocytopenia and impaired liver function.
Furthermore, the modification of spiral arteries occurs during the first and early second
trimester of pregnancy, leading to uteroplacental hypoperfusion and fetal hypoxia. Despite
much work in the last decade, the causes that trigger PE are uncertain and the predictive
value of potential risk factors is poor. Increasing evidence suggests that placental and
systemic oxidative stress plays a crucial role in its development. Indeed, oxidative stress and
disrupting angiogenesis is considered the link bridging the two stages of the disease. Markers
of oxidative stress in women with established PE have shown both increased lipid
peroxidation in placental tissue, along with increased in maternal plasma biomarkers
indicating decreased antioxidant capacity and increased lipid peroxidation. These findings
have contributed to the interest in using antioxidants to prevent the development of PE. The
lack of appropriate early predictors of the disease has determined that the risk groups for
primary prevention of PE should be characterized on the basis of the clinical history of the
patients and from knowing that is possible to establish some risk factors. A large number of
publications suggest a potential role of antioxidant nutrients in the prevention of PE in
women at high increased risk of the disease. Vitamins C and E have been the main
antioxidants agents used for this purpose. Despite the biological properties of these
compounds, exerting ROS scavenging and a down-regulation of ROS, the results of clinical
trials do not support benefits for routine supplementation with vitamins C and E during
pregnancy to reduce the risk of PE.
This chapter examines the role of oxidative stress in the pathophysiology of PE and
reviews the available data on the use of antioxidant compounds, mainly vitamins C and E, to
prevent the development of this disease.
Chapter VII - The biochemical steps linking insulin resistance with the metabolic
syndrome have not been completely clarified. Mounted experimental and clinical evidence
indicates that oxidative stress is an attractive candidate for a central pathogenic role since it
Preface xi
potentially explains the appearance of all risk factors and supports the clinical manifestations.
Indeed, metabolic syndrome patients exhibit activation of biochemical pathways leading to
increased delivery of ROS, decreased antioxidant protection and increased lipid peroxidation.
The described associations between increased abdominal fat storage, liver steatosis and
systemic oxidative stress, the diminished concentration of nitric oxide derivatives and
antioxidant vitamins, and the endothelial oxidative damages observed in subjects with the
metabolic syndrome support oxidative stress as the common second-level event in an
unifying pathogenic view. Moreover, it has been observed that oxidative stress regulates the
expression of genes governing lipid and glucose metabolism through activation or inhibition
of intracellular sensors. Diet constituents can modulate redox reactions and the oxidative
stress extent, thus also acting on nuclear gene expression. As a consequence of the food–gene
interaction, metabolic syndrome patients may express different disease features and extents
according to the different pathways activated by oxidative stress-modulated effectors. This
view could also explain family differences and interethnic variations in determining risk
factor appearance.
Chapter VIII - Elevation of glycemia in diabetic patients may lead to the autooxidation of
glucose, glycation of proteins, and the activation of polyol metabolism. These changes
accelerate the generation of reactive oxygen species (ROS) and increase oxidative
modification of lipids, DNA, and proteins in various tissues. Thus, oxidative stress occurring
in this setting may play an important role in the development of the chronic complications of
diabetes, such as nephropathy, neuropathy, and lens cataracts. Langerhans islets are more
vulnerable to the occurrence of oxidative stress, since they contain low levels of antioxidant
enzyme activities compared to other tissues. High glucose concentrations are known to give
rise to a manifestation named glucose toxicity. Major manifestations of glucose toxicity in
the pancreatic β-cells are defective insulin gene expression, diminished insulin content, and
defective insulin secretion. The link between the clinical complications and oxidative stress-
related parameters has been established by the study of advanced glycation end products
(AGEs). Among the latter, heterocyclic amines, acrylamide, and AGEs are well-known
compounds hypothesized to cause harmful health effects. First, AGEs act directly to induce
cross-linking of long-lived proteins, such as collagen, to promote vascular stiffness, thus
altering the structure and function of vasculature. Second, AGEs can interact with their
receptors to induce intracellular signaling leading to enhanced oxidative stress and
elaboration of key proinflammatory and prosclerotic cytokines. Over the last decade, a large
number of preclinical studies have been performed, targeting the formation and degradation
of AGEs, as well as their interaction with specific receptors. Translational research with
humans is now under way to ascertain whether this protection can be provided to patients
experiencing inadequate glycemic control.
Chapter IX - Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver
diseases characterized mainly by macrovesicular steatosis that occurs in the absence of
alcoholic consumption. NAFLD is closely associated with comorbid conditions, such as
obesity, dyslipidemia, and insulin resistance. It is a medical condition in which the liver is
invaded with fat and excessive amounts of lipids are present within hepatocytes. There is
increasing evidence to consider that fatty liver is the hepatic manifestation of the metabolic
syndrome, a growing problem in the modern western world. NAFLD might worsen into a
xii Joaquin Toro and Ramón Rodrigo
more serious condition, known as nonalcoholic steatohepatitis (NASH), in which fat

accumulation is accompanied by an inflammatory process in the liver.
The clinical relevance of these conditions is given by the high prevalence of NAFLD in
the general population and to the possible evolution of NASH towards end-stage liver
disease, including hepatocellular carcinoma, as well as the need for liver transplantation. The
molecular mechanism whereby NASH might eventually lead to fibrosis, and severe cirrhosis
in some patients, is a process associated with increased production and release of
inflammatory mediators, such as nitric oxide (NO), cytokines, and reactive oxygen species
(ROS) by the cells. Oxidative stress caused by increased ROS plays an important role in the
pathogenesis of NASH. These reactive species would derive from mitochondria, cytochrome
P-450 2E1, peroxisome, and iron overload in the liver with steatosis. Excessive ROS is
considered to cause simple steatosis to progress to NASH. Regardless the origin of hepatic
fat, it could produce a rise of hepatic free fatty acids. The latter, particularly the
polyunsaturated ones, are closely linked to ROS generation by different pathways, including
increased oxidation in different cellular organelles, disruption of mitochondria and
endoplasmic reticulum, microsomal cytochrome P450 activation and ceramide formation. In
addition, increased ROS production could derivate not only in hepatocyte cell death but also
in the activation of liver resident cells, such as Kupffer, stellate and endothelial cells. This
might enhance the original oxidative stress, inflammatory response and subsequent immune
infiltration thus aggravating NASH.
Up to date no absolute effective medical treatment is available for NASH patients.
Therapy is predominantly aimed at controlling the comorbid conditions, such as obesity,
insulin resistance, and dyslipidemia. However the major role of oxidative stress in the
pathogenesis of NASH suggests that the antioxidant treatment would be an effective therapy.
Hence, both several substances with different antioxidants mechanism and effects related
with the redox balance have been assayed in small clinical trials. These agents have shown
the ability of improve the outcome of patients, thus opening the door to new strategies to
manage or treat this disease. This chapter provides the clinical and experimental evidence to
support the role of oxidative stress in the pathophysiology of NAFLD and NASH, as well as
the molecular bases promoting the development of mechanism-based therapeutic
interventions, mainly clinical trials aimed to target specific pathways involved in the
pathogenesis of NASH.
Chapter X - Oxidative stress has been related to the pathogenesis of virtually every
neurodegenerative disease. However, two clinical entities stand out for the major
epidemiological burden they provide, as they are intimately related to our increasingly aging
population. Alzheimer’s disease and Parkinson’s disease are the two most prevalent
neurodegenerative diseases affecting roughly over 5.5 million people in the United States
alone. In spite of this, the understanding of their underlying pathophysiological mechanisms
is scarce, and thus, they have remained difficult to treat, prevent or cure. Early diagnosis is
fundamental, as is in early stages of the neurodegenerative process when therapeutic
interventions in both animal models and clinical trials have proven more beneficial.
However, early detection can be a painstaking procedure due to their subtle, highly
unspecific first clinical features and still rather undeveloped biomarkers. It is in all of these
tasks where the understanding of the roles of oxidative stress in the pathogenesis of such
Preface xiii
conditions has been proved beneficial. Biochemical markers of oxidative stress now seem a
feasible way of early detection of such diseases. We could also give possible explanations for
the mixed results obtained in clinical trials using antioxidant supplementation against
neurodegenerative diseases. The task now is to continue looking deeper at oxidative stress in
the mechanisms of such diseases, but also to develop new therapeutic resources to meet the
needs of a rapidly growing population. This chapter deals with the general pathophysiology
of oxidative stress and its role in the pathogenesis and antioxidant supplementation in the
detection, understanding and treatment of Alzheimer’s disease and Parkinson’s disease.
Chapter XI - Glaucoma constitutes an increasingly serious public health problem,
moreover in developed countries and is an important cause of blindness after cataracts. It is
an optic neuropathy that implies loss of retinal ganglion cells, including their axons, and a
major tissue remodeling, especially in the optic nerve head. Although increased intraocular
pressure is a major risk factor for glaucomatous optic neuropathy, there is little doubt that
other factors such as ocular blood flow play a role as well. Mechanisms leading to
glaucomatous optic neuropathy are not yet clearly understood. There is, however, increasing
evidence that both activation of glial cells and oxidative stress in the axons play an important
role. The involvement of reactive oxygen species (ROS) in the pathogenesis of glaucoma is
supported by various experimental findings, including: (i) resistance to aqueous humor
outflow is increased by hydrogen peroxide by inducing trabecular meshwork (TM)
degeneration; (ii) TM possesses remarkable antioxidant potential, mainly explained by
superoxide dismutase and catalase activities and glutathione pathways, all that is found
decreased in glaucoma patients; and (iii) intraocular-pressure increase and severity of visual-
field defects in glaucoma patients paralleled by the amount of oxidative damage of DNA
affecting TM. Vascular alterations, which are often associated with glaucoma, could
contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM
but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic
nerve in glaucoma. Despite the major pathogenic role of ROS in the pathophysiology of
glaucoma, clinical trials testing the efficacy of antioxidant drugs for its management are still
lacking.
In: Oxidative Stress and Antioxidants ISBN: 978-1-60741-554-1
Editor: Ramon Rodrigo © 2009 Nova Science Publishers, Inc.
Chapter I
Oxidative Stress: Basic Overview
Joaquin Toro1 and Ramón Rodrigo2

1
Molecular and Clinical Pharmacology Program,
Institute of Biomedical Sciences, Faculty of Medicine, University of Chile.
2
Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile
Supported by FONDECYT, grant 1070948
Abstract
Over the last decades, a new concept involving the biological effects of highly
reactive oxygen and nitrogen species in the mechanisms causing disease has filled the
scientific journals. These reactive species, mainly free radicals, are found in normal
physiological condition and can be beneficial when produced at low levels. However,
they are harmful at high concentrations when the endogenous antioxidant defense
systems are overwhelmed (oxidative stress), what has been implicated in disease states.
This paradigm has been widely documented for many settings, and a causal relationship
has been suggested for oxidative stress and some highly prevalent human pathologic
alterations, such as atherosclerosis, hypertension, preeclampsia, diabetes, among others.
This chapter provides a comprehensive review of the basis for the biochemical and
physiological mechanisms involving reactive oxygen species generation and depuration,
as well as their effects on biological molecules. In addition, the defensive response of the
antioxidant defense system in vivo against oxidative damage is also analyzed.
Nevertheless, before extensively examining the specific mechanisms hypothesized for
the diverse pathologies, it is included a basic review of the biochemistry accounting for
the alterations mediated by oxidative stress.
1. Introduction
The oxidation and reduction reactions in biological systems (redox reactions) represent
the basis for numerous biochemical mechanisms of metabolic changes [1]. In biological
2 Joaquin Toro and Ramón Rodrigo
systems, instead of using the terms reducing and oxidant agent, it is more frequent to use the
denominations of antioxidant and pro-oxidant, respectively [2]. A reducing agent, or
antioxidant, is a substance which donates electrons, whereas an oxidant, or pro-oxidant agent,
is a substance that accepts electrons. Cells are constantly exposed to oxidants from both
physiological processes, such as mitochondrial respiration [3] and pathophysiological
conditions such as inflammation, foreign compound metabolism, and radiation among others
[4].
Oxidative stress constitutes a unifying mechanism of injury of many types of disease
processes. This alteration is encountered when there is an imbalance between the production
of reactive oxygen species (ROS) and the ability of the biological system to readily detoxify
these reactive intermediates or easily repair the resulting damage.
Reactive oxygen species are a family of highly reactive species that can be beneficial, as
they are used by the immune system as a way to attack and kill pathogens. Nevertheless,
when these species are found in excess they might cause cell damage either directly or
working as intermediates in diverse signaling pathways. Reactive nitrogen species (RNS)
may have also a similar behavior: While nitric oxide radical (NO) has vasorelaxing and
antiproliferative properties, peroxynitrite anion (ONOO-) increases intracellular ROS
concentration, with deleterious consequences.
This chapter deals first with the basis of physiological mechanisms of ROS generation,
and subsequently with the explanation of their role as toxic molecules in diverse
pathophysiological conditions leading to several common diseases. In addition, the
components of the antioxidant defense system in vivo are described.
2. Oxidative Stress
2.1. Background
The first radical oxygen was discovered by Linus Pauling in 1930s and it was described
as superoxide [5]. Pauling had no knowledge that this radical could be produced biologically
or that it could also be the core of several many disease processes. In the same decade, Mann
and Keilin [6] purified the superoxide dismutase (SOD) protein from bovine blood and liver,
as a copper-binding protein of unknown function. The protein was called “erythrocuprein” or
“hepatocuprein” or later “cytocuprein.” The purification was based solely on copper content.
Until late 1960s, the pathophysiological importance of ROS was completely unknown.
However, several new findings would dramatically lead to a change of this situation:
1. The discovery of McCord and Fridovich in 1968-1969 described the enzymatic

activity of the erythrocyte SOD, which led to eliminate the “Pauling free radical”, or
superoxide anion (O2•–) terminology, and in the same year it was found that SOD
was contained in almost all mammalian cells [7, 8]. The latter finding suggested that
O2•– was a physiological product.
2. In 1969, Knowles et al. showed that the enzyme xanthine oxidase (XO) could indeed
produce superoxide [9].
Oxidative Stress: Basic Overview 3
3. In 1973 Babior et al. [10] showed that the bactericide action of the neutrophil was
associated with large amounts O2•– generation, thereby linking the inflammation
process to ROS generation. It was apparent that some of the tissue damage
associated with the inflammatory process could be attributed to neutrophil-generated
O2•–, and herein SOD would protect cells and extracellular components from
damage [11, 12].
4. In 1980, the discovery of the endothelium-derived relaxing factor allowed
formulation of a novel concept in the pathogenesis of hypertension [13].
Nevertheless, it took long seven years to determine the identity of this factor and to
accept that it corresponds to NO [14, 15].
5. In 1981, Granger et al. [16] showed that tissue damage of ischemia/reperfusion in cat
intestine was caused by increased ROS generation.
From those years until now, hundreds of further researches were needed to achieve our
present knowledge on how oxidative stress is implicated in diverse and seemingly unrelated
diseases.
2.2. Generation of Reactive Oxygen and Nitrogen Species
The generation of ROS is a physiological and normal attribute of any kind of aerobic life.
In mammalian, under physiological conditions, cells metabolize approximately 95% of the
oxygen (O2) to water, without formation of any toxic intermediates. Water if formed
according to the following tetravalent reaction:
O2 + 4H+ + 4e– → 2H2O
The first impressions about oxygen as an element were made by the Swedish researcher
C.W. Scheele in the XVIII century. However, it was only in XX century when it was
demonstrated what Scheele himself had already anticipated that O2 in its pure state at high
pressure and concentration is toxic for animals, and herein for several life forms. The later
was followed by new interesting discoveries, generating the controversy called until these
days as “the oxygen paradox”
Several investigations from the last thirty years were needed to agree that, in normal
conditions, a minimal 5% of O2 is metabolized through univalent reduction, following four
different reactions or stages:
Reaction 1: O2 + e → O2•–
Reaction 2: O2•– + e → H2O2
Reaction 3: H2O2 + e → •OH
Reaction 4: •OH + e → H2O
Indeed, the final product is still H2O. However, through these four reactions three highly
toxic species are formed, two of them being free radicals: O2•– and hydroxyl radical (•OH).
Hydrogen peroxide (H2O2) is still a highly reactive compound, but not a radical in strict
sense.
This four stages model was the first to be discovered, and in fact it explains in general
terms the mitochondrial generation of ROS in normal cellular metabolism. The intermediates
do not leave the complex before the process is finished, but in some pathophysiological
conditions ROS can leave the respiratory burst. On the other hand, once synthesized, NO
might follow different pathways:
1. Diffusion to neighbor cells. The presence of an unpaired electron on its molecule

allows NO to interact with transition metals, derived from different enzymes, to
modulate its activity [17, 18]. The diffusion coefficient of this gas depends on lipids
and proteins on its microenvironment.
2. Autooxidation: Usually it occurs at severe high concentrations of NO. In the
presence of O2 it becomes into dinitrogen trioxide (N2O3) [19, 20]. This reaction
increases when it takes place in hydrophobic sites, such as the inside part of lipid
membranes or proteic nucleus [20, 21]. The molecule of N2O3 is a powerful nitrosant
agent, with great affinity for nucleophilic sites [18].
3. Reaction with superoxide. The half-life of NO and therefore its biological activity
is decisively determined by O2•– concentration [22]. This reaction has a limited-
diffusion kinetic curve, and thus it is thought that it rules the destination of NO in the
presence of O2•– [23, 24]. The final product is ONOO-, a highly oxidant RNS similar
to •OH in terms of toxicity. Therefore, ONOO- formation represents a major
potential pathway of NO reactivity, depending on the rates of tissue O2•– production.
In mammalian cells ROS might be formed through different pathways, either

enzymatically or non-enzymatically. For instance, the generation of O2•–, as well as other
ROS, requires cell activation involving alteration of the cell membrane structure what in turn
activates the generation of lipid peroxidation product molecules. In the context of this
chapter, relevant pathways will be described below.
a. Fenton reaction. This reaction has been known since 1894 and is currently one of
the most powerful oxidizing reactions available. The reaction involves H2O2 and a
ferrous iron catalyst. The peroxide is broken down into a hydroxide ion and a •OH.
The latter is the primary oxidizing species and can be used to oxidize and break apart
organic molecules.
Fe(II) + H2O2 → Fe(III) + •OH + −OH) [25].
It is well known that organic compounds can be easily oxidized. One primary advantage
of the Fenton's Reaction is that it does not produce further organic compounds or inorganic
solids such as permanganate and dichromate, since there is no carbon in the peroxide. This
makes the Fenton's Reaction more appealing than a biological process, if the goal is removal
of organic compounds.
The mechanism of reaction with respect to hydrogen peroxide is very complex and may
change with conditions of the reaction.
b. Haber-Weiss reaction:
The one-electron reduction of hydrogen peroxide by superoxide has also been invoked as
a potential source of •OH:
O2•− + H2O2 → O2 + •OH + OH−
This scheme has been exhaustively investigated and it is now generally accepted that the
Haber-Weiss reaction does not occur in the absence of metal catalysis.
This reaction combines a Fenton reaction and the reduction of Fe(III) by O2•–, yielding
Fe(II) and O2
Fe(III) + O2•− → Fe(II) + O2 [26].
c. Xanthine oxidase: The enzyme xanthine oxidase (XO) catalyzes the oxidation of
hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric
acid, generating O2•−. This enzyme plays an important role in the catabolism of
purines in some species, including humans. Under pathological conditions, such as
tissue ischemia, xanthine dehydrogenase can be converted to XO.
xanthine + H2O + O2 → uric acid + O2•−
d. NADPH oxidase: The enzyme NADPH oxidase (Nox) catalyzes the one electron
reduction of O2 to generate O2•−, using NADPH as the source of electrons. This
enzyme has a complex function that is most easily understood in the context of the
activated neutrophil, wherein it generates large amounts of toxic superoxide anion
and other ROS important in bactericidal function. In addition, it is also functional in
membranes of vascular endothelial and VSMC, and fibroblasts providing a
constitutive source of O2•−. This enzyme consists of several membrane-bound
subunits (gp91, Nox, and p22phox) and cytosolic subunits (p47phox, p67phox,
p40phox, and Rac2). There appear to be at least three isoforms of NADPH oxidase
expressed in the vascular wall.
e. Nitric oxide synthase: NO synthases (NOS) are a family of enzymes that convert
the amino acid L-arginine to L-citrulline and NO. All NOS isoforms are
homodimeric enzymes that require the same substrate (L-arginine), cosubstrates
(molecular oxygen, NADPH) and cofactors such as FMN, FAD, tetrahydrobiopterin
(BH4) and hem group.
Three main isoenzymes exist in mammals that are regulated by distinct genes: a
constitutive neuronal NOS (nNOS or NOS I), an endotoxin- and cytokine-inducible NOS
(iNOS or NOS II) and a constitutive endothelial NOS (eNOS or NOS III). Neuronal NOS
performs an important role in intracellular communication. Inducible NOS uses NO to induce
oxidative stress on pathogens. Endothelial NOS plays a major role in the regulation of
vascular function. For instance, eNOS synthesizes NO by a two-step oxidation of the amino
acid L-arginine thereby leading to activation of guanylyl cyclase (sGC). The resulting second
messenger cGMP in turn activates the cGMP-dependent kinase, which leads to decrease in
intracellular Ca+2 concentrations thereby causing vasorelaxation.
However it has become clear, from studies with the purified enzyme, that eNOS may
become uncoupled in the absence of the NOS substrate L-arginine or the cofactor
tetrahydrobiopterin (BH4). Uncoupled state results in the production of O2•− rather than NO
[27, 28].
The key mechanisms causing eNOS uncoupling are attributed to a decrease in
intracellular BH4 levels caused either by ONOO--induced BH4 oxidation or by decreased
activity of the guanosine triphosphate cyclohydrolase I enzyme and the dihydrofolate
reductase, both related to BH4 synthesis [29].
f. Mieloperoxidase: The mieloperoxidase enzyme (MPO) produces hypochlorous acid

(HOCl) from H2O2 and chloride anion (Cl-) during the neutrophil's respiratory burst.
It requires heme as a cofactor. In addition, it oxidizes tyrosine to tyrosyl radical
using H2O2 as oxidizing agent [30]. Both HOCl and tyrosyl radical are cytotoxic, and
used by the neutrophil to kill bacteria and other pathogens.
g. Cytochrome P450: The membrane-bound microsomal monooxygenase is a
multienzyme system that generally summarizes as cytochrome P450 (C-P450), as the
terminal oxidase and an FAD/FMN-containing NADPH-cytochrome P450 reductase
(CPR). The most common reaction catalyzed by the C-P450 is a monooxygenase
reaction. This might be, for example, the insertion of one atom of oxygen into an
organic substrate (RH) while the other oxygen atom is reduced to H2O
RH + O2 + 2H+ + 2e– → ROH + H2O
One ROS-generating way is given by ferric P450. Once bounded to the substrate, ferric
P450 reduces CPR by accepting its first electron, thereby being reduced. Then, this new
ferrous hemoprotein binds an oxygen molecule to form oxycomplex, which is further reduced
to give peroxycomplex. The input of protons to this intermediate can result in the heterolytic
cleavage of the O–O bond, producing H2O and the ‘oxenoid’ complex, the latter of which
then inserts the heme-bound activated oxygen atom into the substrate molecule. Finally, the
decomposition of this final one-electron-reduced ternary complex results in O2•– release. The
second ROS-producing branch is the protonation of the peroxycytochrome P450 with the
formation of H2O2 [31].
2.3. Pathophysiological Conditions
In pathophysiological conditions, the main sources of ROS include the mitochondrial

respiratory electron transport chain, XO activation through ischemia–reperfusion, the
respiratory burst associated with neutrophil activation, and arachidonic acid (AA)
metabolism.
Activated neutrophils produce O2•– as a cytotoxic agent as part of the respiratory burst
via the action of membrane-bound NADPH oxidase on O2. Neutrophils also synthesize NO
that can react with O2•– to produce ONOO-, a powerful oxidant, which may decompose to
form •OH.
Additionally, in ischemia-reperfusion XO catalyzes the formation of uric acid with the
co-production of O2•–. The enhanced O2•– released results in the recruitment and activation of
neutrophils and their adherence to endothelial cells, which in turn stimulates the formation of
XO in the endothelium, with further O2•– production as a positive feedback model pathway.
Accordingly, allopurinol, a XO inhibitor, has been demonstrated that blocks the O2•–
production in ischemia–reperfusion settings involving organs such as heart [32], liver [33],
kidney [34], and small intestine [35].
2.4. Reactive Oxygen Species as Mediators of Cell Damage
As mentioned previously, ROS have physiological functions that are essential in cells,
such as mitochondrial respiration, prostaglandin production pathways and host defense [36].
Moreover, NO plays an important role in antagonizing the vasoconstrictor effects of
Angiotensin II (Ang-II), endothelins and ROS [37].
However, ROS have well known involvement in common-shared pathophysiological
models causing cell damage, either directly or through behaving as intermediates in diverse
signaling pathways, including DNA damage, protein oxidation and lipid peroxidation
resulting, among others, in membrane damage [38].
2.4.1. DNA Damage

Oxidative DNA modifications are frequent in mammalian and have been suggested as
important contributory factors to the mechanism in carcinogenesis, diabetes and natural
aging. The DNA damages are considered as the most serious ROS-induced cellular
modifications as DNA is not synthesized de novo but copied, perpetuating by this way those
modifications and hence inducing mutations and genetic instability.
The main responsible ROS of DNA damage is •OH, which reacts with all components of
the DNA molecule, damaging both purine and pyrimidine bases and the deoxyribose
backbone. This is explained by the diffusion-limited •OH ability to add to double bonds of
DNA bases, abstracting a hydrogen atom from the methyl group of thymine and each of the
five carbon atoms of 2-deoxyribose [39]. Further reactions of base and sugar radicals
generate a variety of modified bases and sugars, base-free sites, strand breaks and DNA-
protein cross-links.
In addition, RNS such as ONOO- and •NO have also been implicated in DNA damage
[40]. This can be explained by the following mechanisms:
A. Direct Damage to DNA through Reactive Nitrogen Species:
• Endogenous formation of carcinogenic N-nitrosamine molecules:
N-nitrosamines are chemical molecules with known carcinogenic ability, because of their
conversion to strong alquilant agents. They are synthesized through the reaction of N2O3 and
biogenic amines:
RNH2 + N2O3 → RNHO + NO2–
RNHNO → RNNOH → ROH + N2
Endogenous production of these compounds has been demonstrated in immortalized

hepatocytes with the SV 40 apes virus.
• Basis deamination:
Primary amines from N-nitrosamine might generate diazonium ions, which transform to
alcohols, as shown in the following reaction:
RNN+ + H2O → ROH + N2 + H+
The presence of these amines in the main structure of DNA nitrogenated bases shows
that they can be deaminated by NO via N2O3, thereby generating punctual alterations with
mutagenic potential [41, 42]. In vitro experiments have provided evidence suggesting that
bases deamination through this mechanism seems to have an aimed mutation pattern to puric
bases, even though they can also affect pirimidinic ones.
The most common mutations are the guanine to adenine transition and back forward. In
addition, generation of modified bases, such as oxanin derived from guanine, is also a
frequent source of unspecific crossing over between DNA and proteins [43, 44].
Subsequently, DNA is affected either by a mechanism causing genomic instability of the
molecule, given by crossing over, or through a suicide mechanism of substrate for enzymatic
repairing [43].
It has been demonstrated, in vitro, a higher frequency of simple oligonucleotides chains,
rather than double ones. This suggests that the mutagenic mechanism occurs when basis are
unprotected, likely in replication and transcription, in which double spiral is open [42].
• Bases oxidation:
In cultured cells, protocols on activated macrophages show oxidative and deamination

damage of DNA [17]. Further analysis of the NO final onset revealed that most part of it was
transformed to ONOO- [45]. The treatment of DNA plasmids with synthetic ONOO- , and its
insertion into biological systems for replication and further analysis, confirmed a range of
specific mutations, mainly transversions from guanine to timine, and guanine to cytosine
[46].
The oxidant power of ONOO- is also enough to directly damage sugar and creates sites
with no nitrogenated bases on DNA, as well as oxidizing and modifying bases thus
generating hard-reparation class bases [47].
The production of DNA damage through this mechanism also occurs mostly in simple
chain DNA.
B. Indirect Modifying DNA Sequence by Reactive Nitrogen Species
Some authors have suggested that either deamination, oxidation and DNA chain rupture
by RNS requires extremely high concentrations of these species, a situation that would be
exceptionally possible in humans. Moreover, in vivo, some antioxidants molecules such as
ascorbate and reduced glutathione (GSH) are abundant, thus the RNS possibilities of
accumulation at enough concentrations to produce direct DNA damage are extraordinarily
low [48].
One of the suggested hypotheses is based on the inhibition of the DNA repairing
enzymatic systems, thereby making possible indirect damage. The RNS have a high affinity
for the thiol group (-SH) of cysteine [49] and it is believed that those enzymes containing
critic cysteine for their activity might be inhibited through RNS. Other nucleophilic groups,
such as hydroxyl (-OH) from tyrosine [50] and amine (-NH2) of lysine [23] are also
potentially modifiable.
All of the mechanisms exposed before contribute to elucidate from diverse points of view
the mutagenic effects of NO. While being on the right position, these mutations could result
in the inactivation of suppressor tumor genes, and further activation of oncogenes, thus
participating in various stages of carcinogenic process.
The most evident example of this is given by the protein for p53 gen, which is mutated
on nearly 50% of human tumors [51]. Previous researches confirmed in vitro mutations in
p53 gene induced by NO and its methylation [52]. Further investigations also verified that
there is a significant relation between the RNS activities and the mutations on p53 gene in
early staged lung carcinoma [53].
Even though in these studies the functionality of the genetic product was not analyzed,
the hypothesis on the role of NO is clear enough to consider that NO, in stress conditions
such as inflammation, is able to inactivate p53 gene, therefore to create a favorable
environment for tumors emerging and development.
2.4.2. Lipid Peroxidation

It is known that ROS attack cellular components involving polyunsaturated fatty acid
(PUFA) residues of phospholipids, which are extremely sensitive to oxidation [54]. The
overall process of lipid peroxidation consists of three stages: initiation, propagation, and
termination [55, 2]. Once formed, peroxyl radicals can be rearranged via a cyclization
reaction to endoperoxides, being malondialdehyde (MDA) the final product [56]. The MDA
is a minor lipid peroxidation product generated by heating of endoperoxides derived from

arachidonic acid. The main PUFA in tissue is linoleic acid, five times more abundant than
arachidonic acid. Linoleic acid generates only traces of MDA [57], but it is transformed as
easily as arachidonic acid to peroxyl radicals. The F2-isoprostanes are useful to demonstrate
the occurrence of non-enzymatic lipid peroxidation processes, nevertheless they are only
trace products formed through free radicals catalyzed attack on esterified arachidonate,
providing a reliable tool to identify population with enhanced rates of lipid peroxidation [58].
Lipid peroxidation involves low-density lipoprotein (LDL) as well as high-density
lipoprotein (HDL) oxidation. It is well known that the LDL oxidation is a key process in the
pathogenesis of atherosclerosis (chapter 3) [59]. The oxidized cholesterol esters are directly
incorporated into lipoproteins and transferred to endothelial cells via the LDL where they
induce damage and start the sequence of events leading to atherosclerosis.
2.4.3. Protein Oxidation

The side chains of all amino acid residues of proteins are susceptible to oxidation by the
action of ROS [60]. The protein carbonyl group is generated by ROS through many different
mechanisms and its concentration is a good measure of protein oxidation via oxidative stress.
The NO reacts rapidly with O2•– to form the highly toxic ONOO- that is able to nitrosate the
cysteine sulfhydryl groups of proteins, to nitrate tyrosine and tryptophan residues of proteins
and to oxidize methionine residues to methionine sulfoxide [1]. Oxidation of proteins is
associated with a number of age-related diseases and aging [61, 62].
2.4.4. Other Damage

Oxidative damage to the mitochondrial membrane can also occur, resulting in membrane
depolarization and the uncoupling of oxidative phosphorylation, with altered cellular
respiration [63]. This can ultimately lead to mitochondrial damage, with release of
cytochrome c, activation of caspases and apoptosis [64].
2.5. Antioxidant Defense System
All forms of life maintain a reducing environment within the cells. The maintenance of
this status is achieved possibly through the antioxidant defense system, which is in action to
protect cellular homeostasis against harmful ROS produced during normal cellular
metabolism, as well as in the pathophysiological states. The antioxidant system is preserved
by antioxidant substances that maintain the reduced state by a constant input of metabolic
energy.
Antioxidant substances are small molecules that can scavenge free radicals by accepting
or donating an electron to eliminate the unpaired condition. Typically, this means that the
antioxidant molecule becomes a free radical in the process of scavenging a ROS to a more
stable and less reactive molecule. In most cases the scavenger molecule provides hydrogen
radical that combines with the free radical. Consequently, it is generated a new radical that
has an enhanced lifetime compared with the starting one, for instance, due to a conjugated
system [59]. The extended lifetime of this radical enables it to react with a second radical by
formation of a new molecule and thus one scavenger molecule can eliminate two radicals.
Antioxidant molecules can be produced endogenously or provided exogenously through
diet or antioxidant supplements. The main endogenous antioxidant enzymes are SOD,
catalase (CAT), and glutathione peroxidase (GSH-Px). The SOD converts superoxide anion
to H2O2, which is a substrate for CAT and GSH-Px. Catalase metabolizes H2O2 to water and
oxygen and GSH-Px reduces both H2O2 and organic hydroperoxides when reacting with GSH
[65]. Reduced glutathione is present at high concentrations in all mammalian cells, especially
in the renal cells, hepatocytes, and erythrocytes [66]. This tripeptide protects protein thiol
groups from non-enzymatic oxidation or as a co-substrate of GSH-Px [67]. The endogenous
antioxidant defense system is summarized on Table 1.
Exogenous antioxidants, such as vitamins E and C, exist at a number of locations namely
on the cell membrane, intracellularly and extracellularly. They react with ROS to either
remove or inhibit them. The hydrophobic lipid interior of membranes requires a different
spectrum of antioxidants. Fat-soluble vitamin E is the most important antioxidant in this
environment, which protects against the loss of membrane integrity.
Fat-soluble antioxidants are important in preventing membrane polyunsaturated fatty
acids (PUFA) from undergoing lipid peroxidation. Glutathione removes already generated
radical, if no radicals are present, the PUFA cannot be attacked. Therefore, they shield the
membrane rich in PUFA against ROS [68]. In addition, water-soluble antioxidants including
vitamin C play a key role in scavenging ROS in the hydrophilic phase.
Other small antioxidant molecules are also naturally present in the plasma, such as uric
acid and bilirubin. Recently, it was found that fish, fish oils, and some vegetables contain
furan fatty acids that are radical scavengers, partly responsible for the beneficial efficiency of
a fish diet [69].
Table 1-1. Functional characteristics of the antioxidant enzymes
Antioxidant Chemical Scavenged General characteristics

enzyme name oxidant agent
GSH-Px Glutathione H2O2 It is the major endogenous antioxidant molecule
peroxidase It catalyzes the conversion of H2O2 and organic peroxides
into water or alcohols, respectively.
SOD Superoxide O2•- It catalyzes the conversion of O2•− to O2
dismutase and to less-reactive species like H2O2.
Necessary for the release of biologically active NO.
It protects NO from inactivation.
CAT Catalase H2O2 It catalyzes the breakdown of H2O2 to water and molecular
oxygen.
3. Reactive Oxygen Species as Factors

for Diseases Development
In physiological conditions, both enzymatic and non-enzymatic systems preserve the
oxidant/antioxidant status. However, these systems are overwhelmed during oxidative stress,
which is a metabolic derangement due to an imbalance caused either by an excessive
generation of ROS or by a diminished capacity of the antioxidant defense system.
In a simplistic manner, it could be considered that diseases are the result of cell
functioning disorders that may lead or not into systemic alterations as a chain reaction result.
Impairment of cell function might be caused by several factors, typically more than one
acting at the same time, enhancing the same pathophysiological pathway or other. On the
other hand, it has been discussed the most important conditions involving ROS either as
direct cellular damage agents or as mediators implicated in pathophysiological pathways [70,
71].
Reactive oxygen species are thought to contribute to the pathogenesis of a number of
seemingly unrelated disorders, including type 2 diabetes, cancer and aging, heart failure,
hypertension, preeclampsia and atherosclerosis, among others. All of these pathologies were
important causes of morbidity and mortality on the twentieth century, and have been
extensively studied over the past few years. Urbanization, aging and globalized lifestyle
changes combine to make chronic diseases, while in-between them deleterious effect of ROS
seems to be a constant. In order to further explain this, some examples of widely known
syndromes will be briefly described as follow:
3.1. Cardiovascular Disease
Cardiovascular diseases are the first cause of death in the world. Recent estimations
consider that its prevalence will keep on rising for the next decades to come [72], due to an
increase in older population and non healthy lifestyle. Reactive oxygen species have a key
role in the homeostasis of the vascular wall and there is compelling evidence pointing to ROS
as important factors for the development of cardiovascular disease.
3.1.1. Hypertension
Hypertension is probably the most prevalent chronic disease in the world. It is also
known that its incidence is still arising, especially in emergent countries. However, it seems
that this does not apply in developed countries. In U.S.A, for example, the overall prevalence
is 29.3%, or 65 million people, and it has not increased significantly since 1999 [73]. It is a
silent and harmful condition, as it constitutes an asymptomatic disease on early stages but
represents a key risk factor for many other diseases such as heart or brain stroke, cardiac
insufficiency or chronic renal failure, among others.
Over the past fifty years notable therapeutic advances, particularly pharmacological ones,
have been made for the treatment and control of hypertension. Reactive oxygen species are
thought to contribute to the pathogenesis of hypertension through an impairment of
endothelial cells and through uncoupled eNOS. Chronic oxidative stress causes senescence of
endothelial cells. This is characterized by a detachment of endothelial cells or part of the

endothelial cell membrane. With the persistence of oxidative stress, the capacity of
neighboring endothelial cells to repair endothelial injury is limited, and vascular integrity
becomes dependent on the incorporation of endothelial progenitor cells, with lower NO
synthesizing capacity [74]. This is called endothelial dysfunction, which has been clearly
associated with prognosis in patients with heart failure [75], essential hypertension [76], and
peripheral artery disease [77].
In most situations where endothelial dysfunction due to increased oxidative stress is
encountered, the expression of the eNOS has been shown to be paradoxically increased rather
than decreased [78-81]. The mechanisms underlying increased expression of eNOS are likely
to be secondary to increased endothelial levels of H2O2, which increases the expression of
eNOS at the transcriptional and translational level [82]. An interesting marker of endothelial
dysfunction is the asymmetric dimethyl L-arginine (ADMA) that can compete with L-
arginine for eNOS and therefore reduce eNOS-derived NO production. Increased ADMA
levels have been found in patients with risk factors such as chronic smokers, patients with
hypercholesterolemia, and in patients with diabetes and renal insufficiency [83]. There is also
some evidence that ADMA may even cause eNOS uncoupling, thereby switching eNOS from
a NO to O2•− producing enzyme. Several in vivo animal studies have acknowledged that
uncoupled eNOS is a significant O2•− source in diverse pathological conditions, including
angiotensin II hypertension [84], and chronic congestive heart failure [85].
Additionally, an increased synthesis of O2•− reduces NO bioavailability by inactivation,
leading to ONOO- formation. Then, the following ONOO- protonation will break off,
liberating the highly peroxidant •OH. Endothelium is affected by this reaction in two
different ways: 1) Nitric oxide scavenging impairs its vasodilating activity, leading to
permanent high blood pressure and 2) Hydroxyl radical causes damage in endothelial cells
perpetuating by this way, resulting in a vicious cycle [85].
3.1.2. Stroke and Atherosclerosis

The relationship between high stroke risk and chronic oxidative stress has been widely
documented. This is mainly due to endothelial dysfunction. Nitric oxide has potent
antiatherosclerotic properties because once released from endothelial cells, it works in
concert with prostacyclin to inhibit platelet aggregation [86]. Nitric oxide blocks the adhesion
of neutrophils to endothelial cells and the expression of adhesion molecules. It is interesting
to point out that at high concentrations NO also inhibits the proliferation of smooth muscle
cells [87]. Therefore, under all conditions where an absolute or relative NO deficit is
encountered, the process of atherosclerosis is being initiated or accelerated.
In normal conditions, the physiological stimuli for blood vessels to release NO are shear
stress and pulsatile stretch. In order to assess the endothelial function, an intra-arterial
infusion of acetylcholine (ACh) is used in the clinic. Once infused into the brachial artery,
ACh causes a dose-dependent vasodilation. In the coronary artery the inability for a normal
vasodilation response or some degree of vasoconstriction entirely depends on the functional
integrity of the endothelium. In the presence of cardiovascular risk factors and endothelial
dysfunction ACh will cause vasoconstriction due to stimulation of muscarinergic receptors in
the media [83].
3.2. Metabolic Syndrome and Diabetes
The metabolic syndrome (MS) includes a variety of cardio-metabolic abnormalities

associated with a high risk of developing type 2 diabetes and cardiovascular disease. The
pathophysiological basis of MS is a relative insulin deficit, given by a resistance of either
peripheral or hepatic insulin action. Chronic oxidative stress impairs insulin action, as was
demonstrated in type 2 diabetics. This impairment might be due to several factors, such as
membrane fluidity alterations, decreased availability of NO and increased intracellular
calcium content [88]. Oxidative stress is strictly influenced by glycometabolic control either
in type 1 or type 2 diabetics [89, 90]. In type 2 diabetics, if glycemic control improves the
oxidative stress-related parameters, such as thiobarbituric acid reactant substances, if the
latter decrease, the same trend seems to occur for the NO2-/NO3- ratio and cGMP content
[91].
It has also been demonstrated that insulin treatment nearly corrects the oxidative stress in
type 1 diabetics but only improves it in type 2 diabetics [92]. Because the period of insulin
treatment and the HbA1c values were similar, the authors suggested the existence of
metabolic differences between the two types of diabetes. Even if in diabetes mellitus the
postprandial hyperglycemic spikes have been considered to be a critical event in the
pathogenesis of micro and macroangiopathic complications [93]. It is obvious that all the
metabolic pathways associated with these spikes increase free radical synthesis and worsen
the oxidative state. Indeed, in diabetic patients the oxidative stress, physiologically induced
by a standard meal, is enhanced. Diabetic patients show, during the postprandial period, an
increase in MDA plasma levels and a decrease in sulphydryl groups, α-tocopherol and total
radical-trapping antioxidant parameter [94]. The latter observation might be related to the
significantly lower levels of urate and ascorbic acid observed in diabetic patients, although
dietary intake should be considered when explaining changes in plasma antioxidant levels
[95].
3.3. Neurodegenerative Diseases
3.3.1. Alzheimer's Disease

A major obstacle in the research for treatment of Alzheimer's disease (AD) is the lack of
knowledge about the etiology and pathogenesis of selective neuron death. In recent years,
considerable data have been accumulated indicating that the brain in AD is under increased
oxidative stress and this may have a role in the pathogenesis of neuron degeneration and
death in this disorder. The following evidence strongly supports the role of oxidative stress in
AD:
1. Increased brain Fe, Al, and Hg found in AD is capable of stimulating radical species.
2. Increased lipid peroxidation and decreased PUFA in the AD neurons, with a
concomitant increase of 4-hydroxynonenal, an aldehyde product of lipid
peroxidation, is found in AD ventricular fluid.
3. Increased protein and DNA oxidation is a characteristic of an AD patient brain.
4. Diminished energy metabolism and decreased cytochrome c oxidase levels are

observed in AD brains.
5. Advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite,
heme oxygenase-1 and SOD-1 are found in neurofibrillary tangles and AGE, heme
oxygenase-1, SOD-1 in senile plaques of AD brains.
6. A number of studies show that amyloid beta peptide is capable of generating free
radicals.
Supporting indirect evidence comes from a variety of in vitro studies showing that free
radicals are capable of mediating neuron degeneration and death. Overall, these studies
indicate that free radicals are possibly involved in the pathogenesis of neuron death in AD.
Because tissue injury itself can induce oxidative stress, it is not known whether ROS
generation is a primary or secondary event.
Even if free radical generation is secondary to other initiating causes, they are deleterious
and part of a cascade of events that can lead to neuron death, suggesting that therapeutic
efforts aimed to the removal of ROS or prevention of their formation may be beneficial in
ameliorating the development of AD [96].
3.3.2. Parkinson’s Disease

Parkinson’s disease (PD) is the most common neurodegenerative disease, and the
majority of PD cases involve the sporadic form of PD. Although the etiology of the sporadic
form is unknown, mitochondrial dysfunction and oxidative stress are considered to play a
prominent role in its pathogenesis.
The discovery of the genes that are linked to a rare familial form of PD has provided
crucial insights into the molecular mechanisms involved in the pathogenesis of PD. Recent
findings implicate mitochondrial dysfunction associated with oxidative damage and abnormal
protein accumulation (ubiquitin/proteosome pathway) as the key molecular mechanisms
compromising dopaminergic neurons in familial PD. Mutations in Parkin, PTEN-induced
kinase 1 (PINK1) and DJ-1 are found in autosomal recessive forms of PD. Recent studies on
these genes suggest the central importance of mitochondrial dysfunction and oxidative stress
in PD.
The above mentioned 3 proteins may be biologically related to each other and may
protect the mitochondria against oxidative stress and other harmful stimulations. In particular,
Parkin seems to be the most important factor that improves the mitochondrial dysfunction
[97].
3.4. Pre-Eclampsia
Pre-eclampsia is a human pregnancy-specific disorder that adversely affects the mother,

by vascular dysfunction, and the fetus through intrauterine growth restriction. The incidence
of preeclampsia is about 5% of all pregnancies, and it constitutes the leading cause of
maternal mortality in developed countries. Preeclampsia is characterized by vasospasm,
increased peripheral vascular resistance, and thus reduced organ perfusion [98].
The etiology and pathogenesis of this pregnancy syndrome remains poorly understood.
There is substantial evidence to suggest that the diverse manifestations of preeclampsia,
including altered vascular reactivity, vasospasm, and discrete pathology in many organ
systems, are derived from pathologic changes within the maternal vascular endothelium.
The key event leading to the clinical manifestations of preeclampsia is endothelial cell
dysfunction likely caused, among other factors, by an increase in ROS and RNS
concentration [99]. Defective spiral arteries remodeling causes reduced uteroplacental
perfusion, which primarily may contribute to intrauterine growth restriction. In addition,
maternal dyslipidemia or a primary or secondary decrease of antioxidants makes
preeclampsia increasingly to develop. However, the mechanisms involved in induction of
endothelial cell dysfunction remain to be determined.
There is evidence for increased nitrotyrosine formation in the preeclampsia placenta due
to ONOO- production, perhaps arising from local NO production. In addition, increased
xanthine oxidase generation of O2·- and either regionally decreased or inadequate SOD could
also be involved.
Then, oxidative stress may be the point at which multiple factors converge resulting in
endothelial cell dysfunction and the consequent clinical manifestations of preeclampsia [100].
3.5. Glaucoma
Increasing evidence indicates that ROS play a key role in the pathogenesis of primary
open angle glaucoma (POAG), the main cause of irreversible blindness worldwide. Oxidative
DNA damage is significantly increased in the ocular epithelium regulating aqueous humor
outflow. This is demonstrated in the appearance of the trabecular meshwork (TM) of
glaucomatous patients compared to controls. The pathogenic role of ROS in glaucoma is
supported by various experimental findings, including the followings:
a Resistance to aqueous humor outflow is increased by H2O2, which induces TM

degeneration.
b Trabecular meshwork possesses remarkable antioxidant activities, mainly related to
SOD, CAT and glutathione pathways that are altered in glaucoma patients.
c In glaucoma patients, intraocular pressure and severity of visual-field defects
increase in parallel to the amount of oxidative DNA damage affecting TM.
Vascular alterations, which are often associated with glaucoma, could contribute to the
generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal
cells, seems to be involved in the neuronal cell death affecting the optic nerve in POAG.
The highlighting of the pathogenic role of ROS in POAG has implications for the
prevention of this disease. The latter is supported by the growing number of studies using
genetic analyses to identify susceptible individuals, and of clinical trials testing the efficacy
of antioxidant drugs for POAG management. [101]
3.6. Aging and Cancer
Aging has drawn attention to an issue that is of particular relevance to the organization of
health systems: the increasing frequency of multimorbidity. in the industrialized world, as
many as 25% of 65–69 year olds and 50% of 80–84 year olds are affected by two or more
chronic health conditions simultaneously [72]. Aging can be defined as a progressive decline
in the ability of the organism to resist stress, damage, and disease. Although there are
currently over 300 theories to explain the aging phenomenon, it is still not well understood
why organisms age and the reason why the aging process can vary so much in speed and
quality from individual to individual. The oxidative stress hypothesis is one of the prevailing
theories of aging. This theory states that ROS produced during cellular respiration damage
cell lipids, proteins and DNA, accelerate the aging process and increase the risk of disease. It
has been hypothesized that the production of free radicals is dependent upon resting
metabolic rate and this may have an impact on the aging process [102].
Damage of DNA and thus cancer, is pathophysiologically related with ROS, as
mentioned in diverse parts of this chapter.
3.7. Renal Damage
Considerable experimental evidence supports the view that ROS could play a key role in
the pathophysiological processes of renal diseases [103], including chronic renal failure,
hemodialysis, rhabdomyolysis-induced acute renal failure, renal fibrosis, glomerulosclerosis,
kidney stones formation, and hyperlipidemia, among others. The abundance of PUFA makes
the kidney an organ particularly vulnerable to ROS attack [104]. The involvement of ROS in
the mechanism of renal damage is supported by two lines of experimental evidence: (i)
detection of products of oxidant injury in renal tissue or urine, and (ii) experimental
demonstration of a protective effect of metabolic inhibitors of ROS [105], such as antioxidant
vitamins or antioxidant compounds proper of the Mediterranean diet. It is thought that
oxidative stress up-regulates the expression of adhesion molecules, chemoattractant
compounds and inflammatory cytokines [106].
The glomerulus is considerably more sensitive to oxidative injuries than other nephron
segments. Oxidative stress may alter the structure and function of the glomerulus because of
the effects of ROS on mesangial and endothelial cells [107]. Reactive oxygen species are
increasingly believed to be important intracellular signaling molecules in mitogenic pathways
involved in the pathogenesis of glomerulonephritis. In mesangioproliferative
glomerulonephritis, the increase of ROS is thought to be produced by a pronounced
dysregulation of pro-oxidative and anti-oxidative enzymes leading to a net increase in
glomerular ROS levels [108].
4. Conclusions and Perspectives

As a final conclusion, it should be mentioned that oxidative stress plays a major role in
cellular damage, and it is involved in several mechanisms leading to a variety of unrelated
diseases. The data exposed in this chapter supports the view that the different interaction
between oxidant and antioxidant agents, and thus ROS and/or RNS generation, is somehow
implicated in a wide range of disease development. Treatments that enhance the antioxidant
system are expected to effectively ameliorate cell damage. It should be expected an
amelioration on cell damage by means of enhancing the antioxidant system.
The following chapters will discuss the possible mechanisms by which ROS and/or RNS
are linked with the pathogenesis and the development of well known and highly prevalent
human diseases and the studies performed to counteract ROS deleterious effects, by means of
a reinforcement of the antioxidant defense system.
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Chapter II
Hypertension
Ramón Rodrigo
Institute of Biomedical Sciences,
Abstract
Reactive oxygen species (ROS) and reactive nitrogen species play a key role in the
modulation of the vasomotor system. Thus, ROS are recognized as mediators of the
vasoconstriction induced by angiotensin II, endothelin-1 or urotensin-II, among others;
while nitric oxide (NO) is a major vasodilator. In physiological conditions, low
concentrations of intracellular ROS play an important role in normal redox signaling
involved in maintaining vascular function and integrity. In addition, under
pathophysiological conditions ROS contribute to vascular dysfunction and remodeling
through oxidative damage. The fact that ROS play a key role in development of
hypertension is supported by the findings of increased production of superoxide anion
and hydrogen peroxide, reduction of NO synthesis, and a decrease in bioavailability of
antioxidants in human hypertension. In both animal models and humans, increased
blood pressure has been associated with an excessive endothelial production of ROS
(oxidative stress) which may be both a cause and an effect of hypertension.
Antioxidants, whether synthesized endogenously or exogenously administered, are
reducing agents that neutralize these oxidative compounds before they can cause damage
to biomolecules. In the management of hypertension and other cardiovascular diseases,
the primary interest was focused on the therapeutic possibilities of antioxidants to target
ROS, thus avoiding hypertensive end-organ damage. The use of antioxidant vitamins,
such as vitamin E and vitamin C, has gained considerable interest for their role as
protecting agents against vascular endothelial damage, in this way contributing to
ameliorate chronic diseases, beyond its essential function associated to body deficiencies.
However, promising findings from experimental investigations, the results of clinical
trials aimed to demonstrate antihypertensive effects of antioxidant supplementation are
disappointing. Nevertheless, the methodology used in some of these studies makes them
26 Ramón Rodrigo
a matter still to be debated. Some studies reported a potential antihypertensive effect,

particularly when using association of two or more antioxidants. Even more, antioxidant
diets low in fat, have found to be of most significant benefit in hypertensive patients.
Taken together data are consistent with the view that while an antioxidant alone has
not yet demonstrated its efficacy as a therapeutic antihypertensive agent, the synergistic
actions among the various antioxidants appear to be effective to counteract the ROS
effect on the vascular wall. These effects could arise from their complex biological
actions, from their ability not solely to scavenge ROS, but also to prevent their formation
through down regulation of NADPH oxidase and up-regulation of endothelial NO
synthase and antioxidant enzymes.
1. Introduction
Hypertension is considered to be the most important risk factor in the development of
cardiovascular disease worldwide [1]. In recent years, oxidative stress has gained widespread
attention as one of the fundamental mechanisms responsible for the development of
cardiovascular morbidities. Although reactive oxygen species (ROS) have an important role
in the homeostasis of the vascular wall, an excessive ROS contributes to impaired
endothelium-dependent dilation by decreasing nitric oxide (NO) bioavailability, a
pathophysiological condition that leads to hypertension. Increased ROS may be a risk factor
for cardiovascular events such as unstable angina, myocardial infarction and sudden death.
The understanding of the biological processes that generate ROS and the intracellular signals
elicited by ROS is most relevant to gain insight into the pathogenesis of diseases such as
hypertension.
An increasing body of evidence suggests that oxidative stress could be a contributing
factor to the underlying pathophysiological mechanism of hypertension [2-4]. Thus,
increased production of superoxide anion and hydrogen peroxide, reduced NO synthesis, and
decreased bioavailability of antioxidants have been demonstrated in experimental and human
hypertension.
The vasculature is a rich source of ROS, which under pathological conditions play an
important role in vascular injury, as well as in hypertensive end-organ damage. Vascular
ROS are produced in endothelial, adventitial, and smooth muscular cells, and derived
primarily from NADPH oxidase that produces superoxide anion when stimulated by
hormones such as angiotensin II (Ang-II), endothelin-1 (ET-1) and urotensin II (U-II), among
others. In addition, increased ROS production may be generated by mechanical forces, such
as both unidirectional laminar and oscillatory shear stress occurring during elevation of blood
pressure. Reactive oxygen species function as intracellular second messengers to increase
intracellular calcium concentration, a major determinant of vasoconstriction, thereby
contributing to the pathogenesis of hypertension. In addition, induction of other signaling
cascades leads to vascular smooth muscle cell growth and migration, expression of pro-
inflammatory mediators, and modification of extracellular matrix. Significantly reduced
acetylcholine-mediated vasodilation has been partly attributed to elevated ROS and decreased
NO bioavailability [5]. Since the regulation of vasomotor tone is dependent upon a delicate
balance between vasoconstrictor and vasodilator forces resulting from the interaction of the
Hypertension 27
components of the vascular wall and the blood, and both of them can be altered by oxidative
stress, the cellular events triggered by ROS significantly contribute to the mechanism of
hypertension. These findings have stimulated the interest on antihypertensive therapies
targeted against free radicals by decreasing ROS generation and/or by increasing NO
bioavailability. Accordingly, antioxidants may be useful in minimizing vascular injury and
thereby prevent hypertensive end-organ damage. The next sections of this chapter present the
available information pointing to a role of oxidative stress in the mechanism of production of
high blood pressure, as well as data related with the use of antioxidants in the prevention or
treatment of this pathology.
2. Pathophysiology of Hypertension
The role of oxidative stress in the pathophysiology of hypertension will be analyzed on
the basis of the interaction of the vascular wall components and effects of vasoactive
hormones and factors, in settings altering the vascular homeostasis.
2.1. Role of the Vascular Wall Components
The integrity of the vascular wall, composed by endothelium, smooth muscular cells and
adventitia, is critical for the maintenance of vascular homeostasis, including the modulation
of blood pressure. The regulation of vascular tone may be impaired by changes affecting the
interaction between these vascular cells. A description of the physiological role of the
vascular wall components is given below.
2.1.1. Endothelium
The vascular endothelium is formed by a monolayer of cells that separates the blood
from the interstitial compartment and the vascular smooth muscle. It is an autonomous organ
that serves not just as a barrier of the transvascular diffusion but is the largest endocrine
organ in the body. Endothelial cells adhere to one another through junctional structures
formed by transmembrane adhesive proteins that are responsible for homophilic cell-to-cell
adhesion. Adherent junctions and tight junctions are the main types of junction. Another kind
of junction, the gap junction, allows cells to communicate with each other. The endothelium
senses mechanical stimuli, such as pressure and shear stress, and hormonal stimuli, such as
vasoactive substances. In response, it releases agents that regulate vasomotor function, trigger
inflammatory processes, and affect hemostasis. From the physiological viewpoint, the
endothelium is characterized by a wide range of important homeostatic functions. It
participates in the control of blood coagulation and fibrinolysis, platelet and leukocyte
interactions with the vessel wall, regulation of vascular tone and of blood pressure. Many
crucial vasoactive endogenous compounds are produced by the endothelial cells to control
the functions of vascular smooth muscle cells and of circulating blood cells. These complex
systems determine a fine equilibrium which regulates the vascular tone. Impairments in
endothelium-dependent vasodilation lead to the so called endothelial dysfunction. The
28 Ramón Rodrigo
endothelium modulates the balance between opposing mechanisms that are

vasodilatation/vasoconstriction, pro-coagulant/antithrombotic, cell proliferation/apoptosis.
This dynamic tissue layer constitutes a source and/or target of multiple growth factors and
vasoactive mediators involved in regulating the physical and biochemical properties of the
systemic vessels, as well as vascular contractility and cell growth. There is no doubt that
endothelium plays a regulatory and protective role by generating vasorelaxing substances.
However, under pathophysiological processes and circumstances, endothelium-derived
vasoconstricting factors can dominate and contribute to deleterious effects. Under some
pathophysiological circumstances, e.g. in atherosclerosis or hypertension, endothelium
derived vasoconstricting factors can be released and contribute to the paradoxical
vasoconstrictor effects. Apart from the peptides ET-1, Ang-II and U-II, other endothelium-
derived vasoconstricting agents such as superoxide anions, vasoconstrictor prostaglandins,
and thromboxane A2 have been postulated. Indeed, the vascular reactions are the result of a
complex interaction of many vasoactive pathways since there are numerous interactions
between some of the vasoactive agents released from the endothelium. In normal conditions
many factors that stimulate ET-1 synthesis, (e.g. thrombin, Ang-II), also cause the release of
vasodilators such as PGI2 and/or NO, which oppose the vasoconstricting action of ET-1.
Enhanced production of ET-1 also stimulates mitogenic activity on smooth muscle cells
while NO and PGI2 inhibit this proliferative effect [6].
2.1.2. Vascular Smooth Muscle Cells

Functional integrity of vascular smooth muscle cells (VSMC) is essential for good
performance of the vasculature. They can modify the luminal diameter, which enables blood
vessels to maintain an appropriate blood pressure. In addition, VSMC perform other
functions, which become progressively more important during vessel remodeling in
physiological conditions such as pregnancy and exercise, or after vascular injury. In these
cases, VSMC synthesize large amounts of extracellular matrix components and increase
proliferation and migration. Because of these properties, VSMC are fit not only for short-
term regulation of the blood vessel diameter, but also for long-term adaptation, via structural
remodeling by changing cell number and connective tissue composition. The main function
of vascular smooth muscle tonus is to regulate the caliber of the blood vessels in the body.
Excessive vasoconstriction leads to hypertension having physical deleterious effects on the
vascular wall, such as tensile stress caused by pressure and the shear stress caused by flow.
Tensile stress is the force exerted by blood perpendicular to the vessel wall, whereas shear
stress is the dragging frictional force created by blood flow as a tangential pressure on the
vessel wall. As a consequence of both effects, a remodeling is induced in vessel wall by
changing VSMC characteristics. The effects of shear stress are mediated by the endothelium,
which coordinates the response of VSMC to this mechanical stress. In contrast to
endothelium-modulated shear stress, stretch acts directly on the VSMC. Mechanical forces
appear to enhance the expression of both extracellular matrix and contractile proteins by
VSMC in the vessel wall. The molecular mechanisms involved in redox-sensitive cell growth
control are poorly understood. Stimulation of cultured VSMC with xanthine/xanthine oxidase
increases proliferation, whereas stimulation with hydrogen peroxide causes growth arrest of
VSMC. In VSMC, ROS mediate many pathophysiological processes, such as growth,
Hypertension 29
migration, apoptosis and secretion of inflammatory cytokines, as well as physiological

processes, such as differentiation, by direct and indirect effects at multiple signaling levels. In
VSMC it was reported that H2O2 activates phospholipase C through tyrosine phosphorylation
and that this activation has a major role in rapid [Ca2+]i mobilization in this type of cells [7].
2.1.3. Adventitia
The tunica external of blood vessels, also known as the tunica adventitia, is a connective
tissue coat mainly composed by fibroblasts and collagen. It has been shown that vitamin C is
essential for the synthesis of collagen and its deficiency lead to scurvy, an alteration caused
by the fact that collagen cannot maintain the blood vessel walls, as collagen serves to anchor
them to nearby organs. The importance of the vascular adventitia has been recognized in
normal maintenance and homeostasis of vessels as well as in vascular disease. The response
of adventitial fibroblast to injury, stretch, cytokines, and hormones can lead to stimulate
collagen deposition, differentiation, migration, and proliferation. This response is
characterized by increased ROS production by adventitial fibroblast NADPH oxidase,
considered an initiator of vascular disease and remodeling. In addition, another source of
ROS may be generated by some stimuli, such as Ang-II, causing adventitial accumulation of
macrophages enriched in NADPH oxidase, giving rise to a paracrine effect. Therefore, the
adventitia can contribute to hypertension by either reducing NO bioavailability or
participating in vascular remodeling.
2.2. Role of Vascular Active Hormones and Factors
2.2.1. Acetylcholine
Since the discovery in 1980 that acetylcholine (ACh) requires the presence of endothelial
cells to elicit vasodilation, the importance of the endothelial cell layer for vascular
homeostasis has been increasingly recognized. In vascular vessels with healthy endothelium,
ACh induces endothelium-dependent dilation via endothelial muscarinic membrane
receptors, which are specific G-protein-coupled receptors, leading to a sequence of Ca2+-
dependent events that induce the production of endothelial factors, mainly NO by stimulating
endothelial NO synthase (eNOS). Nitric oxide then diffuses to underlying VSMC, where it
activates guanylyl cyclase to produce cyclic GMP, thus inducing vascular smooth muscle cell
relaxation. Administration of exogenous ACh to endothelial cells produces these mediators to
cause vasodilation. However, muscarinic cholinergic vasodilation is impaired in the presence
of endothelial damage, as occur in coronary atherosclerosis. In this setting ACh may promote
smooth muscle-mediated vasoconstriction, a paradoxical vasoconstriction occurring early as
well as late in the course of coronary atherosclerosis suggesting that the abnormal vascular
response to ACh may represent a defect in endothelial vasodilator function, what may be
important in the pathogenesis of coronary vasospasm. In addition, the response to ACh could
be reduced by either inhibitors of NOS or cyclooxygenase [8]. It should be emphasized that
the diminution in NO bioavailability will lead to significantly reduced ACh-mediated
vasodilation [5]. The consequence of an overall increase in ROS is a reduced ability of
endothelium to cause vasodilation, thereby accounting for a role of oxidative stress in the
30 Ramón Rodrigo
elevation of blood pressure. Accordingly, as an example seen in diabetes mellitus, a setting

involving oxidative stress, the relaxation response of aorta to ACh was found to be
significantly decreased compared with control subjects, and the antioxidant resveratrol
restored the response to ACh [9].
2.2.2. Renin-Angiotensin System

The renin–angiotensin system (RAS) plays a key role in the development and
pathophysiology of hypertension and cardiovascular disease. In hypertension small and large
arteries undergo structural, mechanical and functional changes that contribute to vascular
complications and increased cardiovascular risk. The major effects are vasoconstriction,
endothelial damage and cell growth. Angiotensin-II is the end product of the RAS cascade, a
potent vasoactive peptide that can be formed at various sites: in vascular beds rich in
converting enzyme. When Ang-II production increases above normal levels, it induces
vascular remodeling and endothelial dysfunction in association with increases in levels of
blood pressure. As a potent activator of NADPH oxidase, Ang-II through the type 1 Ang-II
(AT1) receptor contributes to the production of ROS which participate in a number of
different pathologies within the circulatory system [10]. In contrast, NO not solely
antagonizes the effects of Ang-II on vascular tone, cell growth, and renal sodium excretion,
but also down-regulates the synthesis of angiotensin converting enzyme (ACE) and AT1
receptors. Thus, inhibition of NO synthesis with Nω-nitro-L-arginine methyl ester (L-NAME)
induces both increase in blood pressure and heart hypertrophy. The development of oxidative
stress may enhance this effect of Ang-II, supported by the finding that Ang-II–dependent
hypertension is particularly sensitive to NADPH oxidase–derived ROS. In addition, the
repeated administration of Ang-II leads to up-regulation of NADPH oxidase activity. In rats
and mice made hypertensive by Ang-II infusion, expression of NADPH oxidase subunits
(Nox1, Nox2, Nox4, p22phox), oxidase activity, and generation of ROS are all increased [11,
12]. To investigate the role of NADPH oxidase–derived ROS production in the pathogenesis
of Ang-II–sensitive hypertension, various mouse models with altered NADPH oxidase
subunit expression have been studied. In p47phox knockout mice and in gp91phox (Nox2)
knockout mice, Ang-II infusion fails to induce hypertension, and these animals do not show a
significant increase in superoxide production, vascular hypertrophy, and endothelial
dysfunction observed in Ang-II–infused wild-type mice [13, 14]. Studies in Nox1-deficient
mice demonstrated that vascular superoxide production is reduced, and blood pressure
elevation is blunted, in response to Ang-II [15, 16], whereas in transgenic mice in which
Nox1 is over expressed in the vascular wall, Ang-II–mediated vascular hypertrophy and blood
pressure elevation are enhanced [17]. Numerous signaling pathways activated in response to
Ang-II and ET-1 are mediated through the increased level of oxidative stress, which seems to
be in causal relation to a number of cardiovascular disturbances including hypertension. On
the other hand, ACE inhibition up-regulates eNOS expression. Captopril and enalapril
prevented blood pressure rise in young spontaneously hypertensive rats. Captopril, probably
due to the antioxidant role of its thiol group, had more effective hypotensive effect than
enalapril [18], further supporting the role of oxidative stress in the mechanism of
hypertension. In Ang-II–infused mice treated with siRNA targeted to renal p22phox, renal
NADPH oxidase activity was blunted, ROS formation was reduced, and blood pressure
Hypertension 31
elevation was attenuated, suggesting that p22phox is required for Ang-II–induced oxidative
stress and hypertension [19]. Treatment with apocynin or diphenylene iodinium, two
pharmacological inhibitors of NADPH oxidase, reduced vascular superoxide production,
prevented cardiovascular remodeling, and attenuated the development of hypertension in
Ang-II–treated mice [11, 20]. In the vasculature, the endothelial as well as adventitial
NADPH oxidase is composed of gp91phox (Nox2) and p22phox, as well as p47phox and
p67phox and the G protein Rac1. In vitro studies in VSMC support Ang-II–stimulated Nox1
expression in a protein kinase C (PKC)-dependent fashion [21]. Use of PKC inhibitor
GF109203X efficiently inhibited PKC activity, decreased Nox1 basal expression, and
abolished Ang-II-induced up-regulation of Nox1 expression. The use of anti-sense Nox1
mRNA in rats completely inhibited Ang-II-induced superoxide production, supporting a role
for Nox1 in redox signaling in VSMC. Thus, increased expression of both the endothelial and
smooth muscle gp91phox homologues in all the layers of the vessel participate in the
superoxide production, which occurs in a PKC dependent fashion [22]. The occurrence of
oxidative stress uncouples eNOS, leading to further enhancement in superoxide production.
The ability of Ang-II to induce endothelial dysfunction is also due to its ability to down-
regulate the downstream target of NO soluble guanylyl cyclase, thereby leading to impaired
NO/cGMP signaling. Recent studies have demonstrated that autoantibodies against Ang-II
type 1 receptor are present in women with preeclampsia. These autoantibodies isolated from
the sera of preeclamptic patients behave as Ang-II agonists inducing vasoconstriction in a
concentration-dependent fashion. The agonistic effect was completely blocked by losartan, an
AT1-receptor antagonist [23]. In addition, the agonistic autoantibodies induce signaling in
vascular cells including activating protein-1 and nuclear factor kappa B (NF-κB) activation
that results in ROS generation [24].
2.2.3. Endothelin-1
Endothelins are potent 21 amino acid vasoconstrictor isopeptides produced in different
vascular tissues, including vascular endothelium. Endothelin-1 is the main endothelin
generated by the endothelium and probably the most important in the cardiovascular system.
When ET-1 is administered in large concentrations, it behaves as a potent vasoconstrictor
capable of exerting an array of physiological effects, including the potential to alter arterial
pressure and circulatory function. Endothelin-1 mediates its effects through two membrane
G-protein coupled receptors, ETA and ETB, which exhibit a wide tissue distribution including
the endothelial cells, VSMC and adventitial fibroblasts [25]. Endothelin-1 acts through ETA,
present only on smooth muscle cells and having mitogenic properties and also mediating
contractions. The ETB receptor is located both on smooth muscle cells, where they evoke
contractions, and on endothelial cells, inducing relaxation. In the peripheral vasculature, ETA
receptors are expressed primarily on the surface membrane of VSMC where they mediate, in
large part, the potent and characteristically sustained vasoconstrictor response associated with
administration of exogenous ET-1 peptides [26]. Administration of exogenous ET-1 to an
intact normotensive animal produces a classic, transient hypotension and vasodilation that is
mediated via ETB receptors through enhanced generation of NO and prostaglandin-related
substances, a response that precedes ETA-mediated vasoconstriction [27]. In the vasculature,
the proendothelin may be released from the non-luminal surface of the endothelial cells and
32 Ramón Rodrigo
converted extracellularly to mature ET-1 by membrane-bound endothelin-converting

enzymes, which are neutral metalloproteinases. Endothelin-1 does not appear to be stored in
endothelial cells, but is rather synthesized de novo in response to several substances
(thrombin, Ang-II, cytokines) or physical stimuli (shear stress, hypoxia). Endothelin-1 is a
potent vasoconstricting agent with long lasting effects. In normal conditions there are
numerous interactions between some of the vasoactive agents released from the endothelium.
Many factors that normally stimulate ET-1 synthesis, (e.g. thrombin, Ang-II) also cause the
release of vasodilators such as PGI2 and/or NO, which oppose the vasoconstricting action of
ET-1. On smooth muscles cells ET-1 also stimulates mitogenic activity, while NO and PGI2
inhibit this proliferative effect. It should be mentioned that ETB receptors involved in the
pressor responses triggered by ET-1 are importantly involved in the plasma clearance of the
endogenous peptide. In addition, the endothelial ETB receptor acts as an important modulator
of ETA receptor-mediated pressor effects [28]. Endothelin-1 stimulates ROS production
through the activation of NADPH oxidase, xanthine oxidase, lipoxygenase, uncoupled NO
synthase, and mitochondrial respiratory chain enzymes. With respect to arterial hypertension
development, NADPH oxidase seems to be the main enzyme responsible for superoxide
production [3]. It was reported that essential hypertension is characterized by increased ET-1
vasoconstrictor tone, an effect that seems to be dependent on decreased endothelial ETB-
mediated NO production attributable to the impaired NO bioavailability. In such conditions
endothelial ETB-induced vasodilation no longer compensates for the direct classical ET-1
vasoconstrictor effect mediated by smooth muscle cell ETA and ETB receptors [29]. Over-
expression of human ET-1 in mice also induces vascular remodeling and impairs endothelial
function, via activation of NADPH oxidase [30]. Infusion of ET-1 increases NADPH
oxidase-dependent superoxide production; however, preventing this increase in ROS
generation does not inhibit development of hypertension in these animals [31].
2.2.4. Urotensin-II
Human urotensin-II (U-II) is a potent vasoactive peptide, indeed the most potent
vasoconstrictor identified. Urotensin-II is a peptide composed of 11 amino acid residues with
a structure similar to somatostatin that was firstly isolated from a fish. Subsequently, human
U-II and its receptor were identified. In rat thoracic aorta U-II triggers powerful
vasoconstrictor activity, with effects on pulmonary artery smooth muscle cells [32]. This
action is brought about via activation of a Gq/11-protein coupled receptor (UT receptor).
Urotensin-II activation of the UT receptor increases inositol phosphate turnover and
intracellular Ca2+concentration. However, the constrictor response to U-II appears to be
variable and highly dependent on the vascular bed examined. Vasoconstriction is not its only
effect; U-II and its receptor have been demonstrated in the central nervous system, where U-
II induces a cardiovascular, behavioral, motor and endocrine response and in the kidney,
where it seems to influence renal hemodynamics but also salt and water excretion, in rat
pancreas where it inhibits insulin secretion, in the heart where it seems to play a role in
cardiac hypertrophy and fibrosis. In humans high plasma or urine levels of U-II have been
described in some pathologic conditions.
U-II has also been shown to act as a potent vasodilator in some isolated vessels; for
example, human small pulmonary and abdominal arteries [33]. In addition to these vascular
Hypertension 33
actions, U-II is a positive inotrope in human right atrial trabeculae and also exhibits
arrythmogenic activity [34]. Human U-II and its UT receptor display greatest expression in
the peripheral vasculature, heart, and kidney [35], although both are found in other tissues,
notably the central nervous system [36]. It also appears that it plays a relatively minor role in
health, as shown by knockout studies in mice and infusion studies in humans. The role of U-
II in disease is not well elucidated. Urotensin-II is expressed in endothelial cells,
macrophages, macrophage-derived foam cells, and myointimal and medial VSMC of
atherosclerotic human coronary arteries. UT receptors are present in VSMC of human
coronary arteries, the thoracic aorta and cardiac myocytes. Lymphocytes are the most active
producers of U-II, whereas monocytes and macrophages are the major cell types expressing
UT receptors. The recent detection of this potent vasoconstrictor in human tissue, and the
identification of its receptor in the spinal cord, heart lungs, blood vessels, and brain, have
made U-II a major focus of current clinical research and a potential target for future human
pharmacotherapy.
2.2.5. Norepinephrine
Vascular smooth muscle is innervated primarily by the sympathetic nervous system
through adrenergic receptors (adrenoceptors), which are G protein–coupled receptors. Three
types of adrenoceptors are present within VSMC: α1, α2 and β2. The main endogenous
agonist of these cell receptors is norepinephrine (NE). Norepinephrine stimulates VSMC
proliferation through α1-adrenergic receptors via the activation of the Ras/mitogen activated
protein kinase (MAPK) pathway, it also stimulates phospholipase D activity in VSMC, an
enzyme that catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and
choline and whose activation by neurotransmitters, hormones, or growth factors has been
implicated in a wide range of cellular responses, including cellular trafficking, inflammatory
and immune response, mitogenesis, cellular differentiation, and apoptosis. In addition, over-
expression of iNOS increases blood pressure via central activation of the sympathetic
nervous system, which is mediated by an increase in oxidative stress. [37]. In turn, Ang-II
enhances sympathetic nervous system activity centrally and peripherally, but the exact
mechanisms of this activation are not well established.
2.2.6. Nitric Oxide

The primary role of the endothelial cells is the modulation of the vascular tone, by
producing vasodilator and vasoconstrictor factors. The endothelial cell-derived relaxing
factor (EDRF), which was originally described by Furchgott and Zawadzki [38], has been
identified as NO and is now known to play an important role as a key paracrine regulator of
vascular tone. Nitric oxide has been found to play many diverse physiological roles ranging
from a neurotransmitter, a vasodilator to a cytotoxic agent. Physiologically, NO inhibits
leukocyte–endothelial cell adhesion, VSMC proliferation and migration, and platelet
aggregation to maintain the health of the vascular endothelium. Therefore it has many
beneficial effects, including inhibiting thrombosis, inhibiting inflammation, promoting
survival of endothelial cells, and inhibiting recruitment of macrophages to the vessel wall.
The decrease in bioavailable NO in the vasculature reduces vasodilatory capacity and
contributes to hypertension. The enzyme that catalyzes the formation of NO from oxygen and
34 Ramón Rodrigo
arginine is NO synthase (NOS), a very complex enzyme containing several cofactors and a
heme group which is part of the catalytic site. Indeed, there is a family of NOS, flavo-heme
enzymes that catalyze a stepwise oxidation of L-arginine to form NO and L-citrulline. The
NOS isoforms differ with respect to the main mode of regulation, the tissue expression
pattern and the average amount of NO produced [39]. Endothelial NOS (eNOS), expressed in
endothelial cells, is the predominant NOS isoform in the vessel wall. Receptor-mediated
agonist stimulation (e.g. bradykinin, acetylcholine, thrombin, histamine) leads to rapid
enzyme activation by depalmitoylation, binding to calmodulin/calcium, displacement of
caveolin and release from the plasma membrane [40]. In addition, shear stress is also an
important modulator of eNOS activity. Endothelial NOS activity is also regulated by
allosteric modulators [41]. Nitric oxide activates guanylyl cyclase by binding to the heme
moiety of this enzyme. Guanylyl cyclase catalyzes the conversion of guanosine triphosphate
(GTP) to cGMP, which in turn activates cGMP-dependent protein kinase. Except the
vasorelaxing and antiproliferative properties per se, NO plays an important role in
antagonizing the effects of Ang-II, endothelins and ROS. It was shown previously that ACE
inhibition up-regulates eNOS expression. The mechanism of this up-regulation is still
unclear. However, it is conceivable that ACE inhibitor-induced accumulation of endogenous
kinins mediates this effect [42]. All NOS isoforms are homodimeric enzymes that require the
same substrate (L-arginine), cosubstrates (molecular oxygen, NADPH) and cofactors such as
FMN, FAD, tetrahydrobiopterin, or heme. Tetrahydrobiopterin (BH4) is bound tightly within
NOS and this enables it to remain bound in NOS through multiple catalytic turnovers; it
reduces the ferric heme-superoxy intermediate that forms during oxygen activation, and
becomes an enzyme-bound BH4 radical in the process [43]. Nitric oxide is released by the
endothelium and is a gas that bubbles from the endothelial cell to the VSMC. Nitric oxide
diffuses to the adjacent smooth muscle where it interacts with different receptor molecules, of
which the soluble guanylyl cyclase (sGC) is the best characterized and presumably most
important one with regard to control of vessel tone and smooth muscle proliferation.
Activation by NO requires sGC heme-iron to be in the ferrous (II) state. Upon NO binding,
cGMP formation will increase substantially. Cyclic GMP in turn activates the cGMP-
dependent kinase I which in turn will increase the open probability of Ca2+-activated K+(BK)-
channels, thereby inducing a hyperpolarization of the VSMC and inhibition of agonist-
induced Ca2+ influx. During a relatively short time period, our knowledge on the role of
endothelium and NO in cardiovascular diseases has tremendously increased. It is accepted
that the normal reduction of NO plays a crucial role in the maintenance of the physiologic
conditions within the cardiovascular system. L-arginine, a substrate for eNOS, seems to be
promising in preserving NO formation. However, L-arginine failed to prevent blood pressure
increase and left ventricle remodeling due to chronic treatment with L-NAME, an inhibitor of
eNOS [44]. Some other effects of L-NAME, besides blood pressure increase and NO
deficiency, could participate in this lack of L-arginine protection. It has been demonstrated
that L-NAME inhibits L-arginine transport to the caveolae containing NOS [45]. Moreover,
L-NAME increased the activity of NF-κB, which may participate in cardiovascular
remodeling independently of the blood pressure increase [46]. The angiotensin converting
enzyme inhibitor captopril completely prevented NO-deficient hypertension, yet without
improving NOS activity. It was suggested that both inhibition of Ang-II formation and
Hypertension 35
enhanced production of PGI2 caused by increased bradykinin level may be responsible for
observed protective effect of captopril. Thiols protect NO from oxidation by scavenging
oxygen-free radicals and by forming nitrosothiols, both effects prolonging NO half-life and
duration of NO action [47, 48]. Interestingly, aldosterone receptor blocker spironolactone
was also able to prevent degradation of thiol groups and to increase the expression of eNOS
protein, two effects associated with blood pressure reduction [49, 50]. It seems that not the
absolute NO production but the relative balance between vasodilators and vasoconstrictors is
decisive. Nitric oxide is able to reduce generation of ROS by inhibiting association of
NADPH oxidase subunits. The balance between NO and Ang-II in the vasomotor centers
seems to play important role in the regulation of the sympathetic tone. Reduced NO levels
can be attributed to oxidative stress that is related to elevated levels of ROS, such as
superoxide and hydrogen peroxide, together with peroxynitrite. Elevated NADPH oxidase
expression and activity leads to high superoxide levels. Superoxide combines with NO to
form peroxynitrite that oxidizes BH4 and destabilizes eNOS to produce more superoxide [51,
52], thus further enhancing the development of oxidative stress (see below). Increased
oxidative stress in the vasculature, however, is not restricted to the endothelium and also
occurs within the smooth muscle cell layer. Increased superoxide production has important
consequences with respect to signaling by the sGC and the cGMP-dependent kinase I, which
activity and expression is regulated in a redox-sensitive fashion [53].
2.2.7. Prostaglandins
Prostacyclin (PGI2), another endothelium-dependent vasodilator, relaxes the underlying
VSMC through activation of adenylyl cyclase and subsequent generation of cAMP.
Constitutively released PGI2 appears to be involved in the regulation of resting vascular tone.
Prostacyclin is released in higher amount in response to ligand binding on the cell surface
such as thrombin, arachidonic acid, histamine, or serotonin. The endothelium has an
important function in maintaining vascular tone, which is mediated in part by the enzymes
prostaglandin H2 synthase that uses arachidonic acid as a substrate, forming prostaglandin H2
(PGH2). Prostaglandin H2 is converted to vasoactive molecules, such as PGI2 and
thromboxane, via specific synthases (prostacyclin synthase and thromboxane synthase,
respectively). Prostaglandin H2 synthase (PGHS) has an inducible isoform (PGHS-2), which
is oxidant sensitive through the activation of NF-κB, a response also shown by inducible
NOS. The isoform PGHS-2 may mediate vascular dysfunction in conditions characterized by
oxidative stress. In addition, enhanced NOS activity in an environment of oxidative stress
would result in scavenging of NO by superoxide anion generated by endothelial cells and
VSMC, forming the potent pro-oxidant peroxynitrite, thus reducing NO bioavailability as a
vasodilator. Peroxynitrite can contribute to the altered vascular reactivity in a variety of
conditions in which the clinical manifestations are mediated by oxidative stress. Thus,
peroxynitrite inhibits the enzymatic activity of prostacyclin synthase, thereby causing
impairment in the PGI2-mediated vasodilation. The mechanism by which peroxynitrite
inhibits prostacyclin synthase activity involves the nitration of tyrosine 430 [54, 55]. As
nitration of this tyrosine residue disrupts the catalytic activity, it has been postulated that this
tyrosine residue is embedded in the heme region and is crucial for electron transfer. The
36 Ramón Rodrigo
physiological and pathophysiological implications of prostacyclin synthase nitration by

peroxynitrite remain to be evaluated by appropriate in vivo experiments.
2.2.8. Endothelium Dependent Hyperpolarizing Factor and Leukotrienes

The endothelium controls vascular tone not only by releasing NO and PGI2 but also by
other pathways causing hyperpolarization of the underlying VSMC. Early experimental
evidence suggested that, beside the cyclooxygenase and the NOS pathways, an additional
endothelial pathway had to be involved to fully explain endothelium-dependent relaxations.
Furthermore, it is of interest to note that the endothelial monolayer behaves as a conductive
tissue propagating an electrical signal along the axis of the blood vessel by means of
homocellular gap junctions and throughout the vascular wall itself by means of myo-
endothelial gap junctions. It has been suggested that endothelium-dependent relaxations,
independent of the production of NO and PGI2, probably play an important role in
cardiovascular physiology in the animal and in the human [56]. Therefore, a yet unidentified
endothelium-derived hyperpolarizing endothelial factor (EDHF) associated with the
hyperpolarization of the VSMC was suggested [57]. Although the nature of EDHF is still
controversial, this additional endothelial pathway, endothelium-dependent hyperpolarization,
has been demonstrated in many blood vessels of different species, including humans. This
factor is the major contributor to endothelium-dependent dilatations induced by agonists such
as ACh and bradykinin in small arteries [58]. Nevertheless, the EDHF cannot be defined as a
single factor or pathway which accounts for all features of EDHF signaling in different
vascular beds and species. This led to the assumption that there are several distinct EDHFs
acting alone, in parallel, or even together. Several candidate molecules/mediators have been
shown to act as EDHF in different tissues and species. These include K+, cytochrome P450
leukotriene metabolites (epoxyeicosatrienoic acids), lipoxygenase products, NO itself,
reactive oxygen species (H2O2), cyclic adenosine monophosphate, C-type natriuretic peptide,
among others. Electrical communication between endothelial and smooth muscle cells
through gap junctions (myoendothelial gap junctions) has also been suggested to be involved
in endothelium-dependent hyperpolarization. Endothelium generates a hyperpolarizing factor,
which is suspected to be an arachidonic acid metabolite produced by cytochrome P450. The
EDHF contributes to vasodilatation by acting on K+ channels. Under conditions of oxidative
stress, a decrease in the bioavailability of NO, as demonstrated in various states associated
with endothelial dysfunction, alleviates this intrinsic inhibition so that the activity of the
production of the vasodilator epoxyeicosatrienoic acids is increased. As a consequence of this
interaction, vascular responsiveness is thought to be at least partially maintained despite the
apparent loss of NO.
2.3. Vascular Oxidative Stress
Oxidative stress constitutes a unifying mechanism of injury of many types of disease

processes, it occurs when there is an imbalance between the generation of ROS and the
antioxidant defense systems in the body so that the latter become overwhelmed [59]. By far
the dominant situation is increased generation due to alterations in mitochondrial metabolism
Hypertension 37
and metabolism of fatty acids and carbohydrates. The ROS family comprises many molecules
that have divergent effects on cellular function, such as regulation of cell growth and
differentiation, modulation of extracellular matrix production and breakdown, inactivation of
NO, and stimulation of many kinases and proinflammatory genes [60-62]. Importantly, many
of these actions are associated with pathological changes observed in cardiovascular disease.
ROS are produced by all vascular cell types, including endothelial, smooth muscle, and
adventitial cells, and can be formed by numerous enzymes. Enzymatic sources of ROS that
are important in vascular disease and hypertension are xanthine oxidase, uncoupled NOS, and
NADPH oxidase. In pathological conditions, ROS production in vascular tissues, particularly
superoxide anions, has been implicated as playing an important role in vascular events such
as inflammation, endothelial dysfunction, cell proliferation, migration and activation,
extracellular matrix deposition, fibrosis, angiogenesis, all important processes contributing to
cardiovascular remodeling in hypertension, atherosclerosis, diabetes, cardiac failure,
myocardial ischemia-reperfusion injury, vascular remodeling after angioplasty and ischemic
stroke [63-65]. These effects are mediated through redox-sensitive regulation of multiple
signaling molecules and second messengers [37, 66, 67]. The elevation of blood pressure has
been associated with ROS abundance and frequently also with an impairment of endogenous
antioxidant mechanisms [3]. Superoxide, the first ROS formed by one electron reduction of
molecular oxygen, and superoxide-derived ROS have multiple pathophysiological actions in
the artery wall, including an impairment of endothelium-dependent vasodilation. In
agreement with this view, in human hypertension, biomarkers of systemic oxidative stress are
elevated [68]. Clinical studies have demonstrated that essential hypertensive patients produce
excessive amount of ROS [69, 70], and have abnormal levels of antioxidant status [71],
thereby contributing to the accumulating evidence that increased vascular oxidative stress
could be involved in the pathogenesis of essential hypertension [4, 72]. Recently, it was
demonstrated a strong association between blood pressure and some oxidative stress–related
parameters [73]; thus, systolic and diastolic blood pressures of hypertensives were negatively
correlated with plasma antioxidant capacity and positively correlated with both plasma and
urine 8-isoprostane, a recognized biomarker of oxidative stress in vivo. In the context of
oxidative stress in the vasculature it is particularly important to note that increased
superoxide reacts extremely rapidly with NO to form peroxynitrite, thereby elevating
vascular resistance and promoting vasoconstriction [74]. Formation of peroxynitrite is a
pathophysiological process, because NO is an essential endogenous vasodilator. Thus,
therapeutic strategies should aim to restore bioavailability of NO, scavenging ROS by
antioxidant agents.
2.4. Sources of Reactive Oxygen Species in the Vascular Wall
A variety of enzymatic and non-enzymatic sources of ROS exist in blood vessels.

Enzymatic sources of ROS include NADPH oxidases located on the cell membrane of
polymorphonuclear cells, macrophages and endothelial cells and cytochrome P450-dependent
oxygenases. The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase
provides another enzymatic source of both superoxide and H2O2 (and therefore constitutes a
38 Ramón Rodrigo
source of the highly reactive hydroxyl radicals) and has been proposed to mediate deleterious
processes in vivo. In addition to NADPH oxidase, the best characterized source of ROS,
several other enzymes may contribute to ROS generation, including NO synthase,
lipoxygenase, cyclo-oxygenases, xanthine oxidase and cytochrome P450 enzymes. It has
been suggested that also mitochondria could be considered a major source of ROS: in
situations of metabolic perturbation, increased mitochondrial ROS generation might trigger
endothelial dysfunction, possibly contributing to the development of hypertension. However,
the use of antioxidants in the clinical setting induced only limited effects on human
hypertension or cardiovascular endpoints.
2.4.1. NADPH Oxidase

The primary biochemical source of ROS in the vasculature, particularly of superoxide,
appears to be the membrane associated nicotinamide dinucleotide (phosphate)
(NADH/NADPH) oxidase enzyme complex [75], the major source of superoxide in the
vascular wall. This system catalyses the reduction of molecular oxygen by NADPH as
electron donor, thus generating superoxide. The function of this enzyme complex is most
easily understood in the context of the activated neutrophil, wherein it generates large
amounts of toxic superoxide anion and other ROS important in bactericidal function.
NADH/NADPH oxidase is also functional in membranes of vascular endothelial and VSMC,
and fibroblasts providing a constitutive source of superoxide anion. This enzyme consists of
several membrane-bound subunits (gp91, Nox, and p22phox) and cytosolic subunits
(p47phox, p67phox, p40phox, and Rac2) [76]. It forms an enzyme complex of VSMC [77].
There appear to be at least three isoforms of NADPH oxidase expressed in the vascular wall
(see chapter 1, for more details). Thus, although endothelial cells and adventitial fibroblasts
express a gp91phox-containing NADPH oxidase similar to that originally identified in
phagocytes, VSMC may rely on novel homologues of gp91phox, namely Nox1 and Nox4, to
produce superoxide. Upon assembly of the subunits in the membrane, this enzyme generates
a burst of superoxide [78]. NADPH oxidase is up-regulated in hypertension by humoral and
mechanical signals, and quantitatively this enzyme makes the largest contribution to ROS
production. Genetic and chemical manipulation of NADPH oxidase and of antioxidant
enzymes causes predictable changes in oxidative stress and endothelium-dependent function
in hypertension. The activity of the enzyme in endothelial as well as VSMC is increased upon
stimulation with Ang-II, the most studied stimulus of the vascular NADPH oxidase, although
other pressor agents such as ET-1 and U-II are also involved, thereby resulting in increased
ROS. This is consistent with the hypothesis that the pathological state of high blood pressure
is associated with loss of balance between status of oxidative stress and level of antioxidants.
In endothelium, adventitia, and cardiomyocytes, the agonist sensitive NADPH oxidase
appears to contain gp91phox. Unlike the phagocytic oxidase, in endothelial cells at least, the
gp91phox-based oxidase is constitutively assembled in a perinuclear location associated with
the cytoskeleton [79]. It is, however, responsive to stimulation with Ang-II, thrombin, and
ET-1, as well as to mechanical forces. Likely the most well known function of NADPH
oxidase derived superoxide is inactivation of NO to form peroxynitrite, leading to impaired
endothelium dependent vasodilation and uncoupling of eNOS to produce additional
superoxide [80]. This scenario, where NADPH oxidase derived superoxide activates other
Hypertension 39
enzymes in turn to produce ROS, may be a general mechanism for enhancing free radical
formation, because it has been shown that NADPH oxidases are upstream of activation of
xanthine oxidase (which also generates ROS) by oscillatory shear stress [81]. Superoxide
combines with NO, which is synthesized by eNOS, to form peroxynitrite. In turn,
peroxynitrite oxidizes and destabilizes eNOS to produce more superoxide [51, 52]. In the
vasculature, NADPH oxidase activation has been strongly associated with hypertension [82].
2.4.2. Xanthine Oxidase

It is an important source for oxygen free radical present in the vascular endothelium [82].
It catalyzes the last two steps of purine metabolism through the sequential hydroxylation of
hypoxanthine to yield xanthine and uric acid. During this process oxygen is reduced to
superoxide. The enzyme can exist in two forms that differ primarily in their oxidizing
substrate specificity. The dehydrogenase form preferentially utilizes NAD+ as an electron
acceptor but is also able to donate electrons to molecular oxygen. By proteolytic breakdown
as well as thiol oxidation xanthine dehydrogenase from mammalian sources can be converted
to the oxidase form that readily donates electrons to molecular oxygen, thereby producing
superoxide and hydrogen peroxide, but does not reduce NAD+. Although xanthine oxidase–
derived superoxide has been studied mainly in the context of cardiac disease, there is
evidence suggesting involvement in vascular dysfunction in hypertension. Spontaneously
hypertensive rats demonstrate elevated levels of endothelial xanthine oxidase and increased
ROS production, which are associated with increased arteriolar tone [84]. This may be
mediated in part through an adrenal pathway, because adrenalectomy reduces xanthine
oxidase expression [85]. In addition to effects on the vasculature, xanthine oxidase may play
a role in end-organ damage in hypertension. The enzyme inhibitor allopurinol can improve
cardiac hypertrophy in spontaneously hypertensive rats but having a minimal impact on blood
pressure [86], thereby supporting a role for xanthine oxidase in hypertensive end-organ
damage rather than in the development of hypertension per se. It was suggested that this
damage may be mediated through direct vascular effects of uric acid [87].
2.4.3. Uncoupled Endothelial NO Synthase

A third potential source of vascular ROS production is eNOS. Endothelial NOS is a
cytochrome P450 reductase-like enzyme that requires cofactors including BH4, flavin
nucleotides, and NADPH for transfer of electrons to a guanidino nitrogen of L-arginine to
form NO. L-arginine and BH4 deficiency are associated with uncoupling of the L-arginine-
NO pathway resulting in decreased formation of NO, and increased eNOS-mediated
generation of superoxide (and peroxynitrite). In agreement with this view, BH4 repletion
improves endothelial function in chronic smokers [88], and augments NO bioactivity in
hypercholesterolemic humans [89]. The BH4 deficiency, in turn, induces eNOS uncoupling,
resulting in the generation of superoxide anions from uncoupled eNOS, which decreases BH4
levels further–a vicious cycle causing endothelial dysfunction [90]. NADPH oxidase is the
initial source of ROS leading to BH4 oxidation. In fact, BH4 is highly sensitive to oxidation,
e.g., by peroxynitrite, and reduced levels of BH4 promote eNOS uncoupling. In addition,
supplementation with BH4 is capable of correcting eNOS dysfunction. Under various
pathological conditions, such as substrate/cofactor availability, eNOS activity becomes
40 Ramón Rodrigo
uncoupled, resulting in the production of superoxide rather than NO, thus contributing to the
development of hypertension. When NOS is uncoupled, electrons flowing from the reductase
domain to the heme are diverted to molecular oxygen instead of to L-arginine, resulting in the
formation of superoxide anion [91]. A number of potential mechanisms are responsible for
uncoupling of eNOS, although the most consistent evidence exists for BH4 deficiency [92].
This is an essential cofactor in the oxygenase domain and is proposed to have multiple roles
in all mammalian NOS isoforms. One of the presently accepted functions of BH4 is to act as a
one-electron donor during reductive activation of the oxyferrous complex of the heme. It was
reported that eNOS uncoupling is not simply a consequence of BH4 insufficiency, rather it
results from a diminished ratio of BH4 vs. its catalytically-incompetent oxidation product,
7,8,-dihydrobiopterin (BH2). The activity of NADPH oxidase is critically important in
producing ROS that ultimately oxidize BH4 in blood vessels of hypertensives. The loss of
BH4 alters the function of eNOS, resulting in diminished NO production and increased
production of ROS from the enzyme. Oral treatment with BH4 or NADPH oxidase deficiency
blunts the increase in blood pressure, suggesting that eNOS uncoupling contributes to the
progression of hypertension. A similar hypothesis to BH4 deficiency that causes eNOS
uncoupling has been also proposed for asymmetric dimethylarginine (ADMA) accumulation.
This compound is a naturally occurring amino acid resulting from proteolysis of methylated
arginine residues in proteins and it behaves as an endogenous inhibitor of eNOS. It competes
with L-arginine to inhibit eNOS for NO production [93]. In the presence of high
concentrations of ADMA, eNOS produces superoxide instead of NO. Thus, elevated levels of
ADMA and oxidative stress in hypertensive patients could contribute to the associated
microvascular endothelial dysfunction and elevated blood pressure. A recent study
demonstrated that hypertensive patients show an improvement of endothelial dysfunction by
treatment with nebivolol, a selective 1-adrenergic receptor antagonist. This effect may be
related to a diminution of circulating ADMA levels. Although the mechanism by which
nebivolol reduces circulating ADMA in these patients remains unclear, it was suggested that
the up-regulation of the expression of the enzyme that selectively degrades ADMA
(dimethylarginine dimethylaminohydrolase 2) may have a role [94].
2.4.4. Mitochondria and Microsomes

The mitochondrion is a major source and target of ROS. Thus, superoxide formation
occurs on the outer mitochondrial membrane, in the matrix and on both sides of the inner
mitochondrial membrane. Whilst the superoxide generated in the matrix is eliminated in that
compartment, part of the superoxide produced in the intermembrane space may be carried to
the cytoplasm via voltage-dependent anion channels [95]. Superoxide is enzymatically
converted to H2O2 by superoxide dismutase (SOD), a family of metalloenzymes (for more
details see chapter 1). The mitochondrial matrix contains a specific form of SOD, with
manganese in the active site (MnSOD), which eliminates the superoxide formed in the matrix
or on the inner side of the inner membrane. The expression of MnSOD is further induced by
agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated
by the oxidative activation of NF-κB. Catalase, a major H2O2 detoxifying enzyme found in
peroxisomes, is also present in heart mitochondria. In addition to cytochrome c, other
electron carriers appear to have a detoxifying role against ROS. Ubiquinol or coenzyme Q
Hypertension 41
has been shown to act as a reducing agent in the elimination of various peroxides in the
presence of succinate [96]. Thus, coenzyme Q is a source of superoxide when partially
reduced (semiquinone form) and an antioxidant when fully reduced. The inner mitochondrial
membrane also contains vitamin E, a powerful antioxidant that interferes with the
propagation of free radical-mediated chain reactions. Complex I produces most of the
superoxide generated by mammalian mitochondria in vitro during reverse electron transport
from succinate to NAD+. Complexes II and IV are not normally significant sites. The high
superoxide production from complex I during reverse electron transport is particularly
sensitive to mild uncoupling. Therefore mild uncoupling very effectively decreases the high
superoxide production that occurs from complex I during reverse electron transport.
Superoxide is reactive, but can be converted into hydrogen peroxide by SOD, then to oxygen
and water by catalase or glutathione peroxidase. However, superoxide that evades these
antioxidant systems (together with the secondary ROS it generates) can damage proteins,
lipids and DNA. Although the hydrogen peroxide produced by SOD is relatively unreactive,
it can form highly reactive hydroxyl radicals in the presence of ferrous ion via Fenton
chemistry and these hydroxyl radicals can initiate lipid peroxidation cascades in membranes.
Furthermore, the products of sugar, protein and lipid oxidation can cause secondary damage
to proteins. Thus mitochondrially-produced superoxide can be a major source of cellular
damage. There are two major side reactions: electrons may leak from the respiratory chain
and react inappropriately with oxygen to form superoxide. Glycerol 3-phosphate
dehydrogenase produces significant amounts of superoxide. Its distribution is limited in
mammals to tissues such as brown adipose and brain, where it is a potentially important site.
Two other enzymes involved in fatty acid oxidation, electron transfer flavoprotein and
electron transfer flavoprotein quinone oxido reductase may also produce superoxide.
2.5. Endothelial Dysfunction
Dysfunction of the endothelium has been implicated in the pathophysiology of different

forms of cardiovascular disease, including hypertension, coronary artery disease, chronic
heart failure, peripheral artery disease, diabetes, and chronic renal failure [97]. Endothelial
dysfunction may be defined as impairment characterized by a shift of the actions of the
endothelium toward reduced vasodilation, a proinflammatory state, and prothrombotic
setting. Endothelial dysfunction is seen early in the development of atherosclerosis (see
chapter 3), before overt vascular and structural changes. It is manifested by impaired
vasorelaxation to endothelium-dependent dilators, such as ACh. The pathophysiology of
endothelial dysfunction is complex and involves multiple mechanisms. It is characterized by
unbalanced concentrations of vasodilating and vasoconstricting factors, the most important
being represented by NO and Ang-II, respectively. Nitric oxide is recognized as one of the
major mediators of the maintenance of vascular homeostasis, and a decrease in NO
bioavailability is associated with endothelial dysfunction. In this context, the causes of
reduced vasodilatory responses in endothelial dysfunction include reduced NO generation,
oxidative excess and reduced production of hyperpolarizing factor. Reduced NO
bioavailability could be due to either reduced formation or accelerated degradation of this
42 Ramón Rodrigo
vasodilator. The mechanism, by which oxidative stress mediates endothelial cell function,
and ultimately vascular reactivity, is not fully understood. Although these mechanisms may
be multifactorial, there is a growing body of evidence that increased production of ROS may
contribute considerably as a causative factor in endothelial dysfunction by reducing NO
bioavailability and uncoupling eNOS. The endothelium, the media and also the adventitia
produce large amounts of ROS, which will attenuate endothelial mediated dilation, although
the mechanisms underlying endothelial dysfunction are located in addition to the endothelium
in the smooth muscle cell layer [98]. Superoxide combines with NO, which is synthesized by
eNOS, to form peroxynitrite. The consequence is an overall increase in ROS and reduced
ability of endothelium-dependent vasodilation. In addition to loss of vasodilation, endothelial
dysfunction is associated with endothelial cell apoptosis, increased binding of leukocytes and
monocytes, enhanced accumulation of lipid and a predisposition to thrombosis. These events
lead to a state of vascular inflammation. Under settings associated with oxidative stress the
vasculature per se produces large amounts of superoxide via elevated expression of NADPH
oxidase [99]. Consequently, a reduction of NO bioavailability occurs by degrading NO and
by forming the highly toxic product peroxynitrite. In addition, ROS formed by activated
mononuclear cells can lead to increased expression of cell surface adhesion molecules on
endothelium that are considered to be markers of inflammation and thus can enhance the
localization and accumulation of additional mononuclear cells, resulting endothelial
dysfunction. Formation of ROS by mononuclear cells and the vessel wall may be a link
between inflammation and atherosclerosis in hypertensive patients.
2.6. Oxidative Stress and Endothelial Dysfunction in Hypertension
A great body of evidence supports the idea that ROS are involved in the pathogenesis of
hypertension. Oxidative stress, characterized by increased bioavailability of ROS, plays an
important role in the development and progression of cardiovascular dysfunction associated
with hypertensive disease. There are many sources of ROS, including neutrophil-like
membrane-associated NADPH oxidase, xanthine oxidase, myeloperoxidase, uncoupled eNOS
and spillover from mitochondrial respiratory chain [100]. In addition, the occurrence of this
disturbance may be caused by decreased antioxidant enzyme activity (SOD, catalase) and
reduced levels of ROS scavengers (e.g. vitamin E, glutathione), acting as contributing factors
to the development of oxidative stress. These findings are based, in general, on increased
levels of plasma thiobarbituric acid-reactive substances and 8-isoprostanes, biomarkers of
lipid peroxidation and oxidative stress [68, 101]. Indeed, ROS of vascular origin contribute
importantly to peripheral vascular resistance and arterial pressure under pathophysiological
conditions such as hypertension [3]. In addition, polymorphonuclear leukocytes and platelets,
rich superoxide sources, also participate in vascular oxidative stress and inflammation in
hypertensive patients [102, 103]. In this setting, the elevation of blood pressure has been
associated with ROS abundance and frequently also with an impairment of endogenous
antioxidant mechanisms. Accordingly, increased markers of oxidative stress are found in
human hypertensive subjects, as well as in various animal models of hypertension [68, 104-
107]. Mouse models with genetic deficient in ROS-generating enzymes have lower blood
Hypertension 43
pressure compared with wild-type counterparts, and Ang-II infusion fails to induce
hypertension in these mice [12, 80]. In addition, in cultured VSMC and isolated arteries from
hypertensive rats and humans, ROS production is enhanced, redox-dependent signaling is
amplified, and antioxidant bioactivity is reduced [108]. It should be mentioned that in
patients with never-treated mild-to-moderate hypertension, lipid peroxidation and oxidative
stress were not found increased [109], suggesting that ROS may not be critical in the early
stages of human hypertension, but could be more important in severe hypertension. In
addition, classical antihypertensive agents such as β-adrenergic blockers, ACE inhibitors,
AT1 receptor antagonists, and Ca2+ channel blockers may be mediated, in part, by decreasing
vascular oxidative stress [110, 111]. It is of interest to note that increased ROS production in
vascular tissues has also effects other than elevation of blood pressure. Particularly
superoxide anions, has been implicated as playing an important role in vascular events such
as vascular remodeling after angioplasty, atherosclerosis, myocardial infarction, and ischemic
stroke [65]. Thus, therapeutic strategies should aim to restore the bioavailability of NO, either
scavenging ROS or through down-regulation of their generation and/or up-regulation of
eNOS activity and antioxidant enzymes.
3. Antioxidants in Hypertension
With the recent advances in our understanding of the complexity of oxidative stress and
redox signaling in the vascular system pointing to a central role of oxidative stress in the
pathogenesis of vascular dysfunction, it has arisen a growing interest regarding the
therapeutic possibilities to target ROS in the management of hypertension and other
cardiovascular diseases. The deleterious effects resulting from the formation of ROS are, to a
large extent, prevented by various antioxidant systems. Theoretically, agents that reduce
oxidant formation should be more efficacious than nonspecific inefficient antioxidant
scavengers in ameliorating oxidative stress. Therefore, it should be expected a beneficial
effect derived from several antioxidants, such as ascorbic acid (vitamin C), α-tocopherol
(vitamin E), glutathione, BH4, and N-acetylcysteine, among others, which have shown to
improve endothelial function and NO bioaction in cultured cells, and in animal and human
clinical studies of vascular reactivity. In support of this view, epidemiological studies suggest
that individuals with higher antioxidant intake have reduced cardiovascular risk. Based on
experimental evidence of the importance of oxidative stress in vascular damage, there has
been great interest in developing strategies that target ROS in the treatment of hypertension
and other cardiovascular diseases. Therapeutic approaches that have been considered include
mechanisms to increase antioxidant bioavailability or to reduce ROS generation by
decreasing activity of superoxide-generating enzymes. Gene therapy targeting oxidant
systems are also being developed, but their use in clinical hypertension remains unclear. This
section presents the available evidence for the potential of antioxidants in the prevention and
treatment of hypertension associated with oxidative stress, as supported by experimental
investigations, observational findings, clinical trials, and epidemiological data with special
reference to the antihypertensive effect of the main antioxidants of human use.
44 Ramón Rodrigo
3.1. Vitamin C
Vitamin C (ascorbic acid) is a potent water-soluble antioxidant in humans. It is a six-

carbon lactone synthesized from glucose in the most mammalian species, mainly in liver, but
not in humans. Vitamin C is an electron donor and therefore a reducing agent. When
ascorbate acts as an antioxidant or enzyme cofactor, it becomes oxidized to dehydroascorbic
acid. The latter can be used by cells to regenerate ascorbate, and directly or indirectly, it can
change the redox state of many other molecules. Vitamin C performs against oxidation of
lipids, proteins and DNA, subsequently protecting their structure and biological function. In
addition, on the vascular wall vitamin C behaves as enzyme modulator exerting up-regulation
on eNOS and down-regulation of NADPH oxidase [112]. Recently, it was demonstrated that
vitamin C inhibits the effects of ET-1 of impairing endothelium-dependent and endothelium-
independent vasodilation and the stimulation of interleukin-6 (IL-6) release in humans in
vivo. This suggests that the mechanism by which ET-1 impairs vascular function and
stimulates release of IL-6 involves increased oxidative stress [113]. Most studies have
demonstrated an inverse relationship between plasma ascorbate levels and blood pressure in
both normotensive and hypertensive populations [68, 114]. In a recent study, a decreasing
trend was observed with vitamin C levels and risk of hypertension in women but not in men
[115]. Vitamin C supplementation is associated with reduced blood pressure in hypertensive
patients, with systolic blood pressure falling by 3.6–17.8 mmHg for each 50 µmol/l increase
in plasma ascorbate [68, 116, 117]. Nevertheless, there are several small and short-term
clinical trials in which the effect of vitamin C supplements on blood pressure have yielded
inconsistent findings [116, 118-120]. The lack of antihypertensive efficacy observed in
studies using supplementation with vitamin C alone could be due to the pharmacokinetics of
vitamin C and/or the decreased bioavailability of NO under conditions of oxidative stress.
The antihypertensive effect of vitamin C is expected to occur at 10 mmol/L, a plasma
concentration unobtainable in humans following oral administration. However, this
concentration is required to compete efficiently with the reaction of NO with superoxide, due
to their high reaction rate constant, which is even higher than the reaction between SOD and
NO [121]. The lack of a therapeutic antihypertensive plasma vitamin C concentration via oral
administration may be due to its renal threshold at doses between 60 and 100 mg/day. The
steady-state concentration of vitamin C is attained at approximately 80 µmol/L, and plasma is
completely saturated at daily doses of over 400 mg [122]. Pharmacokinetic modeling
indicates that, with oral administration, even at very large and frequent doses of vitamin C,
plasma concentrations will only be increased modestly, from 70 µmol/L to a maximum of
220 µmol/L, whereas intravenous administration increases it as high as 14 mmol/L [123].
Thus the antihypertensive effect may only occur in plasma following infusion of high vitamin
C doses. Accordingly, intra-arterial administration of vitamin C has been shown to cause a
decrease in blood pressure in subjects with essential hypertension [124]. The molecular
mechanisms underlying the in vivo antioxidant effects of vitamin C related with blood
pressure modulation are not fully understood. Nevertheless, it was shown that these effects
are mediated in part by the ability of vitamin C to protect BH4 from oxidation and thereby
increase the enzymatic activity of eNOS. It should be noted that BH4 is a cofactor necessary
Hypertension 45
for NO generation via eNOS, otherwise becoming uncoupled, a form now recognized as an
important source of superoxide rather than NO [125], a condition likely to occur under a
prooxidant state. A hypothesis for the mechanisms whereby vitamin C, as well as other
antioxidants, could exert antihypertensive effects is shown in Figure 2-1.
Figure 2-1. Involvement of vitamin C, as well as other antioxidants, in counteracting the elevation of
blood pressure induced by oxidative stress. BH4, tetrahydrobiopterin; oxBH4, oxidized
tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; Ang-II, angiotensin II;
AT1, angiotensin II type 1 receptors; AT1-AA, autoantibodies to angiotensin II type 1 receptors; U-II,
human urotensin II; UT, human urotensin II receptors; ET-1, endothelin-1; ETA, type A endothelin-1
receptors; PGI2, prostacyclin; EDCF, endothelium derived contracting factor; TP, thromboxane-
prostaglandin receptors. The effects of vitamin C in these pathways are indicated as down-regulation
( ) and up-regulation ( ). (Adapted from Rodrigo et al., Fund Clin Pharmacol 2007a; 21: 111-
127)[143].
3.2. Vitamin E
Vitamin E is a major lipid-soluble antioxidant that has received considerable attention.

Epidemiological data support a role of high dietary vitamin E intake and a reduced incidence
of cardiovascular disease [126]. Tocopherols have been shown to increase PGI2 levels in
46 Ramón Rodrigo
endothelial cells via opposing effects on phospholipase A2 and cyclo-oxygenase 2 [127], a

potential beneficial effect against endothelial dysfunction as PGI2 is a prostanoid vasodilator
which is important for maintaining normal vascular function. Increasing evidence indicates
that vitamin E can act as a biological modifier independently of its antioxidant activity.
Experimental evidence available shows that vitamin E is capable of dose-dependently
regulating mitochondrial generation of superoxide and hydrogen peroxide. This effect is
reached through the prevention of electron leakage, by mediating the superoxide generation
systems directly and/or by scavenging superoxide generated. By down-regulating
mitochondrial generation of superoxide and related ROS, vitamin E not only attenuates
oxidative damage but also modulates the expression and activation of signal transduction
pathways and other redox-sensitive biological modifiers [128]. However, intervention trials
have not been convincing, with a number of studies demonstrating no beneficial effect of
vitamin E on cardiovascular disease outcomes [129-132]. Most of these studies have
generally not reported blood pressure outcomes, although a subset of the Primary Prevention
Project (PPP) study [133] did show no effect of vitamin E supplementation on clinic or
ambulatory blood pressure in treated hypertensive patients. Moreover, a meta-analysis has
highlighted an increase in all-cause mortality with high-dose vitamin E supplementation
[134]. In support of this view, other study using supplementation with vitamin E, either as a-
tocopherol or mixed tocopherols, showed a significant increase in blood pressure, pulse
pressure and heart rate in individuals with type 2 diabetes. These increases were observed
despite a reduction in plasma total F2-isoprostanes [135]. It should be noted that although
vitamin E can inhibit LDL oxidation in vitro, it is unlikely to achieve sufficiently high
concentrations in the vascular microenvironment to interfere effectively with all components
of oxidative stress, and has limited activity against superoxide and peroxynitrite driven
processes [136]. Therefore, taken together these data support the view that vitamin E alone
supplements at daily doses over 400 IU may increase all-cause mortality and should be
avoided.
It is of interest to note that the association of vitamins C and E is expected to have an
antihypertensive effect probably due to the fact that this combined therapy provides a
reinforcement of their individual properties through a complementary effect in improving
endothelial dysfunction [137]. Both vitamins C and E not only behave as scavengers of ROS,
but also are able to induce the down-regulation of NADPH oxidase and the up-regulation of
eNOS [112, 138]. Vitamin C may reduce the α-tocopheroxyl radical, thereby abrogating lipid
peroxidation [139] and further supporting an antihypertensive effect for this association. The
use of these association aimed to cause an antihypertensive effect is discussed below.
3.3. Clinical Trials for Association of Vitamins C and E
Despite the biological effects of both vitamin C and E, as shown by experimental models,
long-term clinical trials have failed to consistently support their antihypertensive effects in
patients at high cardiovascular risk. Most of clinical studies have looked at all-cause or
cardiovascular mortality, rarely focusing on blood pressure as a primary end point [140], but
none of the large clinical trials examined the effects of antioxidants specifically on blood
Hypertension 47
pressure [107]. Some short-term trials have shown that supplemental antioxidant vitamin
intake lowers blood pressure [118, 141, 142] but the majority of clinical trials did not find
any antihypertensive effects of antioxidant vitamins. However, most of these studies lack
rigorous exclusion criteria in the selection of subjects to avoid the influence of confounders
[143]. It deserves special mention that regarding cohorts included in large trials, most
subjects had irreversible cardiovascular disease. Some of these alterations could contribute to
perpetuate the increased ROS production by the vascular wall. Thus, in atherosclerotic
arteries there is evidence for increased expression of the NADPH oxidase subunit gp91phox
and Nox4, all of which may contribute to increased oxidative stress within vascular tissue
[144]. In addition, in this setting there is an increase in the expression of the Ang-II receptor
subtype AT1, providing evidence for stimulation of the renin angiotensin system and
simultaneously for an activation of the NADPH oxidase in the arterial wall [145]. Recently, a
randomized double-blind placebo-controlled study was conducted to test the hypothesis that
oral administration of vitamins C and E together, by improving the antioxidant status, causes
a decrease in blood pressure in patients with mild-to-moderate essential hypertension [146].
The results of this study, performed with newly diagnosed hypertensives, without end-organ
damage, showed for the first time a specific association between oxidative-stress-related
parameters and blood pressure, thus suggesting a role of oxidative stress in the pathogenesis
of essential hypertension. Moreover, the concomitant decrease in blood pressure and
oxidative stress raises the possibility that oral administration of vitamins C + E in patients
with essential hypertension may be considered as an adjunct therapy for hypertension in those
patients. In summary, the available data lead us to think in a beneficial antihypertensive
effect of vitamins C and E if administered during the phase of endothelial dysfunction, which
precedes an established vascular damage. In this setting it would be more likely to
successfully reverse, or at least counteract, the deleterious effects of ROS on the vascular
wall. In contrast, it should not be expected an antihypertensive effect in patients having
significant cardiovascular disease, in which case chronic damaging effects of oxidative stress
may be irreversible.
3.4. N-Acetylcysteine
The antioxidant N-acetyl-L-cysteine (NAC), a sulfhydryl group donor, improves renal

dysfunction and markedly decreases arterial pressure and renal injury in Dahl salt-sensitive
hypertension [147]. In an experimental model of hypertension, systolic blood pressure was
significantly higher in rats with 10% glucose feeding for 20 weeks [148]. This was associated
with a higher production of superoxide anion and NADPH oxidase activity in aorta. The
therapeutic effects of NAC in rats with established L-NAME hypertension were less
pronounced than the preventive effects of NAC on the development of L-NAME
hypertension [149]. Similarly, in spontaneously hypertensive rats, chronic administration of
NAC partially attenuated the blood pressure increase in young rats, while its effect was
negligible in adults with fully developed hypertension. These results suggest that the
inhibition of the oxidative stress in hypertensive states contributes to the therapeutic effects
of NAC; it seems that ROS play a more important role in the induction than in the
48 Ramón Rodrigo
maintenance of hypertension. On the other hand, in patients with type 2 diabetes and
hypertension, oral supplementation of NAC + L-arginine for 6 months caused a reduction of
both systolic and diastolic mean arterial blood pressure [150]. NAC administered
intravenously during hemodialysis reduced ADMA levels more significantly than
hemodialysis alone [151]. In relation to the mechanisms accounting for these results, the
effect of NAC may be mediated by an NO-dependent mechanism, probably through the
protective effect of NAC on NO oxidation. In patients with type 2 diabetes NAC improves
NO bioavailability via reduction of oxidative stress and increase of NO production. NAC
augments the levels of reduced glutathione and enhances the activity of NOS, probably by
protecting its essential cofactor BH4 from oxidation by the excess superoxide. Moreover,
NAC has been shown to protect the sulfhydryl groups of NOS from destruction by free
radicals and thus to maintain its activity [152]. These data are consistent with the NAC-
induced enhancement of the hypotensive effect of angiotensin-converting enzyme inhibitors,
as it is an effect at least partially mediated by NO. Therefore, NAC could be considered as an
adjuvant in the pharmacology of antihypertensive drugs having antioxidant properties and/or
acting through an improvement of NO bioactivity.
3.5. Polyphenols
Polyphenols are the most abundant antioxidant in the diet. Their intake is 10 times higher
than vitamin C and 100 times higher than vitamin E or carotenoids. Polyphenols like catechin
or quercetin can directly scavenge ROS, such as superoxide, hydrogen peroxide, or
hypochlorus acid, which can be very deleterious by damaging lipids, proteins and DNA. The
phenolic core can act as a buffer and capture electrons from ROS to render them less reactive.
On one hand, flavonoids are the major constituents of this group with more than 4000
compounds. On the other hand, the non-flavonoids compounds contain an aromatic ring with
one or more hydroxyl group. This group includes stilben (resveratrol), phenolic acids (gallic
acid) saponin (ginsenoside) and other polyphenols like curcumin and tannins. The role of
polyphenols in plants may partly explain the biological properties observed in vitro or in
vivo: they are involved in defense against infection and confer protective effects to the plants
against stress, such as ultraviolet radiation, pathogens and physical damages [153].
Epidemiological studies have shown an inverse correlation between polyphenols enriched
diet and reduced risks of cardiovascular diseases [154]. In humans, 30 min after the
consumption of red wine or polyphenols (1 g/kg body weight), circulating NO concentration
increases to 30 and 40 nM, respectively. Chronic treatment with red wine polyphenols
reduces hypertension and vascular dysfunction through reduction in vascular oxidative stress
in female spontaneously hypertensive rats in a manner independent of the ovarian function
[155]. A reduction of the blood pressure (11 mmHg) and an increase of heart rate are
observed [156]. In hypertensive patients, the use of olive oil can reduce the blood pressure
[157]. Short-term oral administration of red wine polyphenols produces a decrease in blood
pressure in normotensive rats. This haemodynamic effect was associated with an enhanced
endothelium-dependent relaxation and an induction of gene expression within the arterial
wall, which together maintain unchanged agonist-induced contractility [158]. In addition, red
Hypertension 49
wine polyphenols can accelerate the regression of blood pressure and improves structural and
functional cardiovascular changes produced by chronic inhibition of NO synthesis [159]. The
flavonol quercetin, one of the most abundant polyphenolic compounds found in the human
diet, relaxes vascular smooth muscle and its chronic daily treatment reduced blood pressure
and endothelial dysfunction in experimental models of hypertension characterized by an
activation of the renin–angiotensin system, such as in spontaneously hypertensive rats [160,
161], in rats made hypertensive by chronic inhibition of NO synthase [162] or in renovascular
hypertensive rats [163]. In relation to the mechanism whereby polyphenols reduce the blood
pressure, it has been reported an effect on the endothelium mainly due to NO production
[164, 165]. Thus, the beneficial effects of plant polyphenols in prevention of hypertension
may result from their complex influence on the NO balance in the cardiovascular system. The
mechanism of endothelial NO release elicited by polyphenols has been investigated. Red
wine polyphenols can modulate the production of NO through an extracellular Ca2+-
dependent mechanism in endothelial cells. Resveratrol and quercetin have been shown to
induce an increase of the intracellular concentration of Ca2+, by activation K+ channels or
inhibition of Ca2+-ATPases of the endoplasmic reticulum in endothelial cells. Prevention of
both blood pressure increase and cardiovascular remodeling by chronic treatment with the
antioxidant provinol was associated with increased NO synthase activity and enhanced
expression of endothelial NO synthase [46]. It has also been documented that polyphenols of
red wines strongly inhibit the synthesis of ET-1, a vasoactive peptide that is crucial for the
development of coronary atherosclerosis [166]. These data suggest that reduced oxidative
stress due to antioxidant action of provinol, its ability to increase endothelial NO synthase
activity and to decrease ET-1 synthesis may contribute to the polyphenol-induced
antihypertensive effect and protection against cardiovascular remodeling in NO-deficient rats
[167]. Nevertheless, the antioxidant treatment is expected to be more efficient in the
prevention than in the reduction of established hypertension [168].
3.6. Diet
There is sufficient evidence to suggest that dietary approaches may help to prevent and
control high blood pressure. There are dietary approaches regarding the prevention and
management of hypertension: i.e. moderate use of sodium, alcohol, an increased potassium
intake, plant fibers, calcium (and dairy products) and adherence to healthy dietary patterns
such as Dietary Approaches to Stop Hypertension (DASH) [169]. In addition, the study also
presents evidence regarding other nutritional factors which may possibly be associated with
levels of blood pressure, but for which there is as yet insufficient current scientific evidence
to support the issue of specific dietary recommendations. The Mediterranean diet has been
described by the following characteristics: an abundance of plant foods (fruits, vegetables,
breads, other forms of cereals, potatoes, beans, nuts, and seeds); minimally processed,
seasonally fresh, and locally grown foods; fresh fruit as the typical daily dessert, with sweets
containing concentrated sugars or honey consumed only a few times per week; olive oil as
the main source of fat; dairy products (principally cheese and yogurt) only in low-to-
moderate amounts; red meat in low amounts; and wine, usually red wine, in low-to moderate
50 Ramón Rodrigo
amounts, normally with meals. In a Mediterranean population with an elevated fat

consumption, a high fruit and vegetable intake is inversely associated with blood pressure
levels [170]. In the clinical trial DASH, assessing the effects of dietary patterns on blood
pressure [169], it was demonstrated that certain dietary patterns can favorably affect blood
pressure in adults with average systolic blood pressures of less than 160 mmHg and diastolic
blood pressures of 80 to 95 mmHg. Specifically, a diet rich in fruits, vegetables, and low-fat
dairy products and with reduced saturated and total fat lowered systolic blood pressure by 5.5
mmHg and diastolic blood pressure by 3.0 mmHg more than a control diet. More recently, it
was suggested that adhering to a Mediterranean-type diet could contribute to the prevention
of age-related changes in blood pressure [171].

There is considerable evidence supporting the view that oxidative stress is involved in
the pathophysiology of hypertension. Indeed, ROS are mediators of the major physiological
vasoconstrictors, such as Ang-II, ET-1, and U-II. In turn, oxidative stress, characterized by
increased bioavailability of ROS, plays an important role in the development and progression
of cardiovascular dysfunction associated with hypertensive disease. There are many sources
of ROS, including the enzymatic components of the vascular wall itself. In addition, the
effects of classical antihypertensive agents such as β-adrenergic blockers, ACE inhibitors,
AT1 receptor antagonists, and Ca2+ channel blockers may be mediated, in part, by decreasing
vascular oxidative stress. However, despite the consistent and promising findings from
experimental investigations, the clinical trials, and epidemiological data suggesting the use of
antioxidants as antihypertensive agents, data are inconclusive. It appears difficult to reconcile
these negative studies in view of the large body of evidence supporting the role of oxidative
stress in cardiovascular disease. First, despite scavenging free radicals is indeed the best way
to decrease ROS bioavailability, in the particular case of the cardiovascular system the
efficacy of this intervention is limited by the high rate reaction constant between superoxide
anion and NO. Second, most of the clinical trials were performed with heterogeneity of
studied populations, inappropriate or insensitive methodologies to evaluate oxidative state,
and incorrect antioxidant therapies. Third, patients with significant cardiovascular disease
were enrolled; therefore, these patients were with established vascular damage in such way
that it could contribute to perpetuate the increased ROS production by the vascular wall. The
use of either vitamin C or vitamin E alone has not proved antihypertensive therapeutic
efficacy; but their association, as well as with other antioxidants has been more promising,
likely due to a synergistic effect. The most relevant effect has been shown by the use of diets
rich in antioxidants provided by fruits and vegetables and low fat. Although the role of
antioxidant therapy for primary prevention remains an open question, it could be concluded
that all these interventions would be expected to be more efficient in the prevention than in
the reduction of established hypertension.
Hypertension 51
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Chapter III
Atherosclerosis
Víctor Molina1 and Ramón Rodrigo2

1
2
Institute of Biomedical Sciences, Faculty of Medicine,
University of Chile
Abstract
Atherosclerosis is a major source of mortality, being the underlying cause for most
cases of cardiovascular diseases such as ischemic heart disease and cerebrovascular
disease. Reactive oxygen species (ROS) can regulate several cellular processes, having a
key role in the homeostasis of the vascular wall. There is compelling evidence pointing
to ROS as important factors for the development of atherosclerosis. Many of the
proatherogenic actions of ROS occur through the generation of oxidized LDL. Also,
ROS can contribute to the development of endothelial dysfunction through the
consumption of nitric oxide and generation of peroxynitrite. Endothelial dysfunction
constitutes an early feature of atherogenesis, preceding the alterations that later
perpetuate the lesion formation. Atherogenesis includes several processes, such as
accumulation and oxidation of LDL in the subendothelial space, expression of adhesion
molecules and chemoattractant mediators, adhesion of monocytes, generation of foam
cells, production of inflammatory mediators and proliferation of certain cell types. Since
most of these processes can be modulated by ROS, supplementation with antioxidants is
expected to exert some degree of protection against atherosclerosis. Several lines of
evidence support a role of antioxidant supplementation in attenuating some of the
processes involved in atherogenesis. However, clinical trials have failed to consistently
prove a protective effect. The potential role of antioxidant supplementation against
atherosclerosis development or progression remains an open question.
64 Víctor Molina and Ramón Rodrigo
1. Introduction
Atherosclerosis is a major source of mortality, being the underlying condition for most
cases of ischemic heart disease and cerebrovascular disease that constitute the leading causes
of death worldwide [1, 2]. Only in USA, these two conditions caused 589,266 deaths in 2005
[3]. Cardiovascular diseases (CVD) are expected to be the first cause of death in almost every
country in the upcoming years [2, 4].
The process of atherosclerosis is characterized by the accumulation of macrophages
within the wall of large and medium sized arteries, and proliferation of certain cell types. The
consequence is the formation of a lesion known as atheromatous plaque that starts early in
life, even in late childhood [5], and progressively occludes the vessel lumen. Among the
complications that this plaque can suffer it is a rupture leading to thrombosis and acute
impaired blood supply in certain organs such as heart and brain, resulting in heart attack and
stroke, respectively.
Reactive oxygen species (ROS) have a key role in regulating several cellular processes as
well as homeostasis of the vascular wall. As shown in this book, oxidative stress has been
implicated in the pathogenesis of multiple highly prevalent diseases. Accordingly, there is
compelling evidence pointing to ROS as important factors in the development of
atherosclerosis. Understanding the pathophysiological basis of ROS involvement in
atherogenesis is mandatory for the design of future therapeutic approaches aimed to prevent
or treat this disease. This chapter focuses on the available data that supports a role of
oxidative stress in the mechanism of production and perpetuation of atherosclerosis, as well
as the current evidence regarding the use of antioxidants in the prevention or treatment of this
pathology.
2. Pathophysiology of Atherosclerosis
The pathophysiology of atherosclerosis constitutes an extensive subject closely related to
the current available evidence of ROS involvement in atherogenesis. This section starts by
referring to the known risk factors for atherosclerosis, followed by the morphologic features
of the atherosclerotic lesion and the hypotheses that have been postulated to explain its
initiation. Finally, the role of the different components involved in the atherogenesis process
and their relation to ROS is reviewed in more detail.
2.1. Risk Factors
Several variables have been associated with an increased chance of developing CVD.
Most of these factors have arisen from the analysis of poblational studies relating the
incidence of CVD to the presence of other conditions. One of the major sources of
information on this topic is the Framingham Heart Study, a cohort follow up that started in
1948. Recognizing these risk factors has made possible the selection of those with a stronger
association and the development of cardiovascular risk prediction charts that facilitate the
Atherosclerosis 65
patient assessment in the clinical practice [6, 7, 8]. Also, it has led to the design of guidelines
for the prevention of CVD, focusing on these factors [8, 9].
Age
Age is one of the strongest risk factors for CVD, and, although it is a non modifiable one,
it has a special relevance due to its unavoidability. Based on the Framingham Heart Study
data, the average risk of developing coronary heart disease during the following 10 years is
3% for a 30-34 years old male, increasing to 14% for a 50-54 years old and to 21% for a 60-
64 years old [6].
Gender
Males exhibit a higher risk in relation to same age females [6].
Serum Cholesterol
High levels of total cholesterol and LDL cholesterol have been consistently associated
with higher cardiovascular risk [6]. The treatment with cholesterol lowering drugs diminishes
the risk in these patients, especially with drugs such as statins, highly effective in lowering
levels of LDL cholesterol [10]. On the contrary, high levels of HDL cholesterol are
cardioprotective, whereas low levels associate with an increased cardiovascular risk [11, 12].
Blood Pressure
Increased blood pressure is considered a major risk factor for CVD [13].
Pharmacological reduction of blood pressure is consistently associated with a reduction in
total cardiovascular mortality [14].
Diabetes Mellitus
Macrovascular complication of diabetes, including diseases of coronary arteries, carotid
arteries and peripheral vessels, are an important cause of mortality by CVD in diabetic
patients [15]. There are several lines of evidence supporting a role of diabetes mellitus in the
pathogenesis of atherosclerosis [15].
Obesity
Obesity, and particularly abdominal obesity, increases cardiovascular risk, either by
predisposing to other risk factors, such as diabetes mellitus or hypertension, or as an
independent predictor of CVD [16].
Smoking
Cigarette smoking is an important risk for CVD. Smoking cessation results in a reduction
of this risk [17].
2.2. Morphologic Features of Atherosclerosis
The normal arterial wall is formed by three concentric layers surrounding the arterial
lumen. The layer closest to the lumen is called the intima, the middle layer is known as the
media and the most external layer is the adventitia. There are also two concentric layers of
elastin that separate these three structures. The internal elastic lamina separates the intima
from the media, and the external elastic lamina separates the media from the adventitia. The
luminal surface of arteries is formed by a single layer of endothelial cells, delimitated by its
basal membrane, which is in contact with the internal elastic lamina. Endothelial cells are the
main cell type present in the intima, although vascular smooth muscle cells (VSMC) and
macrophages can be found occasionally. The thickness of the intima is not uniform and can
be expressed as the intima:media ratio, which is normally between 0,1 to 1 [18]. Besides
establishing a structural barrier for the blood flow, the endothelium is implicated in the
regulation of many processes such as vascular tone, thrombosis and inflammation, whose
relevance in the pathogenesis of atherosclerosis will be discussed later in this chapter. The
media is composed mainly by layers of VSMC, the number of which increases with the
arterial size. Vascular smooth muscle cells are held together by an extracellular matrix of
elastic fibers and collagen. The adventitia is a loose matrix of fibroblasts, VSMC and
collagen; its potential role in the pathogenesis of atherosclerosis through the production of
ROS will be discussed later.
The morphologic manifestation of atherosclerosis is the presence of a lesion known as
atheromatous plaque or simply plaque. The developing lesion evolves during many years
before it can become symptomatic. Atherosclerotic lesions have been extensively
characterized and classified [19, 20] in six types, according to morphologic and histologic
features that reflect a progression in severity. These lesions form first in some regions of
arteries, such as bifurcations, that show a physiologic increase of the intimal thickness known
as adaptative intimal thickening, particularly its eccentric form [18]. Accordingly, these
susceptible regions have been called atherosclerosis-prone areas [18]. Atherosclerotic
lesions are divided in initial lesions (type I-II), intermediate lesions (type III) and advanced
lesions (type IV-VI). Type I lesions are the first microscopically and chemically detectable
lipid deposits in the intima, characterized by the presence of small and isolated groups of
lipid loaded macrophages (foam cells). Type II lesions include “fatty streaks” as a
macroscopic lesion and are characterized by a greater number of foam cells and by the
presence of intimal VSMC with lipid inclusions. Type III lesions are intermediate lesions
between type II lesions and advanced lesions, being characterized by the presence of isolated
pools of extracellular lipid deposits. Type IV lesions are known as atheroma, and are
considered advanced lesions. They present a dense accumulation of extracellular lipid called
the lipid core, formed by confluence of extracellular isolated lipid deposits present in type III
lesions. The lipid core is separated from the arterial lumen by a thin tissue layer. Type V
lesions are characterized by the presence of a prominent fibrous connective tissue formation.
They present a thick fibrotic cover separating the lipid core from the arterial lumen, known as
the fibrous cap. Type VI lesions present a more complex morphology, with surface
disruption, hematoma, hemorrhage and/or thrombosis.
Atherosclerosis 67
As described in the previously exposed classification, mature plaques have a lipid core
that consists in accumulation of extracellular lipid. The lipid core is separated from the
arterial lumen by the fibrous cap. The region where the fibrous cap contacts the normal
arterial wall is called the “shoulder”. This region of the plaque is more cellular than other
areas, presenting a high number of macrophages, VSMC and T-lymphocytes. The shoulder
region is where the majority of the plaque ruptures occur. In relation to the possibility of
rupture, mature plaques can be classified in stable and unstable. Stable plaques have a thicker
fibrous cap, smaller lipid core and shoulder region with a less inflammatory component than
unstable plaques. In consequence, unstable plaques are weaker structurally and more prone to
rupture.
2.3. Hypotheses for the Development of Atherosclerosis
Several hypotheses have been proposed to explain the pathogenesis of atherosclerosis.

During the nineteenth century Rudolf Virchow proposed that atherosclerotic lesions were the
result of an injury to the arterial wall, involving inflammatory and proliferative responses that
preceded the development of advanced lesions [21]. A modification of Virchow hypothesis
by Ross and Glomset led to the establishment of the original response-to-injury hypothesis
[21, 22]. These authors proposed that an initial step in the developing of atherosclerotic
lesions would be an injury that resulted in the desquamation of endothelial cells from the
arterial wall. There could be several potential injury sources such as chronic hyperlipidemia,
chemical factors, immunological injury or mechanical injury. The endothelial denudation and
exposure of the subjacent connective tissue would lead to the adhesion of platelets and
platelet aggregation, releasing platelets constituents to the arterial wall. The platelets-derived
factors along with lipoprotein and other plasma constituents would lead to migration and
proliferation of VSMC in these sites of injury.
The original response-to-injury hypothesis suffered several modifications. One of the
first was that it was not necessary a denudating injury of the endothelium to initiate the
development of an atherosclerotic lesion. Instead, an injury could trigger the process by
producing endothelial dysfunction [23]. One of the initial manifestations of endothelial
dysfunction would be the accumulation of lipid and lipoprotein particles under the
endothelium due to increased endothelial permeability [23, 24]. Another feature of
endothelial dysfunction would be an enhanced leukocyte and platelet adhesion. These cell
types release cytokines and growth factors that promote an inflammatory response, with
migration and proliferation of VSMC [25]. Recruitment of macrophages, and the
accumulation of lipids inside of them, leads to the formation of foam cells, the hallmark of an
initial atherosclerotic lesion [25].
Other two hypotheses are focused on the role of LDL as an initiator of the development
of atherosclerotic lesions [19, 25]. Under certain conditions, non modified LDL particles
could accumulate beneath the intima and initiate the process of atherogenesis, in which it
could be of importance the interaction of apolipoprotein B-100 (apoB-100) and extracellular
matrix proteoglycans [26]. This evidence supports the hypothesis of an accumulation of
subendothelial LDL as an initial factor for atherogenesis. On the other hand, extensive
evidence has been achieved sustaining the role of oxidized LDL in the pathogenesis of
atherosclerosis. In brief, the oxidative modification hypothesis states that oxidative
modification of LDL in atherosclerosis-prone areas leads to its uptake by macrophages and
formation of foam cells [27, 28]. The role of LDL in the pathogenesis of atherosclerosis will
be reviewed more extensively later in this chapter.
The different hypotheses for the initiation of the development of an early atherosclerotic
lesion are not mutually exclusive. On the contrary, evidence supporting each one of them
shows that most probably they are complementary in explaining the early processes involved
in atherogenesis. A more detailed review of the main actors involved in atherosclerosis
pathophysiology is presented in the following paragraphs.
2.4. Role of the Different Components of the Atherogenesis Process
2.4.1. Low-Density Lipoproteins

Low-density lipoproteins (LDL) are the main cholesterol carriers in plasma. The LDL
particles are defined as lipoproteins with a density between 1.019-1.063 g/ml [29]. The main
components of LDL are phospholipids, triglycerides, cholesteryl esters, unesterified
cholesterol and apoB-100 protein. High plasmatic LDL levels have been considered a risk for
the development of CVD [6]. Accordingly, accumulation of cholesterol in macrophages
within the intima is considered an early feature of the atherogenic process [23]. Most
macrophages internalize LDL through the classic LDL receptor [30]. However, this receptor
is down-regulated when intracellular levels of cholesterol increase [30]. So, although
macrophages can uptake LDL through this mechanism, the increase in intracellular
cholesterol should down-regulate LDL receptors and this way prevent the accumulation of
cholesterol and formation of foam cells. In fact, a diminished LDL receptor expression has
been detected in foam cells of human atherosclerotic lesions [31]. In addition, patients
suffering from homozygous familial hypercholesterolemia, a disease characterized by a total
or near total absence of LDL receptors, develop severe atherosclerosis and macrophage
derived foam cells are detectable [32]. This evidence suggests that a different mechanism of
LDL uptake by macrophages must be present. Brown and Goldstein demonstrated that
acetylation of LDL permitted its accumulation in macrophages, establishing the possibility
that modified forms of LDL were involved in the pathogenesis of atherosclerosis [32]. It was
demonstrated that this uptake is mediated by a specific scavenger receptor called the acetyl-
LDL receptor [32, 33]. Also, it was demonstrated that isolated endothelial cells could modify
LDL, allowing its uptake by macrophages, at least in part through the acetyl-LDL receptor
[34]. Later, it was established that a modification associated with an increased uptake of LDL
by macrophages was oxidation, proposing that free radicals should be involved in this
process [35, 36]. Oxidation of LDL is the main oxidative modification in pathophysiology of
atherosclerosis, and constitutes the basis of the original hypothesis of oxidative modification
[25].
Oxidized lipids have been detected in atherosclerotic lesions of different stage of
evolution. There is evidence regarding the presence of oxidized derivatives of fatty acids,
such as hydroxyoctadecaenoic acids, and oxisterols, the oxidized derivatives of cholesterol
Atherosclerosis 69
[37]. Also, products of protein oxidation have been detected in human atherosclerotic plaques
[38]. If oxidized LDL (oxLDL) is in fact participating in atherogenesis, it must be detected in
atherosclerotic lesions in vivo. Experiments in rabbits and humans have shown that oxLDL is
present in atherosclerotic lesions and absent in normal artery wall [39], and that LDL
extracted from that lesions shares many biological properties with in vitro oxLDL [40]. Even
more, evidence suggests that the uptake of lesion LDL occurs via scavenger receptors [40].
It is important to notice that oxLDL does not refer to a specific molecular species, but to
a spectrum of heterogeneous forms of oxLDL [27]. An early form of oxLDL is referred to as
minimally modified LDL (MM-LDL), which can still be recognized by LDL receptor and is
not recognized by the scavenger receptor. It is not likely that LDL oxidation occurs in
plasma, due to the abundance of antioxidants present in it [41]. Instead, the site of oxidation
could be the arterial wall. There are several potential sources of ROS in the arterial wall that
could participate in LDL oxidation. As it was explained in chapter 1, NADPH oxidase is a
source of superoxide anion radical (O2-·). There is evidence of ROS production in the
vascular wall through NADPH oxidase activity [42, 43]. Apart from macrophages, the
presence of NADPH oxidase has been described in endothelial cells, VSMC and fibroblasts
[44]. NADPH oxidase in vascular wall can increase its activity in response to several stimuli
such as angiotensin II, platelet derived growth factor, TNF-α and, in endothelial cells,
exposure to mechanical forces [44]. Xanthine oxidase (XO), another source of O2-·, has also
been described in endothelial cells, with an increased activity in response to shear stress [45].
Another potential source of O2-· in the vascular wall is uncoupled endothelial nitric oxide
synthase (later in this chapter). However, in consistence with its low reactivity, O2-· has a
limited capacity to oxidize LDL [46]. Instead, it could be the precursor of more reactive
oxidants [25]. Reactive nitrogen species (RNS), such as peroxynitrite resulting from the
reaction of O2-· with nitric oxide (NO), can also be a source for oxidized LDL. Low-density
lipoproteins isolated from human atherosclerotic lesions show higher levels of 3-nitrotyrosine
in relation to plasma LDL of healthy subjects, which is consistent with LDL oxidation by
RNS [47]. There is evidence supporting a possible role of lipoxygenases (LOs) in LDL
oxidation. 15-lipooxygenase (15LO), an enzyme that is expressed in atherosclerotic lesions,
is capable of oxidize LDL [48, 49]. In addition, inhibition of 15LO showed a protective effect
against atherogenesis [50]. Myeloperoxidase (MPO) is another enzyme potentially involved
in the pathogenesis of atherosclerosis [51]. Evidence shows that active MPO is present in
atherosclerotic lesions [52]. In addition, 3-chlorotyrosine, a specific marker of MPO-
catalyzed oxidation, is present in high levels in LDL isolated from atherosclerotic lesions
[53].
Besides promoting foam cell generation, a number of other proatherogenic properties of
oxLDL have been described and extensively reviewed [54, 55]. Oxidized LDL is
chemoattractant for monocytes [56] and inhibits tissue macrophage motility [57]. Thus,
oxLDL could initially recruit monocytes to the arterial wall. Later, after differentiation of the
monocyte to a tissue macrophage, oxLDL would inhibit its migration, “trapping” it in the
subendothelial space. Also, MM-LDL can increase the expression of monocyte
chemoattractant protein-1 (MCP-1) [58] and macrophage colony-stimulating factor (MCSF)
[59] in endothelial cells. Oxidized LDL increases the expression of Vascular Cell Adhesion
Molecule-1 (VCAM-1) in endothelial cells [60], facilitating monocyte adhesion. In addition,
oxLDL is mitogenic for macrophages [61] and VSMC [62]. Another effect of oxLDL is
inhibition of NO induced vasodilation [63].
2.4.2. Endothelium
The endothelium is a single layer of endothelial cells that covers the luminal surface of
vessels. Although originally it was thought to be only a structural barrier between the blood
and the arterial wall, many other functions have been described, such as regulation of
vascular tone, modulation of inflammation, regulation of vascular growth and modulation of
platelet aggregation and coagulation [64]. Endothelium alterations have been implicated in
the pathophysiology of atherosclerosis for a long time, as in the original response to injury
hypothesis for atherogenesis, implicating a denudating endothelial injury. Endothelial
dysfunction has a central role in atherogenesis, although the exact cellular and molecular
processes implicated have not been completely elucidated. There is increasing evidence of a
role of ROS in the developing of endothelial dysfunction.
Regulation of vascular tone is one of the main functions of the endothelium (for more
details see chapter 2). The hallmark of endothelial dysfunction is alteration of endothelium
dependent vasodilation, which is mediated by NO. Nitric oxide is produced in endothelial
cells by endothelial nitric oxide synthase (eNOS) from L-arginine (later in this chapter).
Nitric oxide diffuses to VSMC where it activates the soluble guanylyl cyclase, increasing
cyclic guanosine monophosphate (cGMP) production. The latter activates the cGMP-
dependent kinase I which in turn increases the opening probability of Ca2+-activated K+
channels, thereby inducing a hyperpolarization of the VSMC and inhibition of agonist-
induced Ca2+ influx. This leads to VSMC relaxation and, in consequence, vasodilation. Apart
from its role in the regulation of vascular tone, NO has a number of other functions in
vascular homeostasis [65] including suppression of abnormal VSMC proliferation, control of
hemostasis and fibrinolysis, and regulation of platelet and leukocyte interactions with the
arterial wall. Therefore, impaired production or activity of NO leads to events or actions,
such as vasoconstriction, platelet aggregation, VSMC proliferation and migration, and
leukocyte adhesion, that promote atherosclerosis [66]. In consequence, any process leading to
a decreased bioavailability of NO could be potentially proatherogenic. Assessment of
endothelial dysfunction can be made through several techniques, such as the coronary
response to the use of acetylcholine, flow mediated dilatation with brachial artery imaging,
forearm plethysmography, finger-pulse plethysmography and pulse curve analysis [64, 67].
By this means, endothelial dysfunction has been detected in clinical conditions known to be
risk factors for CVD such as hypercholesterolemia, hypertension, smoking, diabetes, and a
positive family history of premature CVD [67, 68]. There is evidence supporting a role of
ROS in reducing NO bioavailability. Endothelial dysfunction is associated with an increase
production of ROS. Once formed, O2-· reacts with NO to form peroxynitrite. This reaction
consumes NO, decreasing its bioavailability. Furthermore, under certain conditions eNOS
can become uncoupled (later in this chapter), resulting in production of O2-·, which
perpetuates the process [69].
Another stimulus for endothelium dependent vascular relaxation is shear stress. Shear
stress is defined as the lateral force exerted on the endothelial cells by the passage of a
semiviscous fluid over them. It is now well established that areas of the vasculature that
Atherosclerosis 71
experience unusual shear stress are particularly vulnerable to endothelial dysfunction, such as
bifurcations, branch points and tortuous vessels [67]. Also, laminar flow has been proven to
induce the expression of antioxidant response element-mediated genes that have a protective
role against oxidative stress in endothelial cells [70].
There are other endothelium-derived vasodilators that could have a role in atherogenesis,
including prostacyclin and bradykinin [66]. Prostacyclin acts synergistically with NO to
inhibit platelet aggregation. Bradykinin stimulates the release of NO, prostacyclin, and
endothelium-derived hyperpolarizing factor, another vasodilator, which contributes to
inhibition of platelet aggregation. Bradykinin also stimulates the production of tissue
plasminogen activator, and thus may play an important role in fibrinolysis.
Activation of the endothelium by inflammatory stimuli results in the expression of a wide
range of proteins that alter its function. Most notable among these are vascular cell-adhesion
molecules. Several adhesion molecules are over-expressed on endothelial cells in
atherosclerosis, such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin
and VCAM-1 [71, 72, 73]. Intercellular adhesion molecule-1 binds to integrins present on all
white cells. E-selectin binds to leucocytes expressing sialylated Lewis antigens, including
neutrophils, monocytes, and memory T cell. Vascular cellular adhesion molecule-1 binds to a
ligand present on lymphocytes, eosinophils, and monocytes. There is evidence of an
antioxidant-sensitive mechanism involved in the expression of VCAM-1 in endothelial cells
[74], which is concordant with an increased expression of this adhesion molecule after
exposition to oxLDL [60].
2.4.3. Inflammation Cells and Mediators

The role of inflammation in atherosclerosis has been well established, leading to the
concept that atherosclerosis in an inflammatory disease [24, 75]. Endothelial dysfunction
leads to an increase of the expression of adhesion molecules in endothelial cells, such as
VCAM-1, especially in regions with unusual shear stress (low average shear stress but high
oscillatory shear stress). This leads to adherence of monocytes and T-lymphocytes. After
adhesion, leukocytes migrate into the underlying intima in response to chemoattractant
stimuli, including chemokines such as MCP-1. This inflammatory process stimulates
migration and proliferation of VSMC that become intermixed with the area of inflammation
to form an intermediate lesion. If inflammation continues, an increased number of monocytes
and lymphocytes accumulate in the arterial wall, due to emigration from the blood and
multiplication in the lesion, perpetuating the inflammation process [24].
Monocytes – Macrophages – Foam cells

After adhesion to the endothelium and migration to the subendothelial space, monocytes
mature into macrophages under the influence of MCSF, which is over-expressed in the
inflamed intima [76]. Macrophage differentiation is a necessary step for atherosclerosis and is
associated with up-regulation of pattern recognition receptors for innate immunity, including
scavenger receptors and toll-like receptors (TLRs) [76]. As previously discussed,
macrophages internalize oxLDL via scavenger receptors. The accumulation of cholesteryl
esters in the cytoplasm leads to the formation of foam cells. Toll-like receptors bind certain
ligands and initiate a signal cascade leading to macrophage activation [75]. Besides ligands
such as bacterial toxins, TLRs can be activated by oxLDL and heat shock protein 60
(HSP60), which is highly expressed in atherosclerotic lesions of increasing severity [77].
Macrophage activation in atheroma leads to release of vasoactive molecules, ROS and
metalloproteinases that may degrade matrix components. The loss of matrix components may
subsequently lead to destabilization of plaques involving increased risk for plaque rupture
and thrombosis.
T-Cells
T-cells are present in atherosclerotic lesions, with a majority of CD4+ T-cells over CD8+
T-cells. Major histocompatibility complex (MHC) class II–expressing macrophages and
dendritic cells can be detected close to these T cells. This implies a possible immune
activation of T-cells in atherosclerotic lesions through processing and presentation of
antigens by macrophages. Also, the atherosclerotic lesion contains cytokines that promote a
T-helper 1 response, inducing activated T cells to differentiate into T-helper 1 effector cells.
T-cell activation results in the secretion of cytokines, including interferon-γ and TNF- α and
β that amplify the inflammatory response [24].
Markers of Inflammation in Atherosclerosis

A crescent interest in establishing the utility of biomarkers for inflammation in
atherosclerosis has developed in recent years. This is in part due to the potential utility of
these markers for the assessment of early or advanced atherosclerosis in the clinical practice.
Biomarkers include adhesion molecules such as VCAM-1; cytokines such as TNF-α, IL-1,
and IL-18; proteases such as MMP-9; the messenger cytokine IL-6; platelet products
including CD40L and myeloid-related protein 8/14; adipokines such as adiponectin; and
acute phase reactants such as C-reactive protein (CRP), plasminogen activator inhibitor-1,
and fibrinogen [78]. Among these, CRP has proved to be a useful marker, with a relatively
easy and standardized detection. CRP is a strong and independent predictor for
cardiovascular events, and can be used in addition to LDL levels for assessment of
cardiovascular risk [79].
2.4.4. Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) are the main cell type present in the media of
arteries. It is well known that the central cellular feature of atherosclerosis is the
accumulation of certain cell types, including VSMC, in the intima of arteries. Although the
main localization of VSMC in vascular wall is the medial layer, it is possible to find VSMC
in the intima of arteries in atherosclerosis-prone areas. These areas are characterized by
intimal thickening that occurs mainly by accumulation of VSMC and proteoglycans secreted
by them [18]. Given that atherosclerosis-prone areas are the preferential site for development
of advanced atherosclerotic lesions, a possible role of VSMC in the initial development of
atherosclerotic lesions has been proposed [80]. In advanced lesions VSMC migrate from the
media and proliferate within the intima, forming part of the cellular component of
atherosclerotic lesions.
Vascular smooth muscle cells in the media of arteries have a contractile function,
predominantly expressing proteins such as smooth muscle myosin heavy chain or smooth
Atherosclerosis 73
muscle actin. However, intimal VSMC associated with atherosclerotic lesions have a
different phenotype, with lower expression of these proteins, higher proliferative index and
greater synthetic capacity for extracellular matrix, proteases, and cytokines [81]. These two
phenotypic states have been called contractile state and synthetic state, respectively [81].
Altered VSMC phenotype migrates and proliferates more readily and can synthesize more
collagen. In addition, they express a greater proportion of VLDL, LDL and scavenger
receptors allowing more efficient lipid uptake. Thus, VSMC can internalize and accumulate
LDL and generate foam cells. Also, VSMC in atherosclerotic lesions express adhesion
molecules VCAM-1 and ICAM-1, being able to bind and retain monocytes within the
developing atherosclerotic lesion. Besides retaining monocytes, there is evidence supporting
a protective effect of VSMC against monocyte apoptosis [80]. Vascular smooth muscle cells
are capable of producing a wide range of cytokines, such as MCP-1, IL-8 and IFN-γ [82].
Among other functions, cytokines produced by VSMC attract and activate leukocytes, induce
proliferation of VSMC, promote endothelial cell dysfunction and stimulate production of
extracellular matrix components [80]. One of the main functions of VSMC in normal arteries
and atherosclerotic lesions is the production of extracellular matrix (ECM). Extracellular
matrix of healthy arteries consist mainly in type I and type III collagen, whereas
atherosclerotic lesions contain mainly proteoglycans, type I collagen and fibronectin [80].
Apolipoprotein B-100 present in LDL can bind to proteoglycans, this way retaining LDL in
the subendothelial space, where it can be oxidized [26]. Also, proteoglycans and fibronectin
in ECM promote VSMC proliferation [80].
Predominantly in vitro studies have shown that rat and mouse VSMC can switch between
the contractile and synthetic phenotypic states in response to a variety of atherogenic stimuli
including extracellular matrix cytokines, shear stress, and lipids [80]. There is evidence
supporting a role of ROS in the induction of a synthetic phenotype [83, 84] and of a
contractile-differentiated phenotype [85, 86]. Despite this apparent contradiction, it has been
established a role of ROS in promoting VSMC growth (hypertrophy and proliferation) and
migration, expression and activation of metalloproteinases secreted by VSMC, and
expression of inflammatory genes such as MCP-1 and interleukin-6 [87], further supporting a
role of oxidative stress in the pathophysiology of atherosclerosis.
2.4.5. Platelets
The presence of platelets in sites of endothelial injury has been known for a long time.
Platelets have an important role in the thrombotic vascular occlusion following the erosion of
a plaque. However, platelets may have a role also in the lesion formation [88]. There is
evidence showing that platelets can adhere to non denudated endothelium in regions of
activated endothelial cells [89]. Adhesion causes activation of platelets, involving the
secretion of a variety of chemokines that potentiate the inflammatory response [88]. Also,
platelets can promote monocyte transformation into foam cells [88]. The effects of ROS in
platelet function are not clear since there are studies supporting a role of ROS in promoting
platelet aggregation and studies that show an opposite effect. It appears that exposure to low
levels of oxidants may promote aggregation and high levels of oxidants may inhibit it [90].
However, the role of oxidative stress in platelet function still has to be elucidated.
2.4.6. Adventitia
For a long time the adventitia was considered to have only a structural function, with no
participation in pathologic vascular processes. However, in recent years novel functions of
adventitia have been described, and its possible role in vascular homeostasis has been
addressed. During atherosclerosis, infiltration of the adventitia by inflammatory cells can
occur. The presence of NADPH oxidase activity in the adventitia allows this structure to be
another source of ROS that can reduce NO bioavailability [91]. Also, oxidants can have a
role in promoting fibroblast proliferation, this way participating in vascular remodeling [92].
2.4.7. Urotensin II
Urotensin II (U-II) is the most potent mammalian vasoconstrictor identified. Although an
important role of U-II in hypertension pathophysiology has been established (see chapter 2),
a variety of potential proatherogenic features have also been described [93, 94]. U-II is
expressed in several cell types in atherosclerotic lesions, and its receptor is present in VSMC.
U-II is chemotactic for monocytes and stimulates foam cell formation. Also, U-II promotes
proliferation of endothelial cells and VSMC, induces the expression of NADPH oxidase and
collagen 1 in VSMC, but decreases that of metalloproteinase-1. This way, U-II could be a
link between hypertension and atherosclerosis. Accordingly, there is evidence that suggests
an effect of U-II in atherosclerotic plaque formation and, moreover, U-II plasmatic level is an
independent risk factor for the development of carotid atherosclerotic plaque in essential
hypertensive patients, showing an association even stronger than that for some widely
accepted risk factors, such as age or systolic blood pressure, among others [95].
2.5. Role of Oxidative Stress in the Pathogenesis of Atherosclerosis
2.5.1. Sources of ROS in the Vascular Wall

There are several potential sources of ROS in the vascular wall, most of which have been
proved to intervene in some degree in the atherogenesis process. This section exposes the
evidence supporting the role of the main possible ROS sources in atherosclerosis.
2.5.1.1. NADPH Oxidase

NADPH oxidase is a membrane-associated enzyme that catalyze the 1-electron reduction
of oxygen using nicotinamide dinucleotide (NADH) or nicotinamide dinucleotide phosphate
(NADPH) as the electron donor, this way generating O2-·. NADPH oxidase is considered to
be the main source of ROS in the vascular wall [44]. This enzyme was first described in
phagocytes and later in non-phagocytic cells such as the fibroblast [96]. In relation to
atherosclerosis, there is evidence supporting its presence in VSMC, endothelial cells,
adventitial fibroblasts and macrophages [97]. NADPH oxidase consists of several membrane-
bound subunits (gp91, Nox and p22phox) and cytosolic subunits (p47phox, p67phox,
p40phox and Rac2). In phagocytes gp91phox contains the putative binding sites for NADPH,
heme and FAD, and together with p22phox, supports the flow of electrons from NADPH to
O2. Although endothelial cells and adventitial fibroblasts express a gp91phox-containing
NADPH oxidase similar to that originally identified in phagocytes, VSMC contain
Atherosclerosis 75
homologues of gp91phox, namely Nox1 and Nox4 [97]. NADPH oxidase activity has been
linked to atherogenesis, since NADPH-derived ROS have a role in atherosclerotic lesion
formation [98].
There are several stimuli that can increase NADPH activity, including angiotensin II,
endothelin-1, U-II, growth factors such as thrombin and vascular endothelial growth factor
(VEGF), cytokines such as TNF-α, metabolic factors such as increased glucose and insulin,
oxidized lipids, oscillatory shear stress, hypoxia/reoxygenation, and nutrient deprivation [97,
99]. Angiotensin II, endothelin-1 and U-II are more related to hypertension pathophysiology,
although they can be the pathophysiological link between hypertension and atherosclerosis
(see chapter 2). Growth factors and cytokines are strongly expressed in atherosclerotic
lesions, due to the characteristic inflammatory response previously referred in the chapter. As
it was discussed previously, atherosclerosis prone areas are characterized for being exposed
to altered blood flow which results in oscillatory shear stress instead of laminar shear stress.
Laminar shear stress causes a transient increase in NADPH activity associated with an
increase in superoxide dismutase (SOD) expression. In contrast, oscillatory shear stress
causes a sustained increase of NADPH activity, with no increase in SOD levels [100]. This
way, elevated ROS production by NADPH oxidase in these areas can have a role in initial
lesion formation. Oxidized LDL can enhance the activity NADPH oxidase in leucocytes and
endothelial cells through an induction of gp91phox expression, thereby potentiating the
atherogenesis process [101].
2.5.1.2. Xanthine Oxidase

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are the two forms of
xanthine oxidoreductase (XOR) enzyme. XDH is the original translational product of the
XOR gene and XO derives from XDH through posttranslational modification [102].
Exposure of XDH to proteases leads to an irreversible proteolytic conversion to XO. Instead,
a reversible conversion of XDH to XO can occur through oxidation of certain thiol groups
[103]. XOR is generally recognized as the terminal enzyme of purine degradation in man,
catalyzing the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. Both XDH
and XO catalyze the terminal steps in the metabolism of purines, but XDH binds NAD+ and
XO does not. In consequence, XDH mediated reaction produces NADH, whereas XO
transfers electrons directly to molecular oxygen, leading to the formation of O2-· [102].
Although the metabolic function of these enzymes has been known for a long time, a
great interest in them has been developing in recent years, due to their capacity to generate
ROS and their possible involvement in oxidative stress-related pathologies. In relation to
atherosclerosis, XO is part of the multiple ROS sources potentially involved in atherogenesis.
There is evidence supporting the presence of XO in atherosclerotic plaques [104]. Also, XO
expression and activity is present in endothelial cells and increases in response to oscillatory
shear stress. This effect requires the presence of NADPH oxidase [45]. Furthermore, in
several experimental models the use of XO inhibitors attenuates endothelial dysfunction
[105].
2.5.1.3. Nitric Oxide Synthases

Nitric oxide synthases (NOS) are a group of enzymes that catalyze reactions resulting in
NO production. These enzymes are dimeric in their active form and for their activity require
the presence of cofactors including tetrahydrobiopterin (BH4), flavin adenine dinucleotide
(FAD), flavin mononucleotide (FMN) and iron protoporphyrin IX [106]. Nitric oxide
synthases catalyze the reaction of L-arginine, NADPH, and oxygen to NO, citrulline and
NADP. There are three identified nitric oxide synthase isoforms: nNOS (type I, NOS-I or
NOS-1), iNOS (type II, NOS-II or NOS-2) and eNOS (type III, NOS-III and NOS-3). nNOS
is found in neuronal tissue, iNOS is an inducible isoform present in several tissues and eNOS
is found in vascular endothelial cells.
Inducible NOS is expressed in macrophages and smooth muscle cells in atherosclerotic
lesions, promoting the formation of peroxynitrite [107, 108]. However, the role of iNOS in
vascular pathology is variable and poorly defined in atherosclerosis [109]. There is evidence
suggesting a possible protective effect of iNOS inhibition against atherosclerosis
development [110].
The role of eNOS in atherogenesis has been largely studied. Physiologically, eNOS is
regulated by caveolin-1 that binds to calmodulin and inhibits it. The binding of calcium to
calmodulin-1 leads to displacement of caveolin-1 and activation of eNOS [66]. Asymmetric
dimethylarginine (ADMA) is an endogenous competitive inhibitor of eNOS. It has been
reported an increase level of ADMA in hypercholesterolemic patients, establishing a
potential role in atherogenesis [111]. Endothelial NOS uses BH4 as a cofactor for transfer of
electrons from a heme group within the oxygenase domain to L-arginine to form L-citrulline
and NO. If either BH4 or L-arginine is absent, eNOS switches to an uncoupled state, in which
the electrons from heme reduce oxygen to form O2-·. Nitric oxide can react with O2-· to form
peroxynitrite, thereby decreasing NO bioavailability and promoting atherogenesis. Infusion
of BH4 can improve endothelial function in hypercholesterolemic patients [112].
Furthermore, oxidation of BH4 by peroxynitrite or by NADPH oxidase-derived ROS can lead
to eNOS uncoupling [69, 113].
2.5.1.4. Mitochondria
Mitochondria produce adenosine triphosphate (ATP) through a process called oxidative
phosphorylation. Oxidative phosphorylation is the process by which ATP is formed as
electrons are transferred from NADH or FADH2 (generated through the Krebs cycle) to
molecular oxygen, through a series of electron transport carriers localized in the inner
mitochondrial membrane. The electron transport carriers include: complex I (NADH-
ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III
(ubiquinol-cytochrome c reductase), and complex IV (cytochrome c oxidase). The majority of
electrons transferred by the electron transport chain are coupled with the production of ATP.
However, 1-2% of electrons leak out to form O2-· [114]. During mitochondrial oxidative
phosphorylation under pathophysiological conditions the electron transport chain may
become uncoupled, leading to increased O2-· production [115]. Furthermore, elevated
production of ROS in mitochondria damages lipids, proteins, and mitochondrial DNA,
increasing mitochondrial dysfunction. Of these, it is likely that mitochondrial DNA is the
most sensitive to physiologically relevant ROS-mediated damage. In humans and apoE (-/-)
Atherosclerosis 77
mice, the extent of mitochondrial DNA damage correlates with atherosclerosis progression
and can even precede atherosclerosis in young apoE (-/-) mice [116]. Mitochondrial
dysfunction and increased mitochondrial ROS production can promote atherogenesis through
several mechanisms including impairment of endothelial function and induction of VSMC
proliferation or apoptosis [117].
2.5.1.5. Myeloperoxidase
Myeloperoxidase (MPO) is a tetrameric heme protein present in monocytes,
macrophages and neutrophils. This enzyme catalyzes the conversion of chloride to
hypochlorous acid (HOCl), a potent chlorinating oxidant. Myeloperoxidase is the only human
enzyme known to generate HOCl. Therefore, chlorinated biomolecules are considered
specific markers of oxidation reactions catalyzed by MPO. There is evidence supporting a
role of MPO in atherogenesis by promoting oxidation reactions in atherosclerotic tissues [51].
Active MPO is present in atherosclerotic lesions [52]. In addition, 3-chlorotyrosine, a
“molecular fingerprint” of MPO-catalyzed oxidation, is present in high levels in isolated
lesion LDL [53]. Also, MPO can generate in vivo RNS. Nitric oxide can autooxidize to nitrite
(NO2–) and nitrate (NO3–). Nitrite and hydrogen peroxide can be used as substrates by MPO
for generating RNS that can nitrate tyrosyl residues [118].
2.5.1.6. Lipoxygenases
Lipoxygenases (LOs) comprise a family of enzymes capable of mediating selective lipid
oxidation. These enzymes facilitate the stereospecific addition of oxygen to cis unsaturated
fatty acids, resulting in the formation of hydroperoxy fatty acids. Classically, LOs are
subdivided into the 5, 8, 12, and 15 LOs according to the positional specificity of their
oxidation of the common fatty acid substrate, arachidonic acid [49].
There is evidence supporting a possible role of LOs in atherosclerosis development. The
enzyme 15-lipooxygenase (15LO) is capable to oxidize LDL and is expressed in
atherosclerotic lesions [48, 49]. It was found that 12/15-LO deficient mice show lower rates
of VSMC growth, migration and ECM production, and reduced extension of atherosclerotic
lesions [119, 120]. Accordingly, inhibition of 15LO attenuates atherosclerosis development
in rabbits fed with a high cholesterol diet [50]. A growing interest in a potential role of 5-
lypooxigenase (5LO) in atherosclerosis has recently developed [121]. There is evidence of an
increased density of 5LO expressing cells in the intima of progressively more severe lesions
[122]. Also, LDL receptor-null mice show an important reduction of atherogenesis when they
lack of a copy of the 5LO gene [123].
2.5.2. Pathogenic Role of ROS in Atherosclerosis

The effects of ROS in atherogenesis have been reviewed previously in this chapter in
relation to each one of the components and processes involved in the development of
atherosclerosis. As it was discussed, most of the actions of ROS that promote atherosclerosis
occur through the generation of oxLDL and through a decreased NO bioavailability that leads
to endothelial dysfunction. These include impaired vasodilation, expression of adhesion
molecules and chemoattractant proteins, adhesion of monocytes, accumulation of oxidized
lipoproteins, generation of foam cells, proliferation of VSMC and fibroblasts, and promotion
of an inflammatory response. The effects of ROS in atherosclerosis pathogenesis are depicted

in figure 3-1.
Figure 3-1. Scheme illustrating the hypothesis of the involvement of reactive oxygen species (ROS) in
atherosclerosis. CVD, cardiovascular diseases; oxLDL, oxidized LDL.
3. Antioxidants in Atherosclerosis
The great amount of evidence supporting a role of oxidative stress in the pathogenesis of
atherosclerosis suggests that interventions consisting in the supplementation of antioxidants
should have a protective effect against the development of atherosclerosis. A number of
studies have evaluated the effect of a variety of antioxidants in atherosclerosis
pathophysiology in vitro and in vivo. Most of these studies focus on vitamin E, because of its
strong lipophilicity, whereas other natural or synthetic antioxidants, such as vitamin C,
probucol and β-carotene, are supposed to play a minor role [124].
Atherosclerosis 79
Vitamin E is the collective name for molecules that exhibit the biological activity of α-
tocopherol. Both naturally occurring and synthetic forms of vitamin E are present. Naturally
occurring forms of vitamin E include four tocopherols and four tocotrienols (α, β, γ and δ).
Alpha-Tocopherol is the principal and most potent lipid-soluble antioxidant in plasma and
LDL [125]. One LDL particle contains between 5 and 12 molecules of α-tocopherol. There
are several lines of evidence suggesting a potential protective role of α-tocopherol against
atherosclerosis [125, 126]. Supplementation with α-tocopherol results in a decreased
susceptibility of LDL to oxidation [127]. Furthermore, in a placebo-controlled, randomized
trial, it was established that the supplementation of α-tocopherol at a dose of 400 IU/d or
more was effective in decrease LDL oxidation [128]. The use of α-tocopherol reduces the
expression of adhesion molecules in endothelial cells, the adhesion of monocytes to
endothelial cells, and the production of O2-· and IL-1β by monocytes [129, 130, 131].
Moreover, α-tocopherol prevents oxLDL-induced endothelial dysfunction in vitro and
reduces endothelial dysfunction in vivo in hypercholesterolemic smokers [132, 133]. Alpha-
tocopherol may have an effect in the inhibition of VSMC proliferation [134]. Also, α-
tocopherol can inhibit platelet adhesion [135].
Vitamin C (ascorbic acid) is a six carbon lactone that is synthesized from glucose in the
liver of most mammalian species, but not in humans. Consequently, when humans do not
ingest vitamin C in their diets, a deficiency state occurs that manifests as scurvy. Vitamin C is
an electron donor and therefore a reducing agent. However, vitamin C itself is oxidized in the
process, generating ascorbyl radical, which has a very low reactivity [136]. Ascorbic acid has
an important capacity for preventing lipid peroxidation and LDL oxidation [137, 138]. The
supplementation with ascorbic acid for 10 days was reported to reduce monocyte
adhesiveness to endothelium in smokers [139]; however, another study showed no effect of
ascorbic acid in monocyte adhesiveness [140], although supplementation in this case lasted
for a shorter period of time (2 hours). Also, there is evidence supporting a beneficial role of
ascorbic acid in endothelium-dependent vasodilation, this way preventing endothelial
dysfunction [138]. In rabbits, supplementation with ascorbic acid has been proved to prevent
hypercholesterolemia-induced atherosclerosis [141].
Carotenoids are the pigments responsible for the yellow to red color of some fruit and
vegetables. The main carotenoids present in human diet are lycopene, lutein, α-carotene, β-
carotene, β-cryptoxanthin, and zeaxanthin [142]. Various biological effects have been
attributed to carotenoids, including antioxidant activity due to their capacity for scavenge
ROS [143]. Supplementation with β-carotene has been shown to increase the content of this
carotenoid in LDL and to inhibit endothelial cell-mediated LDL oxidation [144]. Moreover,
high plasma carotenoid levels have been associated with a decreased risk for subsequent
coronary heart disease events [145].
3.1. Clinical Trials
Initial observational, epidemiological and case control studies showed conflicting results,
but a possible protective effect of vitamin E was established [124]. Despite all the evidence
supporting the antiatherogenic effects of antioxidant supplementation, clinical trials have
failed to consistently prove a protective effect of antioxidants against atherosclerosis. A large

number of randomized trials have been carried out, evaluating the effect of supplementing a
single antioxidant or combination of them, including vitamin E, vitamin C and β-carotene.
The results of these trials are contradictory. Although some of them attribute beneficial
effects to antioxidants, most of them show no protective effect [124, 146, 147]. Even more, in
some of these trials, an increase in mortality was reported in patients receiving antioxidant
supplementation, which is also supported by meta-analyses, especially in relation to high
doses of vitamin E [148, 149]. These conflicting results can be conditioned by a number of
factors. It is important to take in notice the heterogeneity of the clinical groups studied,
including patients with prior CVD, at risk of CVD, smokers, hipercholesterolemics and
submitted to invasive interventions. There is also variation in the doses of antioxidants and
the duration of the supplementation. Antioxidants may have a protective effect in initial
phases of atherosclerosis, which could be lost in more advanced stages when the processes
that perpetuate the development of the atherosclerotic lesion are established. This way, it
could be expected a more consistent protective role of antioxidants in primary prevention.
Therefore, further investigation in needed to consistently establish the efficacy of
antioxidants in preventing atherosclerosis.

There is a great body of evidence supporting a role of ROS in the pathogenesis of
atherosclerosis. Most of the proatherogenic actions of an increased production of ROS are
achieved through the production of oxLDL and through the consumption of NO that leads to
endothelial dysfunction. Almost all of the processes involved in atherogenesis can be
modulated by ROS, including attraction and adhesion of leucocytes, formation of foam cells,
proliferation of several cell types and induction of an inflammatory response. There are
several sources of ROS in the vascular wall, including enzymes such as NADPH oxidase and
xanthine oxidase that could be potentially regulated in therapeutic interventions. All this
evidence suggests that interventions consisting in supplementation of antioxidants should
have a protective effect against atherosclerosis development. Consistently, there are several
lines of evidence that show the multiple antiatherogenic effects of antioxidant
supplementation. However, clinical trials have failed to prove a consistent protective effect.
Although many clinical trials have been carried out, comparisons are difficult due to a great
heterogeneity among studied populations and different supplementation duration and doses. It
is possible that antioxidants could have a stronger protective effect in early stages of
atherogenesis attenuating initial endothelial dysfunction, before the processes that perpetuate
the formation of the atherosclerotic lesion are established. Further investigation is required to
fully clarify the role of antioxidants supplementation in atherosclerosis.
Atherosclerosis 81
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Chapter IV
Postoperative Atrial Fibrillation
José Vinay1 and Ramón Rodrigo2

1
Institute of Biomedical Sciences, Faculty of Medicine, University of Chile.
2
Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Abstract
Atrial fibrillation is an arrhythmia occurring frequently within the first few days in
10% to 65% of patients after major cardiothoracic surgery (postoperative atrial
fibrillation, POAF). It is associated with increased morbidity and mortality and longer,
more expensive hospital stays. Despite the use of strategies to prevent POAF through the
prophylactic use of agents such as β-adrenergic blockers, amiodarone, or others, a
considerable percentage of the patients still presents the arrhythmia. The involvement of
oxidative stress in the mechanism of POAF is supported by an increasing body of
evidence indicating that the formation of reactive oxygen species (ROS) released
following extracorporeal circulation are involved in the structural and functional
myocardial impairment derived from the unavoidable ischemia–reperfusion cycle of this
setting. ROS behave as intracellular messengers mediating pathological processes, such
as inflammation, apoptosis and necrosis, thereby participating in the pathophysiology of
POAF. Consequently, myocardial electrical and structural remodeling associates with the
appearance of functional impairment consistent with alterations in electrical conduction.
Therefore, it seems reasonable to assume that the reinforcement of the antioxidant
defense system should protect the heart against functional alterations in the cardiac
rhythm in this setting. Interestingly, exposure to low to moderate doses of ROS could
trigger a cellular defensive response characterized by a prevailing effect of survival over
apoptotic pathway, what should be considered a therapeutic target. The present chapter
examines the molecular basis accounting for the contribution of oxidative stress to the
development of POAF. In addition, it is presented the clinical and experimental evidence
to support a new paradigm based in the prophylactic reinforcement of the antioxidant
defense system toward reduction in the susceptibility of cardiomyocytes to ROS-induced
injury.
92 José Vinay and Ramón Rodrigo
1. Introduction
Atrial fibrillation (AF) represents the most common arrhythmia in clinical practice and is
associated with poor clinical outcome. In the general population, it affects approximately 2.3
million people in the USA and increasing in fivefold the risk for stroke [1]. The efficacy of
currently available treatments is sub-optimal. In turn, AF is the most common complication
associated with coronary artery bypass graft surgery and other surgical procedures performed
with extracorporeal circulation (postoperative atrial fibrillation, POAF). It occurs frequently
within the first few days in 10% to 65% of patients after major cardiothoracic surgery and
results in increased morbidity and length of hospital stay, having enormous cost implications
in these patients. Its appearance increases with age and with the presence of known risk
factors as arterial hypertension, coronary heart disease, diabetes mellitus and valve disease,
among others. Management of POAF is often frustrating, and strategies vary widely from
institution to institution. Despite all the efforts put into preventing POAF, including the use
of β-blockers and amiodarone, a considerable percentage of the patients still presents the
arrhythmia [2-4]. Its genesis and pathophysiology have been heavily studied in the last years,
however the exact mechanisms behind POAF appearance and perpetuation, have not been
clearly described so far. The involvement of oxidative stress in the mechanism of POAF is
supported by an increasing body of evidence indicating that the formation of reactive oxygen
species (ROS) released following extracorporeal circulation are involved in the structural and
functional myocardial impairment derived from the ischemia–reperfusion cycle. Reactive
oxygen species behave as intracellular messengers mediating pathological processes, such as
inflammation, apoptosis and necrosis, likely followed by fibrosis, thereby participating in the
pathophysiology of POAF. Consequently, myocardial electrical and structural remodeling
associates with the appearance of functional impairment consistent with alterations in
electrical conduction. The lack of efficient and relative risk-free treatments has supported the
search for novel drugs or agents that can cover the needs of these patients. In this context, a
relative new line of study that associates POAF to oxidative stress is arising [5-7]. Numerous
studies have suggested the pathophysiological link between POAF and oxidative stress, being
the latter substantially present in the unavoidable ischemia/reperfusion cycle of this setting,
thus giving rise to the involvement of ROS as pathogenic factors of the functional and
structural impairment known to occur.
The new paradigm that puts ROS as main players in the pathogenesis of POAF also
supports the concept that pharmacological treatments that could intercept the mechanisms
behind ROS production, propagation or action, at the same time could prevent or potentially
treat this rhythm disorder. In this group, it can be found drugs with intrinsic antioxidant
power as vitamin C, vitamin E, N-acetylcysteine (NAC) and statins; all which gather
biological and pharmacological properties that make them excellent candidates in the
treatment of this pathology [8-11]. Nevertheless, it should also be considered that agents
causing up-regulation of antioxidant enzymes, such as catalase, superoxide dismutase and
glutathione peroxidase, would be expected to have a beneficial effect against the deleterious
action of ROS on cardiomyocyte function. The role of oxidative stress in the pathogenesis of
AF and POAF, and their possible attenuation by antioxidants will be analyzed in the
following sections.
Postoperative Atrial Fibrillation 93
2. Pathophysiology
Normal heart electrophysiology requires the correct function of the four intrinsic
properties of cardiac cells: excitability, conductivity, contractility and automatism. Both
structural and/or functional impairment of any of these properties could lead to heart disease,
especially to a rhythm disorder. In healthy individuals, the cells with the higher intrinsic
frequency of depolarization are located in the right atria (the heart pacemaker), and are
denominated as a whole as sinus node. In this specific location, the cardiac depolarization
wave starts. Firstly, the depolarization wave travels to the right and left atria, leading to their
contraction. At the same time, other depolarization wave is travelling to the atrioventricular
node, finally reaching the Hiss-Purkinge network and depolarizing both ventricles, leading to
their contraction and the posterior ejection of blood into both aorta and pulmonary arteries.
The most common disease, related to heart electric conduction, is AF [1]. Clinically, this
tachyarrhythmia presents with a cardiac frequency over 90 pulsations per minute, involving
the co-existence of two pivotal pathogenic events: increased cardiac cell automatism and the
presence of reentry foci (the level of influence of each one, will determine the type of AF)
[12-15]. Increased automatism, represented by the generation of rapidly discharging foci,
means that cardiomyocytes located in different areas of the atria that should be overshadowed
by the heart pacemaker, which intrinsically has a higher depolarization rate, start to act like
new pacemakers, thus leading to an incorrect electric conduction and therefore a poor atrial
contraction. Increased automatism is particularly clear in patients with focal AF, which have
ectopic rapidly discharging foci usually near the pulmonary veins [12, 13]. This high
frequency depolarization wave can not be properly conducted through the atria tissue and
could convert into extra systoles, causing the atrium to fibrillate. On the other hand, the
existence of reentry foci means that those new impulses generated in the context of increased
cardiac cell automatism, are perpetuated by new re-entry wavelets. This is usually the
consequence of a chronic heart injury, such as hypertension, coronary or valve disease,
leading to dilation and fibrosis, all of which alter the electrophysiological properties of the
heart, thus helping to perpetuate AF [16].
For the heart to experiment those pathogenic events has to suffer a constant stress for a
long time, leading to a remodeling, which has two faces. For one hand the atria undergoes
and electric remodeling based on electrophysiological changes, like shortening of the
refractory period, decrease in the action potential and activation of cardiomyocytes
automatism properties, thereby contributing to the genesis of ectopic discharging foci and re-
entry wavelets [5, 17, 18]. On the other hand, the atria suffers a structural remodeling, based
on atrial dilation and fibrosis, secondary to the activation of different inflammatory and pro-
fibrotic mediators as angiotensin II, transforming growth factor β1 and tumor necrosis factor
alpha1; leading to changes in heart electric conduction properties, thus helping to the
perpetuation and generation of new re-entry foci [19, 21]. Therefore, both electric and
structural remodeling generates the two main events necessary to the genesis and
perpetuation of AF: ectopic automatism and re-entry wavelets.
However, the mechanisms accounting AF genesis and perpetuation are quite different
when analyzing different subpopulations. For example, POAF is the result of several heart
injuries that co-exist in the post-operative state. Among them, the increase in the adrenergic
tone [22], the activation of the renin-angiotensin system [23, 24], inflammation, ischemia and
preoperative injuries associated with cardiac diseases (ventricular hypertrophy, atrial dilation
and fibrosis, hypertension and necrotic zones secondary to atherosclerotic events) are the
most important. The increase in the adrenergic tone and the activation of the renin-
angiotensin system deserve special mention because of the multiple mechanisms accounting
for heart damage. Angiotensin II acts on vascular smooth muscle cells leading to general
vasoconstriction; at the same time aldosterone generates renal sodium retention. Those two
events, acting together, lead to hypertension, which increases heart oxygen consumption and
ventricular stress, which in the long-term could end with cardiomyocyte hypertrophy and
ventricular failure. Also, angiotensin II exerts an action directly over the ventricular tissue,
causing extracellular matrix remodeling, which is reflected in atria dilation, hypertrophy
and/or fibrosis [23, 24].
The increase in the adrenergic tone is a pivotal link in the pathophysiological chain
accounting for AF genesis and perpetuation. The existence of ectopic rapidly discharging foci
in many individuals can go unnoticed. However, when other risk factors are present, in this
case, increased adrenergic tone, normal heart electric conduction is impaired: new
discharging foci appear and the first ones perpetuate. This leads to asynchronous atrial
electrical activation, and therefore to loss of atrial contractility. At the same time, new reentry
foci emerge, generating multiple re-entrant wavelets that help to the perpetuation of the
conduction abnormality [12, 13].
2.1. Oxidative Stress
Oxidative stress has been found to play a crucial role in the pathogenesis of several
cardiovascular diseases. One of the most studied has been AF and particularly POAF [6, 7,
25]. Following cardiac surgery, and especially with extracorporeal circulation, ischemic
phenomena and posterior reperfusion are mandatory. This leads to the synthesis of high
concentration of ROS, which could impair the normal operation of several physiological
processes in the organism [26].
Before being determined the specific molecular pathways through which ROS exert their
actions, the first evidences accounting for this hypothesis were the high levels of serum
myocardial oxidation biomarkers (peroxide, derivatives of reactive oxidative metabolites of
oxygen and/or nitrogen) in AF presenting patients in relation to healthy individuals [7, 26,
27]. There is also evidence for oxidative injury in atrial tissues from AF patients [26]. On this
line, it was found that patients developing POAF had increased levels and expression of
NADPH subunit Nox2 and in NOX-derived superoxide generation [28, 29]. Together with
NADPH oxidase, it has been found that other pro-oxidative enzymes are in higher activity in
the context of POAF; this is the case of xanthine oxidase and uncoupled nitric oxide synthase
(NOS) [28]. Hence, it has been objectified that oxidative stress is present in this setting. ROS
production is far from being a simple process; on the contrary, it is a complex mechanism
involving pre- and post-transcriptional regulation. In the next paragraphs it will be presented
the experimental data and theoretical bases of the different targets of ROS action, accounting
for AF and POAF production and perpetuation.
2.1.1. Electric Remodeling

Electric remodeling is one of the most important mechanisms by which ROS disturbs the
normal electric conduction of the heart. Fibrillating atria is characterized by a diminished
action potential and effective refractory period, due to changes in several currents that
normally maintain the cardiomyocytes electric potential [17]. Between those currents, the L-
type voltage-gated Ca2+ current has been found to be the principal target of ROS action. This
current has been found to be diminished in cells extracted of fibrillating atria, as a result of
cardiomyocytes calcium overloading [5, 17]. To completely understand how ROS produce
calcium overloading and therefore diminish L-type voltage-gated Ca2+ current it is important
to take into account the normal cardiomyocyte calcium homeostasis. Calcium influx into the
cytosolic space is mediated largely by the ryanodine receptor Ca2+ channel (RyRC), which
moves calcium between the sarcoplasmic reticulum (SR) into the cytosol. Physiologically,
RyRC release calcium as a response to the arrival of an action potential to the cardiac cell
[30]. However, experiments using canine SR vesicles demonstrated the existence of a
superoxide activated calcium releases from RyRC [31]. Hence, ischemia-reperfusion
dependent ROS could activate the RyRC and produce calcium overloading. As a
consequence, L-type current is reduced, thus leading to the electrophysiological changes, like
shortening of the refractory period, involved in the initiation and perpetuation of POAF.
Finally, it has to be mentioned the effect of ROS in the disruption of cardiomyocytes
connexins. Connexins are a set of proteins assembled between two adjacent cardiac cells,
forming the structure known as gap junction. This structure participates in the efficient and
rapid conduction of the electric potential through the cardiac tissue. Disruption of connexin
43 has been correlated with increased propensity for tachyarrhythmias [32]. Under conditions
of oxidative stress, following an ischemia/reperfusion cycle, increased ROS directly interact
with the connexins, particularly with connexin 43, thereby disrupting its organization, leading
to electrical remodeling and therefore to propensity to present AF [33,34]. The exact
molecular mechanism by which ROS disrupt normal connexin distribution has not been
completely identified. However, the ROS-mediated activation of protein kinase C gamma,
unique isoform present only in neural and optic tissue, leads to the phosphorylation and
posterior disassembly of connexin 43 [35].
2.1.2. Muscle Mechanical Impairment

Myofibrillar creatine kinase (MM-CK) has a crucial role in muscle energetic metabolism.
MM-CK buffers ATP concentration during the turnover happening in muscle contraction and
relaxation. ROS may be involved in MM-CK oxidation, and therefore in the reduction of its
activity as seen in AF developing patients undergoing Maze procedure in relation to non-AF
presenting patients undergoing cardiac surgery [26, 36]. These findings are of vital
importance, because set the precedent that atrial fibrillation does not reflect exclusively an
electrophysiological issue, but on the contrary it is the result of several co-existent elements,
among which it could be found the mechanical muscle impairment, as a result of a energetic
deficit that may be involved in the lack of synchronously atrial contraction and therefore help
to perpetuate fibrillation.
2.1.3. Mitochondrial Damage

Mitochondria are the energetic cellular organelles, so any injury that may suffer could
cause cellular energetic impairment that could lead, depending on the intensity of the injury,
to apoptosis or different levels of cellular damage. Mitochondrial DNA (mtDNA) is a
potential target for ROS to produce oxidative damage. Through quantitative PCR technique,
it was shown that the mtDNA of AF patients had more deletions than the mtDNA of patients
in sinus rhythm. This is based on the high concentration of oxidative DNA damage products
found in the first ones [37]. The damage done at this level has to be put in the context of
oxidative stress perpetuation. For this, is important to remember that ROS production is
frequently a reflection of cellular energetic impairment due to hypoxia and/or intracellular
organelle damage, among others, where as there is a lack of oxygen, the electron transport
chain cannot function correctly and therefore ROS are heavily produced. The damage done to
mitochondria, involved in the majority of the cell energetic processes, may lead to an
increment of ROS production rates, and those ROS at the same time will perform oxidative
damage on mtDNA, impairing mitochondrial function, and therefore closing the vicious
cycle. In brief, mtDNA ROS-mediated damage performs a positive feedback on ROS
production that, at the same time, perpetuates mitochondrial damage and ROS synthesis.
2.1.4. Transcriptional Modulation

It is of great importance to be acquainted with the notion that ROS not only exert their
actions by directly modifying the constitution of different organic molecules. In addition,
ROS also are involved in the regulation of several genes expression. These are ROS-sensitive
genes, as they respond to changes in the cellular oxidative state. Many trials have reported
effects of ROS in redox sensitive gene expression. It has been described that the presence of
oxidative stress in AF patients results in changes accounting for a shift from the synthesis of
antioxidant proteins to pro-oxidant ones [38]. Trials studying patients undergoing coronary
artery bypass grafting or valve procedure described significant differences in genomic
response between the patients that presented POAF and the ones that maintained in sinus
rhythm; the first also showing the highest oxidative stress related parameters [27]. Microarray
studies have demonstrated the existence of genes specifically associated with both AF and
sinus rhythm patients. Among the first, the authors described molecules related to
inflammation and different ion channels [39]. In total, there are described over 100 genes
modulated between AF and sinus rhythm specific genes, and it is plausible to believe that
ROS are involved in the modulation cascade of the AF intracellular transcriptional events.
It is important to highlight the ROS-mediated activation of transcriptional factors, such
as NF-κB and AP-1. These factors stimulate the transcription of several protein mediators,
like proinflammatory cytokines that activate several cell death pathways, through apoptosis
and/or necrosis [40]. The heart tissue, being subjected to this chronic injury, responds with a
pathological regeneration, which contributes with the electric and structural remodeling of
the tissue.
For many years the studies involving oxidative stress and ROS have focused in the direct
mechanism by which ROS altered the structure and function of different cell molecules. In
the last years the study of the genetic mechanism by which ROS are involved have opened a
completely new line of study that could help to intercept the different molecular locations
where ROS produce damage, and therefore the new locations where novel therapeutic tools
may help to treat or prevent different types of oxidative stress-mediated disorders. More
studies to analyze the function of these genes are still lacking to date.
2.1.5. Oxidative Stress and Inflammation Nexus

The pathogenesis of AF and POAF is a complex web of events that involves the
activation of many processes, being inflammation one of the majors [24, 51]. Thus, it was
shown that white cell count [42], as well as the levels of C- reactive protein is more elevated
in the postoperative period, at day 2, in patients that experience POAF than in those that do
not [43]. The role of cytokines, chemokines, leukocytes and acute-phase proteins, like high-
sensitivity C-reactive protein in the pathogenesis of POAF has been reported in several
studies [44, 45]. At first sight, it could be thought that inflammation does not have anything
in common with oxidative stress and that its origin, mediators and targets are completely
specific for each process. But the reality cannot be more different from this concept, since
oxidative stress, ROS and inflammation are a continuous that is very difficult to dissect.
These phenomena have important molecular bridges that are activated in presence of ROS
[46], leading to the activation of multiple mechanisms that end up causing heart tissue
remodeling and therefore enhancing the susceptibility to present rhythm disorders. Among
those molecules, the most studied has been the transcriptional factor NF-κB, a factor that
responds to changes of the cellular oxidative state, ischemia-reperfusion and inflammatory
molecules [47].
When NF-κB is activated, for example in presence of ROS, by phosphorylation of its
inhibitory cofactor, it bonds to a DNA response element and promotes the transcription of
genes involved in inflammatory and pro-fibrotic response, interleukin-6, transforming growth
factor beta and tumor necrosis factor alpha [48]. Those molecules act in various tissues, but
particularly at the heart, producing extracellular matrix remodeling and fibrosis (structural
remodeling), which changes the electrophysiological properties of the heart making it
susceptible to generate new re-entry foci and therefore perpetuate conduction abnormalities
generated from rapidly discharging foci.
Several studies have associated NF-κB activation with cardiac dysfunction, ventricular
hypertrophy and maladaptive cardiac growth [19]. Different inflammation markers have been
found in increased levels in serum and atria biopsies of AF and POAF patients [5, 7, 24, 44].
Therefore it is reasonable to assume that oxidative stress and inflammation response act in a
synergic way in the underlying mechanisms of AF and POAF, giving the foundation for
studies involving anti-inflammatory AF therapy. A schematic representation of the events
associated with AF genesis and perpetuation is presented in figure 4-1.
Figure 4-1. Schematic diagram illustrating a hypothesis based on the main contributory factors involved
in the genesis and perpetuation of atrial fibrillation. AF, atrial fibrillation; NF-κB, nuclear factor
kappaB; ROS, reactive oxygen species; AT II, angiotensin II; IL-6, interleukin-6; hsCRP, high sensitive
C-reactive protein; RyRc, ryanodine receptor Ca2+ channel; ERP, effective refractory period; MM-CK,
myofibrillar creatine kinase.
3. Prevention of Postoperative
Atrial Fibrillation by Antioxidants
Based on the numerous evidence supporting the hypothesis that oxidative stress is a
cornerstone in the pathogenesis underlying POAF it could be noted that the use of
antioxidants as therapeutic tools appears to be a rational line of study. Substances with
antioxidant properties such as statins, N-acetylcysteine, and specially vitamins C and E have
probed to be efficient in not only decreasing the serum oxidative levels in patients
undergoing cardiac surgery, but also diminishing the occurrence of POAF [10,49-52].
Furthermore it has been hypothesized that one of the mechanisms whereby classic anti-AF
drugs act is related with the ability to scavenge ROS and protect against membrane lipid
peroxidation [53]. However, with all the evidence gathered to date, vitamin C (ascorbate) and
vitamin E (α-tocopherol) highlight among other antioxidants, gathering several biochemical
and empiric evidence that makes them excellent candidates to be used in the treatment and/or
prevention of AF and POAF. The available evidence supporting the use of each one of these
agents will be heavily discussed below.
3.1. Statins
Statin drugs have both antioxidant and anti-inflammatory properties and several studies
argue that their cardiovascular protection ability is part of their pleiotropic effect and goes
beyond the cholesterol lowering effect alone [54,55]. The pleiotropic effect involves an
improvement of endothelial function, enhancement in the stability of atherosclerotic plaques,
decrease of oxidative stress and inflammation, and inhibition of the thrombogenic response.
With regard to POAF, it has been observed that preoperative statins diminished the incidence
of POAF in patients undergoing cardiac surgery [10, 54, 55]. Furthermore, statins attenuates
AF promotion by atrial tachycardia in dogs [58]; and has been reported a decreased in the
latter appearance of AF in patients undergoing electric cardioversion [56,57]. Recently, a
meta-analysis of over 30000 patients showed that POAF incidence when using preoperative
statins diminished from 29.3% (in the no-statins groups) to 24.9% [11]. All evidence point
towards statins capacity to prevent AF and POAF, based on their antioxidant effect.
3.2. N-Acetylcysteine
N-acetylcysteine (NAC) is a drug used for multiple purposes in clinical medicine [59]. In
the last years, its antioxidant ability has called attention, leading to subsequent research in the
prevention and/or treatment of AF and POAF. A prospective, randomized, placebo-controlled
trial was conducted to study the potential anti-arrhythmic effect of NAC [10]. In this study of
115 patients undergoing coronary artery bypass and/or valve surgery, 58 patients received
pre-operative NAC and 57 patients received placebo (both groups received also standard
medical therapy, including β-blockers). The results showed that POAF incidence was 5.2% in
the NAC group and 21.1% in the placebo group. These data demonstrated that an antioxidant
agent, such as NAC, used in combination with classic anti-arrhythmic drugs, could contribute
to prevent the appearance of arrhythmias like POAF. More studies using NAC are still
lacking to assess the actual potential of this agent.
3.3. Biological Properties and Synergism of Vitamins C and E
Vitamin C and vitamin E are essential antioxidants that perform their roles in different
cell locations, while the first one acts in water-soluble components the second one does it in
lipid-soluble zones (mainly biological membranes or lipoproteins). Therefore all cell
components could be protected against oxidative damage when both vitamins are used
together [60, 61]. The most studied mechanism whereby they act is partly based on their
property to directly reduce ROS. In addition to its ROS scavenging functions, these two
antioxidants exerts their action in a synergistic way: when α-tocopherol losses and electron
and is left like α-tocopheroxyl radical, vitamin C reduces it, so it can maintain its antioxidant
properties [51, 52]. In the absence of efficient reducers, vitamin E cannot be recycled into its
antioxidant form, leading to the formation of tocopheryl quinone, molecule that could
compete in mitochondrial respiratory chain reactions. Hence, the therapeutic strategy
presented in this chapter is based in the associated administration of both ascorbate and α-
tocopherol, ensuring the efficient recycling of vitamin E radicals [62, 63].
3.4. Endothelial Modulation
Besides their ROS scavenger actions, vitamins C and E exert a complex modulation of
numerous enzymes involved in ROS production, endothelial dysfunction, platelet aggregation
and smooth muscle cell tone [27, 64, 65]. The four most important mechanisms in which
antioxidant vitamins modulates the endothelial function are: NADPH down-regulation, and
up-regulation of eNOS, phospholipase A2 and antioxidant enzymes.
NADPH oxidase, the most important superoxide source in the cardiovascular system, can
be directly down-regulated by vitamins C and E. The mechanism behind this effect has not
been completely elucidated. It has been reported that ascorbate and α-tocopherol could be
involved in NADPH oxidase transcriptional and post-transcriptional modulation. Moreover,
studies describing a possibly direct effect to the NADPH oxidase synthesis have also been
presented. Vitamin E could be involved in inhibiting the enzyme subunits aggregation, based
in the location (membranous organelle) in which this process takes place [66].
In the presence of oxidative stress, eNOS is mostly in its uncoupled form, participating in
superoxide production and NO synthesis impairment, all which leads to endothelial
dysfunction. In this context, antioxidant vitamins have shown to increase eNOS activity, by
enhancing the intracellular availability of tetrahydrobiopterin (one of its cofactors) and by
inhibiting the p47phox subunit expression. Therefore, ascorbate and α-tocopherol increase
NO synthesis, reduce ROS formation and contribute to the vascular tone regulation [66-69].
In relation to antioxidant enzymes up-regulation, some studies have demonstrated a
positive correlation between antioxidant vitamins and antioxidant enzymes activity,
particularly SOD. The mechanisms underlying these findings are not well elucidated, but it is
plausible to hypothesize the existence of transcriptional and post-transcriptional events
involved in the up-regulation of those antioxidant enzymes [65].
Finally, vitamin E also modulates the vascular prostanoid synthesis by up-regulating
phospholipase A2 expression and therefore arachidonic acid (precursor of prostanoids, and
leucotrienes) release; and down-regulating cyclooxygenase-2 expression. The final result is a

net increase in vasodilator prostanoids, which contribute to the regulation of the vascular tone
[70]. A schema with the proposed effect of antioxidant agents in the reinforcement of the
myocardial antioxidant defense system is depicted in figure 4-2.
Figure 4-2. Schema with the proposed paradigm for the effect of antioxidant agents in the reinforcement
of the myocardial antioxidant defense system. NAD(P)H oxidase, reduced nicotine adenine dinucleotide
phosphate oxidase; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; NAC, N-
acetylcysteine.
3.5. Empiric Evidence Supporting the Beneficial Effects

of Vitamins C and E
3.5.1. In Vitro Studies and Animal Trials

Vitamins C and E have demonstrated intrinsic abilities in preventing cell apoptosis,
necrosis and cardiac dysfunction. Several studies have established their role in preventing
oxidative damage in in vitro cardiomyocytes. Thus, when isolated cardiomyocytes were

exposed to singlet oxygen oxidative damage, which lead to irreversible hypercontracture of
95% of the cells, the pre-treatment with vitamins C and E reduced the hypercontracture
percentage in direct correlation with the vitamin concentration. This effect was enhanced
when using both vitamins simultaneously [71].
Cardiomyocyte apoptosis has also been prevented by administration of antioxidant
vitamins, which was also correlated with the diminution of oxidative stress biomarkers [72,
73]. Electrophysiological changes, secondary to hypoxia mediated injuries in guinea pigs
cardiomyocytes, were prevented upon ascorbate administration. Vitamin C generated an
important attenuation in the hypoxia related sodium current disturbance [74]. There also exist
available data supporting vitamins anti-arrhythmic specific properties. In isolated rat hearts
undergoing ischemia-reperfusion injuries, vitamin E showed an effective prevention in the
appearance of reperfusion arrhythmias [8].
Furthermore, several animal models have been used to assess the favorable effects of
vitamin C and E in the prevention of necrosis-apoptosis events, oxidative damage, calcium
overloading and cardiac dysfunction [75-77]. Antioxidant vitamin anti-necrosis properties
were established considering that cardiomyocytes necrosis events, of rats submitted to
stimulation of myocardial infarction was prevented by the administration of vitamins C and E
[73].
Myocardium fibrosis and remodeling play an important role in AF and POAF genesis
and perpetuation. In this regard, α-tocopherol has shown important effects in preventing
cardiac remodeling in spontaneously hypertensive rats, based on the inhibition of
cardiomyocyte hypertrophy [78]. In addition, cardiac dysfunction attenuation through vitamin
administration was demonstrated using rabbit models. Antioxidant vitamins were
administered after pacing-induced cardiac dysfunction; subsequently, it was found a decrease
in myocardial oxidation biomarkers, an attenuation of the pacing-induced cardiac dysfunction
and a reduction in cardiomyocyte necrosis biomarkers [70, 72].
Both in vitro and animal trials have helped to understand the actual potential that
antioxidant vitamins could have in preventing AF. Although the molecular basis and the in
vitro evidence that supports their use in the prevention and/or treatment AF and POAF has
been accumulating over the last years (evaluating cardiomyocyte contractility, apoptosis,
electrophysiology, and isolated hearts arrhythmia appearance). It is necessary to gather all
efforts in performing clinical trials, based importantly in the innocuousness of ascorbate and
α-tocopherol administration.
3.5.2. Clinical Trials to Prevent Postoperative Atrial Fibrillation

Antioxidant vitamins and AF related clinical trials have not been heavily studied. The
advances made in this direction are presented in the following paragraphs.
One of the most paradigmatic studies involving ascorbate anti-arrhythmic properties was
conducted to test not only the effects of vitamin C supplementation in POAF incidence, but
also to assess the biochemical changes in oxidative and electric status after canine atrial
pacing. In the first part, 43 patients subjected to coronary artery bypass were given 2 g of
vitamin C the day before the surgery, followed by 500 mg until the fifth post-operative day.
The POAF incidence in the ascorbate treated group was 16% v/s 35% in the control group. In
the other part of the study, eleven dogs were subjected to rapid atrial pacing, which led to
shortening of the effective refractory period (ERP), associated with accumulation of 3-
nitrotyrosine, a peroxynitrite oxidative marker, and decreased levels of ascorbate compared
with non paced controls. Ascorbate treatment attenuated the ERP shortening and diminished
the 3-nitrotyrosine concentration found after atrial pacing [9]. This study showed, on the one
hand that antioxidant vitamins could decrease the incidence of POAF, and on the other hand
showed that this effect could be a reflection of a stabilization of the electrophysiological
properties of the heart, that are impaired in individuals presenting this arrhythmia. It should
be remembered that one of the mediators accounting for the shortening of the refractory
period is superoxide (which leads to the cardiomyocytes calcium overloading), therefore it is
plausible to occur an attenuation of the shortening ERP in presence of antioxidants.
The effects of ascorbate administration in relation to AF have been tested under different
contexts. A trial studied 44 patients subjected to electrical cardioversion of persistent AF, all
received standard treatment, but one group received vitamin C during 7 days, while the other
received only ordinary drugs. Within a week, AF recurred in 4.5% of the ascorbate treated
group and in 36% of the control group [79]. In addition, antioxidant vitamins have been
studied in the prevention of post-thrombolysis AF; comparing two groups subjected to
therapeutic alteplase thrombolysis, one receiving antioxidant vitamins and the other placebo,
the results showed that the first one developed AF after reperfusion in 6% while the placebo
group presented the arrhythmia in 44% [80]. Post-thrombolysis AF is the gold standard
example for ROS induced AF. Before the re-vascularization procedure, the heart tissue was
experimenting high levels of hypoxia; whereas after the administration of the thrombolytic
therapy (in this case alteplase), the heart suffered an acute restoration of blood and oxygen,
which lead to calcium overloading of cardiomyocytes, activation of cell death pathways and
production of high levels of ROS, phenomenon known as ischemia/reperfusion. Through all
those mechanisms, electric properties of the heart were deregulated, and heart tissue was
incapacitated to correctly conduct the electric impulses and therefore AF was observed.
Recently, it was shown that oral vitamin C in association with β-blockers was more
effective in preventing POAF than β-blockers alone. This study consisted in 100 patients
undergoing coronary artery bypass grafting, separated in a β-blockers group and a β-
blockers/ascorbate group, which received 2 g of ascorbic acid on the night before the surgery
and 2 g daily for 5 days after surgery. The POAF incidence was 4% in ascorbate group and
26% in the control group [81]. Consequently, antioxidant vitamins not only have shown
favorable anti-arrhythmogenic results compared with non-vitamin patients, but also with
patients receiving classical anti-AF drug treatment. This concept has major relevance, as the
study for alternative therapeutic tools was originated because of the lack of effective and risk-
free treatment for AF.
The future task is to continue testing antioxidant therapies under different protocols and
contexts, to assess their real potential in preventing and/or treating AF and POAF.

Oxidative stress plays a key role in the development of atrial fibrillation (AF), the most
common arrhythmia in the general population. Increased production of reactive oxygen
species (ROS) in myocardial tissue occurs in the unavoidable ischemia-reperfusion cycle
produced during cardiac surgery with extracorporeal circulation. On this line, it is also
conceivable the contribution of ROS in the development of postoperative AF (POAF), a
frequent complication associated with poor clinical outcome of patients. Therefore, the
deleterious effect of ROS could be counteracted by a reinforcement of the myocardial
antioxidant defense system, involving either its non-enzymatic or enzymatic components.
This paradigm lead to consider the administration of antioxidants, before cardiac surgery, in
order to diminish the vulnerability of the heart to present the arrhythmia, thus avoiding, or at
least mitigating the electrical and structural tissue remodeling caused by ROS exposure.
Accordingly, evidence from both experimental studies and clinical trial has given a clue to
the potential role of antioxidants, particularly vitamins C and E, in diminishing the incidence
of POAF. After an initial approximation to the subject, the preconceived concept that ROS
main function is to destroy and alter biological molecules, such as membranes and proteins, is
replaced with the new paradigm that presents ROS as multi-tasking mediators that perform its
actions through multiple mechanisms such as pre and post-transcriptional modulation,
electric and structural tissue remodeling, energetic impairment and activation of parallel
processes like inflammation. All those mechanisms together account for the high rates of AF
developing in patients that are submitted to procedures that are intrinsically linked with ROS
production, such as cardiac surgery, extracorporeal circulation and re-vascularization
procedures. The fact that oxidative stress has been found to play an essential role in the
pathological events related to this rhythm disorder, it is crucial to the future of therapeutic
research in this field. Available pharmacologic treatments for AF based on ion channel
blockade have demonstrated limited efficacy, underlining the relevance of the development
of a prophylaxis and/or novel treatment for this disorder. In the light of the current advances,
the future of antioxidant vitamin based POAF preventive therapy looks promising. The
studies made in this field, that gathers in-vitro, animal and clinical trials, all point to potential
benefits of the antioxidant vitamins to at least prevent or likely treat oxidative stress related
disorders. As an amelioration of ischemia/reperfusion tissue injury could be expected to
contribute to the success of organ transplantation, future research should aim to find the
experimental and clinical support to this view, based on, an under explored measure to
optimize the quality of a living organ allograft.
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Chapter V
Acute Renal Failure
Joaquín Toro,1 Víctor Molina2 and Ramón Rodrigo3

1
2
3
Institute of Biomedical Sciences, Faculty of Medicine, University of Chile
Abstract
Acute renal failure (ARF) is a condition characterized by a rapid decrease in renal
function, leading to an imbalance in water and solutes metabolism. It constitutes a major
cause of morbidity and mortality in hospitalized patients worldwide, mainly in elderly
population. Despite the medical advances, over the past fifty years the mortality of ARF
has not diminished. This is often attributed to increased risk factors prevalence, mainly
those derived from changes in our lifestyle. However, it is also possible that the
therapeutic methods used until these days are not aiming on the right direction, probably
due to lack of knowledge about some of the mechanisms leading to the development and
progression of ARF.
Over the last decades a large body of evidence has emerged supporting a role of
oxidative stress in the pathogenesis of a variety of diseases, including ARF. Indeed, both
reactive oxygen and nitrogen species are thought to enhance tubular damage caused from
either renal ischemia or direct toxic injury. Nevertheless, the role of oxidative stress in
ARF pathogenesis has not been fully established and some evidence is even
contradictory. A better understanding regarding the real contribution of oxidative stress
to ARF development and progression is required for the design of potentially preventive
interventions, such as antioxidant supplementation. Indeed, clinical trials on this matter
have been carried out with promising results.
This chapter presents an update of the current evidence supporting a role of
oxidative stress in ARF pathophysiology, and the potential role of antioxidants in the
prevention and treatment of this disease.
112 Joaquín Toro, Víctor Molina and Ramón Rodrigo
1. Introduction
Acute renal failure (ARF) is a condition characterized by a fast declination, from hours to
days, of renal function. Consequently, the plasma concentration of nitrogenated compounds
is increased (azotemia), hydroelectrolitical and acid-base disorders develop, and extracellular
volume (ECV) alterations arise [1, 2].
Approximately 1% of hospitalized patients have ARF at the time of admission, and its
estimated incidence during hospitalization is 2-5% [3]. Acute renal failure is said to occur in
anywhere from 1% to 25% of critically ill patients [4, 5]. In intensive care settings, the
mortality rate of ARF is 70-80%. Current prevention strategies are inadequate and available
treatment options besides renal replacement therapy are nonexistent [6].
Despite the medical advances, the mortality of ARF has not diminished in the last forty
or fifty years, remaining in about 40-50%. Likely, this is related to an increasing association
of this condition with aggravating factors, including increased age, presence of comorbidities,
association with multiple renal injuries, inflammatory systemic response syndrome and
multiorganic dysfunction syndrome. The lack of reduction in mortality rates might be due to
the fact that the underlying mechanisms of ARF, and its final damaging pathway, have not
yet been fully elucidated. Indeed, critically ill patients who develop ARF experience a high
mortality rate that is not entirely explained by sepsis, advanced age, or underlying morbid
conditions [7, 8].
It is well known that elderly patients have increased ARF death risk in comparison to
young patients [9]. Moreover, it has been demonstrated that critically ill patients with ARF
present an excess of plasma protein oxidation [10]. Since oxidative stress is strongly related
to ageing, it could be expected that excessive production of reactive oxygen species (ROS) or
impairment in the endogenous ROS scavenging system could play a key role in ARF
pathophysiology in these patients. Ischemia and nephrotoxic damage arise as two important
causes of ARF, both resulting in oxidative stress.
The aim of this chapter is to present an update of the role of oxidative stress in ARF
pathophysiology. Also, the mechanisms by which antioxidants supplementation could modify
the clinical outcome of these patients are explained.
2. Pathophysiology of Acute Renal Failure

Before analyzing the involvement of ROS in ARF pathogenesis, a brief review of basic
renal physiology is presented.
The kidney is an organ that performs two major functions that are essential for survival:
1. It participates in the maintenance of a relatively constant extracellular environment

that is necessary for normal cell functioning. This is achieved through the excretion
of waste products of metabolism, such as urea, creatinine and uric acid, and of water
and electrolytes, derived mainly from dietary intake. The adequate balance is
maintained by keeping the rate of excretion equal to the sum of net intake plus
endogenous production, if this occurs.
Acute Renal Failure 113
2. It produces hormones, enzymes and factors that participate in the regulation of

systemic and renal hemodynamics, such as renin, angiotensin II (Ang-II), and
prostaglandins. Erythropoietin and calcitriol are of major relevance as renal secretion
products. Erythropoietin is related with red blood cell production, and calcitriol is an
essential hormone for calcium and phosphate homeostasis.
The kidney also performs a number of miscellaneous functions such as catabolism of

peptide hormones and synthesis of glucose under fasting conditions, which is known as
gluconeogenesis.
The morphophysiological unit of the kidney is the nephron. The number of nephrons is
estimated in a million per each healthy kidney. For better understanding, the nephron can be
divided in two main parts:
• Glomerulus: It comprises two zones: the vascular pole and the urinary pole. At the
vascular pole, the afferent arteriole (AA) forms the capillary tuft, after which the
efferent arteriole (EA) is formed and leaves the glomerulus. The luminal surface of
the capillaries is formed by a fenestrated endothelium. The continuous glomerular
basement membrane anchors the endothelium to the visceral layer of Bowman’s
capsule. This layer is formed by specialized epithelial cells called podocytes, which,
along with their numerous extensions (the foot processes) cover the capillaries.
• Tubules: The urinary space is continuous with the lumen of the proximal tubule.
The tubular system is responsible for the reabsorption and secretion processes.
Urine formation begins with filtration of a protein-free plasma, or ultrafiltrate, into the
urinary space. The movement of water and associated dissolved small molecules
(crystalloids) is determined by hydrostatic and oncotic pressures. Glomerular capillaries are
about a hundred times more permeable to water and crystalloids than muscle capillaries. This
filtration raises the plasma oncotic pressure as fluid moves along the capillaries, due to net
loss of water into Bowman's space. The filtrate is modified as it passes through the nephron
by tubular reabsorption and/or tubular secretion.
Normal glomerular filtration rate (GFR) is approximately 180 L per day, or 125 mL per
minute. Of this enormous amount, only 1-2 L per day are excreted as urine, implying that
99% of the filtered volume is reabsorbed.
In pathophysiological terms, ARF is defined as an abrupt decrease in GFR. Impairment
of renal function leads to a rise in serum nitrogenated compounds (azotemia), such as
creatinine and urea, being the latter frequently measured as blood urea nitrogen (BUN).
However, immediately after a kidney injury, BUN or creatinine levels may be normal and the
only sign of renal function impairment may be decreased urine output. Moreover, several
conditions might alter these parameters, including medications, protein loading or
gastrointestinal bleeding. Therefore, creatinine and BUN levels must be interpreted in the
context of each patient in order to determine whether or not an alteration of renal function is
present.
Retention of creatinine and urea is accompanied by the accumulation of a variety of
substances generically named as “uremic toxins”. The effects of these toxic substances
account for many of the symptoms and signs associated with end-stage renal disease.
Examples of these uremic manifestations include pericarditis, altered mental status and
peripheral neuropathy, among others. Inadequate potassium and sodium excretion are also
commonly seen, leading to hyperkalemia and edema, respectively. At this time, the GFR is
likely to be found between 5 and 10 mL/min.
For reasons that are not well understood, the intrarenal adaptations that allow the
maintenance of fluid and electrolyte homeostasis are more likely to occur in chronic renal
disease than in ARF. At the same reduction of GFR, patients with ARF are more likely to
develop edema, hyponatremia, and hyperkalemia.
The terms ARF and acute tubular necrosis (ATN) are often mistakenly exchanged. Acute
tubular necrosis is a form of ARF that is caused by an ischemic or toxic injury to the tubular
epithelial cells [11]. Acute renal failure may be caused by several etiologies, which can be
classified in three large groups:
Pre-Renal Causes
Pre-renal causes include a variety of clinical settings that associate with a decreased renal
perfusion, as occurs in a diminution of effective arterial volume (EAV) with structurally
intact nephrons, giving rise to an adaptive kidney response.
Renal Causes
Renal causes are related to cytotoxic, ischemic, or inflammatory insults to the kidney,
leading to structural and functional damage. Structural injury to the kidney is the
characteristic of intrinsic ARF. The most common form is ATN, either ischemic or cytotoxic.
Postrenal Causes
Postrenal causes include all the conditions in which an obstruction to the passage of urine
occurs anywhere along the urinary tract, between the renal pelvis and the urethra.
Despite the fact that many pathophysiological features are shared among these different
categories, they differ in several topics, such as clinical presentation, functional integrity of
the tubule, response to therapy and specific diagnosis tests. Then, this classification is useful
when establishing a differential diagnosis.
The pathophysiological events leading to the death of tubular cells are complex and
incompletely understood. Nevertheless, the central hallmarks of either ischemic or toxic ARF
are injury, apoptosis and necrosis of tubular cells. As follows, we will discuss the major
structural and biochemical features thought to be important for ATN and its consequences.
2.1. Renal ischemia
The kidney is an organ highly responsive to changes in EAV. As a consequence of

reduced renal plasma flow (RPF), renal ischemia may give rise to metabolic changes causing
a deep impairment in the processes responsible for tubular transport. In addition, structural
effects can alter the viability of epithelial tubular cells. Renal ischemia is a disturbance that
has generated conflicting experimental data and different pathophysiological explanations.
This is reflected in the variety of names given to this condition, including traumatic
nephrosis, lower nephron nephrosis, vasomotor nephropathy, post-ischemic ARF and
ischemic nephropathy, among others.
We will define renal ischemia as the deficiency of blood in one or both kidneys. The
impairment of renal perfusion might be due to several causes, mainly arising from functional
constriction or genuine obstruction of a renal artery. Other systemic situations leading to
renal ischemia include volume loss from internal or external hemorrhage, heart failure,
hepatorenal syndrome and shock. The diminution of RPF leads to decreased GFR, being the
latter a less marked change. This effect results from a prevailing vasoconstriction of the EA
mediated by Ang-II that contributes to the maintenance of the hydrostatic pressure within the
glomerular capillaries. Consequently, a rise of the filtration fraction is developed, producing a
relative diminution of hydrostatic pressure and a rise in the oncotic pressure at the level of the
peritubular capillaries. All of these changes create a favorable condition for sodium
reabsorption at the proximal tubule. The diminution in GFR, together with the increased
sodium reabsorption, contributes to the elevation of plasma urea and creatinine concentration,
producing renal azotemia.
In physiological conditions, the kidney has a disproportionately high blood flow in
relation to its oxygen consumption [12]. Blood samples from the renal vein have an oxygen
tension considerably higher than the mixed venous blood draining other organs. The high
renal blood flow is commonly seen as designed to maximize flow-dependent clearance of
wastes [13]. Nevertheless, the kidney has also an important functional oxygen reserve. This
reserve should protect it from potential ischemic challenges, making it less likely to be
damaged by decreased RPF. However, what really occurs is exactly the opposite: the kidney
is an organ remarkably susceptible to hypoperfusion, as mentioned before. ¿How is this
possible?
Initially, it was suggested that a non homogeneous distribution of blood flow exists
inside the kidney. Furthermore, several studies have demonstrated that oxygen delivery to the
kidney is complex, heterogeneous, and gradient-limited, suggesting the possibility of
selective regional hypoxia as a potential major source for localized injury during renal
hypoperfusion. Then, although the overall balance of oxygen consumption is relevant, a
special attention must be paid to the segment of the nephron that has more chance of being
harmed. In general terms, the medullar part of the kidney is more likely to be injured by
hypoxia. The most severe damage takes place in the straight part of the proximal tubule,
known as the S3 segment.
The model that has been proposed for explaining the pathophysiology of renal ischemia
is focused in alterations occurring in the tubular epithelium. Early morphologic changes
observed after ischemia include the formation of blebs in the apical membranes of proximal
tubule cells, with loss of the brush border [14, 15]. Afterwards, proximal tubule cells lose
their polarity, and the integrity of their tight junctions is disrupted [16], a process that is
thought to arise from alterations in the actin and microtubule cytoskeletal organization [17,
18]. As a consequence, some cellular-membrane proteins are transferred to unusual sites. For
instance, the Na+/K+ - ATPase redistributes from the basolateral to the apical membrane [19]
thus reducing or even more, reversing the unidirectional sodium transport from tubular lumen
to peritubular interstitial space. The increased sodium delivery to the distal tubule triggers the
tubule-glomerular feedback, which leads to a vasoconstriction of the AA, with the
consequent decrease of GFR. This is the most relevant mechanism of the maintenance phase
of ARF.
When ischemic damage occurs, integrins, a group of proteins involved in intercellular
adhesion, move to the apical surface of the tubular epithelium [20], and facilitate its adhesion
with cells that have been shed due to apoptosis or necrosis, thereby forming conglomerates
that cause obstruction in the tubular lumen [21]. Then, the desquamation of tubular
epithelium leads to a raise in intratubular hydrostatic pressure. In addition, backleak of filtrate
occurs as a consequence of structural alterations affecting tubular integrity, further
contributing to the diminution of urine output currently present in this setting.
Several changes occur due to the lack of ATP caused by oxygen deprivation, particularly
in the most metabolically active tubular cells. After the occurrence of hypoxia, but before cell
membrane damage, the elevation of intracellular sodium concentration contributes to the
development of an increased intracellular calcium concentration [22]. In turn, intracellular
calcium activates phospholipase A2 that hydrolyze phospholipids of the plasma membrane,
releasing fatty acids and lisophospholipids. It was reported that peroxidation of membrane
lipids due to ischemia-reperfusion enhances the susceptibility of membranes to phospholipase
A2 (PLA2) [24]. Additionally, arachidonic acid, a product of PLA2, is converted into
eicosanoids that produce vasoconstriction and are chemotactic for neutrophils [25].
Calcium can also contribute to epithelial cell toxicity through its ability to activate
proteases, break down the cytoskeleton, and interfere with mitochondrial energy metabolism.
However, there is still controversy regarding the in vivo intracellular calcium concentration
required to cause ischemic tubular cell injury, and if it is possible to reach this concentration
in tubular cells [26].
In ischemic ARF there is also a neutrophil infiltration in the kidney. The migration of
leucocytes to neighbor tissues is possible through the binding of neuthrophil integrins to
adhesion molecules present in the vascular endothelium. This migration to the interstitial
space leads to cell damage by increased ROS production and the activation of enzymes such
as collagenases, elastases and myeloperoxidases, thereby promoting the migration of further
inflammatory cells. Although leucocytes appear to have an important role in AFR
pathogenesis, neutropenic patients can also develop severe forms of ARF. Then, it seems that
leucocytes are not essential for the development of acute tubular disease. However, their role
in ARF has yet to be fully established.
2.2. Nephrotoxic Damage
Nephrotoxic damage exerted by toxins accounts for the second more important
mechanism of ARF development. It is important to notice that the mechanisms whereby the
toxins cause tubular necrosis share many pathophysiological features with ischemic ARF
[27]. Moreover, ischemia and toxins often combine to cause ARF in severely ill patients with
conditions such as sepsis, hematological disorders, cancer or acquired immunodeficiency
syndrome (AIDS) [28, 29]. The mechanisms by which drugs can cause ARF are detailed
next.
2.2.1. Direct Tubular Cell Damage

Aminoglycoside antibiotics and radiocontrast agents are the most common toxins that
cause ARF and both induce damage frequently in proximal tubule. Furthermore, vancomycin,
cisplatin, immunoglobulin and mannitol may also generate proximal damage leading to
impaired tubular function. Typically, tubular cells lose polarity, develop vacuoles and
eventually separate from the basement membrane. Marked disturbance of electrolyte
homeostasis may also occur due to effects on water reabsorption in the distal tubule [30].
Acute anuria has been reported in critically ill patients treated with high dose
immunoglobulin for Guillain-Barré syndrome, probably due to acute renal dysfunction
caused by proximal tubular cell damage, a mechanism known to occur also in mannitol
nephrotoxic damage [31]. On the other hand, non-steroidal anti-inflammatory drugs
(NSAIDs), angiotensin converting enzyme (ACE) inhibitors, cyclosporin A, lithium, and
cyclophosphamide induce direct damage essentially in the distal tubule. Consequently, a
disturbance of sodium, potassium, hydrogen ion and water balances is produced. Non
steroidal anti-inflammatory drugs, ACE inhibitors and cyclosporin A, all alter potassium
balance, resulting in hyperkalemia. Chronic administration of lithium may initially result in
transitory and then in permanent inability to thrive, causing insipidus nephrogenic diabetes.
Acute lithium intoxication causes a similar tubular effect which may be reversible. In
contrast, high doses of cyclophosphamide may result in hyponatremia due to an impaired
ability to excrete water.
In general terms, direct cellular damage is dose-dependent and is enhanced when
occurring in hypoxic conditions. For instance, amphotericin B is capable of inducing damage
in both proximal and distal tubules. The enhanced membrane permeability produced by
amphotericin B triggers an increase in active sodium transport and oxygen demand. In
consequence, a more severe damage occurs if a reduced supply of oxygen is associated [32,
33].
Renal damage arising from the nephrotoxic effects of radiocontrast agents is becoming
increasingly common. Radiocontrast media is used in relatively high doses for computed
tomography scans and some types of vascular surgery. The risk of renal damage is
particularly high in patients who already have impaired renal function or those with diabetes
mellitus [34]. In patients with both diabetes and impaired renal function, the incidence of
further renal failure following use of radiocontrast agents is over 50%. In these cases, both
vasoconstriction and direct tubular damage occur. Preventive measures are limited to saline
diuresis prior to radiocontrast administration.
There are a number of other drugs such as tacrolimus, methotrexate, foscarnet, and
pentamidine that are known to be potentially nephrotoxic [35]. Additionally, several other
substances can cause direct tubular damage including organic solvents, heavy metals (e.g.
mercury) and carbon tetrachloride. Finally, direct tubular damage can also be caused by some
plant and animal toxins.
2.2.2. Reduction in Renal Perfusion through Alteration of Intrarenal

Hemodynamics
In volume depleted states some drugs can also induce ARF through alterations in
intrarenal hemodynamics. This is the case of ACE inhibitors and angiotensin receptor
blockers. It is remarkable to notice that these drugs are otherwise safely tolerated and
beneficial in most patients with chronic kidney disease. Angiotensin II acts directly within
the glomerular circulation. The use of ACE inhibitors, not only inhibits Ang-II production but
also interferes with bradykinin, which has an important role in the circulatory control of the
glomerulus.
Non-steroidal anti-inflammatory drugs may induce a decrease in GRF through a selective
inhibition of cyclo-oxygenase that normally acts as a vasodilator in the AA, thereby
inhibiting the compensatory mechanisms that protect the kidney from reduced plasma flow in
volume depleted states. Cyclosporine A has a similar effect in the AA. Both of these drugs
and their effects are potentiated by hypovolemia, low cardiac output, sepsis, liver disease, and
pre-existing renal failure.
Arteriolar vasoconstriction leading to ARF may also occur in hypercalcemic states, with
the use of radiocontrast agents, amphotericin B, calcineurin inhibitors, norepinephrine, and
pressor agents, among others.
2.2.3. Intratubular Obstruction by Precipitation of the Agent, Its Metabolites

or by-Products
Drugs that may directly or indirectly cause tubular obstruction include acyclovir,
sulfonamides, ethylene glycol, chemotherapeutic agents, and methotrexate.
Patients with Pneumocystis pneumonia as a result of AIDS and other immunosuppressive
disorders are increasingly being treated with high doses of sulphonamides. Such treatment is
associated with increased incidence of crystalluria resulting in tubular obstruction and renal
dysfunction. Adequate salt and water loading should preserve the tubular filtrate flow,
thereby preventing the precipitation of drug and hence renal failure. The most common used
anti-viral agent, acyclovir, and the protease inhibitor indinavir, a pillar in AIDS treatment,
have both similar toxic actions.
Treatment of patients with high dose chemotherapy for hematological malignancies can
result in rapid cytolytic effect resulting in a greatly increased uric acid load arriving to the
kidney. In such patients, acute crystalluria may develop unless adequate urine flow and
sodium diuresis is maintained.
Ethylene glycol, a known antifreeze liquid, can be a cause of ARF when it is accidentally
taken, such as occurs in children poisoning. Its metabolism results in a large oxalate load
which may crystallize in the tubule, thereby causing obstruction.
2.2.4. Allergic Interstitial Nephritis

Acute renal failure due to acute interstitial nephritis is most often caused by an allergic
reaction to a drug [36]. In strict sense, every drug might induce interstitial nephritis.
Nevertheless, the drugs that are more likely to produce this syndrome are: penicillins,
cephalosporins, sulfonamides, rifampicin, ciprofloxacin, vancomycin, NSAIDs, thiazide
diuretics, furosemide and allopurinol. Less commonly ranitidine, cimetidine and phenytoin
may also cause similar damage.
2.2.5. Heme Pigment-Induced Tubular Toxicity (Rhabdomyolysis)

Rhabdomyolysis-induced ARF is a condition that will be further described below in this
chapter. It is caused by drugs such as cocaine, ethanol and statins, particularly lovastatin,
which may induce ARF. Rhabdomyolysis is more likely to occur when lovastatin is given in
combination with cyclosporine [37].
2.2.6. Hemolytic–Uremic Syndrome

Certain drugs can cause hemolytic-uremic syndrome, including cyclosporine, tacrolimus,
mitomycin, cocaine, quinine and conjugated estrogens. Non-drug related causes include
autoimmune diseases (e.g. lupus, Wegener granulomatosis), infiltrative diseases (e.g.
sarcoidosis), hematologic diseases (e.g. myeloma through light-chain proteins) and infectious
agents (e.g. legionnaire’s disease and Hantavirus infection) [38-40].
2.3. Role of Oxidative Stress in the Mechanism of Renal Damage
There is evidence supporting a role of ROS in kidney cellular injury. This includes the
demonstration of an accentuation of renal injury by oxidants and by antioxidants deficiency.
Accordingly, Himmelfarb et al. [10] measured the concentrations of a group of oxidative
stress biomarkers in the setting of ARF. In their retrospective analysis of PICARD study
(Program to Improve Care in Acute Renal Disease) samples, they determined the plasma
protein thiol content, which is a marker of total antioxidant capacity, and the plasma protein
carbonyl content, which is an index of oxidative injury, in critically ill patients with and
without associated ARF, patients with end stage renal disease and healthy controls. Critically
ill patients with associated ARF displayed a significant decrease of thiol content and an
increase of carbonyl content, in relation to all the other groups.
In the kidney, as well as other organs, ROS can react with proteins, carbohydrates,
nucleic acids, and cell membrane lipids. This results in organic radical formation, enzyme
inactivation, glutathione oxidation, lipid peroxidation, and renal cell destruction [41].
Therefore, consequences of ROS activity include proteinuria, disturbances in GFR and
morphological changes in the glomerulus [42].
Acute renal failure itself is recognized as an additional stimulus for oxidative stress [43,
44]. This is a consequence of the dysregulated inflammatory response in these patients, which
basically consists in stimulated phagocytic cells, leading to excess cytokines production.
Indeed, these cells are major producers of ROS.
Furthermore, oxidative stress is considered an important pathogenic mechanism for the

development of ischemic and toxic renal tubular injury [45-47]. As follows, the involvement
of ROS in the diverse mechanisms leading to renal damage will be analyzed separately.
2.3.1. Ischemia-Reperfusion
Increasing evidence has accumulated over the last few years indicating that ROS could
play a crucial role in a variety of pathogenic mechanisms, including ischemia-reperfusion
injury in several human organs.
In ischemic tissue conditions, such as myocardial infarction or prerenal ARF, most of the
cell injury is not inflicted during the period of ischemia, but after the blood flow to the
damaged tissue is restored. This is called reperfusion injury. Ischemia shifts cellular
metabolism from aerobic to anaerobic with rapid depletion of intracellular ATP stores and
increased hypoxanthine concentrations [48]. During reperfusion, the oxygen delivery enables
the activity of xanthine oxidase (XO), an enzyme that catalyzes the conversion of
hypoxanthine to xanthine and uric acid, resulting in an intensification of superoxide anion
(O2•–) and hydrogen peroxide (H2O2) generation [49]. Indeed, the production of these two
highly reactive species starts only when oxygen is widely available [50]. The respiratory burst
also activates polymorphonuclear leukocytes and monocytes that penetrate the glomerulus
and interstitium to become another source of large quantities of ROS [51]. Another pathway
for the production of ROS during reperfusion following ischemia is cyclooxygenase and
lipoxygenase activation [52]. This excessive production of ROS causes oxidative stress that
results in several changes, including impairment of mitochondrial oxidative phosphorylation,
ATP depletion, increase in intracellular calcium, and activation of proteases and
phosphatases. These changes lead to the breakdown of membrane phospholipids and cellular
cytoskeleton, resulting in loss of cellular integrity [26, 53-56].
Although the contribution of early generation of reactive nitrogen species (RNS) to the
development of renal failure has yet to be fully established, it is tempting to speculate that the
generation of RNS, rather than hydroxyl radical, is more important for the injury associated
with ischemia-reperfusion damage [57].
In normal kidney functioning, endothelium-dependent vasodilators, such as acetylcholine
and calcium ionophore A23187, act by stimulating endothelial nitric oxide synthase (eNOS)
activity, thereby increasing endothelium-derived NO production. In contrast, other
vasodilators such as nitroprusside and nitroglycerin induce vasodilation by directly releasing
NO in vascular smooth muscle cells, this way acting through an endothelium-independent
mechanism. Nitric oxide produced by eNOS, as well as released by these NO donor agents,
induces vasodilation by stimulating the production of cyclic guanosine monophosphate
(cGMP) in vascular smooth muscle cells. Other substances, like atrial natriuretic peptide
(ANP), are also endothelium-independent vasodilators but do not act through a mechanism
involving NO. ANP directly stimulates an isoform of guanylyl cyclase in vascular smooth
cells, inducing vasodilation [58]. Over the last decade, several studies have agreed that eNOS
activity is impaired following ischemia-reperfusion cycle (for more details see chapter 2).
Impaired production of NO contributes to the vasoconstriction associated with established
ARF. There is evidence showing that, in isolated erythrocyte-perfused kidney, ischemia-
reperfusion injury is associated with intrarenal vasoconstriction that is reverted by

endothelium-independent vasodilators, but not by endothelium-dependent vasodilators. This
data suggests that endothelium-derived NO production is impaired following ischemic injury
and that the inhibition of eNOS activity can contribute to the vasoconstriction associated with
ARF [59]. Although impaired NO production can contribute to renal damage through
vasoconstriction, NO itself can also be involved in an injury mechanism related with ROS.
During ischemia-reperfusion NO can react with ROS, this way decreasing its bioavailability
and leading to vasoconstriction. However, this reaction also leads to the production of
peroxynitrite, a highly reactive oxidant molecule. Evidence supporting this mechanism shows
that in a model of isolated proximal tubules the injury due to hypoxia-reoxygenation can be
prevented through the inhibition of nitric oxide synthase activity and by the addition of
hemoglobin, an NO scavenger. Moreover, L-arginine, the nitric oxide synthase substrate, and
nitroprusside, a NO donor, can enhance tubular injury under these conditions [60].
In physiological conditions, sulfhydryl groups react with NO in the presence of oxygen
to produce S-nitrosothiols, which are stored in cells as S-nitrosoglutathione [61]. It is thought
that renal tissue nitrosothiols release NO when renal blood flow is altered, oxygen tension
falls to zero, and NO synthesis is ceased. In this situation, S-nitrosothiols decompose slowly
to release NO [62]. If released NO is not inactivated by oxygen or oxyhemoglobin, its
concentration should increase progressively until reaching a maximum [63].
Some in vitro studies have shown that there exists a synergistic interaction between PLA2
and ROS in ischemia-reperfusion injury of the kidney. Membranes exposed to ROS are
peroxidized and become more susceptible to PLA2 action. This synergy occurs also in
mitochondria, where PLA2 acts in concert with ROS to uncouple oxidative phosphorylation
[64]. Indeed, it has been reported that renal ischemia-reperfusion results in increased PLA2
activity of the cytosolic, mitochondrial, and microsomal subcellular fractions of the kidney
[65, 66]. This has led to the observation that mitochondrial PLA2 activation could play a
major role in post-ischemic cellular injury. Furthermore, it was demonstrated that hyperbaric
oxygen does not induce a significant change in PLA2 activity in the non ischemic kidney,
indicating that this type of oxidative stress alone does not cause PLA2 activation in the
mitochondria. However, when hyperbaric oxygen was combined with ischemia-reperfusion,
mitochondrial PLA2 activity was markedly enhanced. This suggests that the activation of
PLA2 caused by ischemia-reperfusion is enhanced by ROS. Moreover, a study demonstrated
that the exposure to high oxygen concentration resulted in a significant decrease in
superoxide dismutase (SOD) activity in the post-ischemic rat kidney, probably due to
consumption by excessive amounts of ROS [67].
Finally, surgical interventions, such as renal transplantation, can also associate with
ischemia-reperfusion injury. This may constitute an important factor predisposing to organ
rejection [68].
2.3.2. Rhabdomyolysis
Myoglobinuria plays a key role in the pathophysiology of acute renal failure in clinical
settings that are characterized by muscle tissue injury [50]. The term rhabdomyolysis refers to
disintegration of striated muscle, which results in the release of muscular cell constituents into
the extracellular fluid and the circulation. One of the key compounds released is myoglobin,
an 18,800-Dalton oxygen carrier. It resembles hemoglobin, but contains only one heme
moiety. Apart from myoglobin, during rhabdomyolysis potentially toxic myocyte contents are
released into the systemic circulation. The renal consequences of this disturbance have been
attributed to both intense vasoconstriction and renal tubular necrosis.
Normally, myoglobin is loosely bound to plasma globulins and only small amounts reach
the urine. However, when massive amounts of myoglobin are released, the binding capacity
of the plasma globulins is exceeded. Myoglobin is then filtered by the glomerulus and reaches
the tubules, where it may cause obstruction and renal dysfunction [69]. The intratubular
degradation of myoglobin results in a massive generation of ROS that overwhelms the
scavenging capacity of the antioxidant system, thereby generating renal damage. In fact, it
has been proved that myoglobin can induce proximal tubular cell death through the
generation of H2O2 [70].
2.3.3. Dialysis
Dialysis procedure is commonly related with chronic renal failure. Nevertheless, dialysis
also represents a therapeutic option for ARF when drugs and other treatments have failed.
Indeed, it constitutes a common therapeutic method in hospitalized patients with intrinsic
ARF. The procedure is repeated as many times as necessary until the patient recovers its renal
function.
Oxidative stress contributes to morbidity in hemodialyzed patients. In order of
importance, three possible sources of ROS can be present in hemodialysis: the uremic state,
the dialyzer membrane, and bacterial contaminants from the dialysate [71].
In general terms, favorable conditions for oxidative stress development are generally
present in uremic patients on maintenance hemodialysis. In this setting, increased generation
of oxidants is associated with chronic antioxidant deficiency [67, 72]. The generation of ROS
during hemodialysis sessions can be measured by basal whole blood chemiluminescence
(CL) [73, 74]. It has been demonstrated that the production of ROS by phagocytic cells is
strictly dependent on the cellulosic nature of the dialysis membrane [73], and is closely
related to the amount of the C5a and C3a complement fractions [75]. It has been reported that
the increased intracellular ROS production in both neutrophils and monocytes from dialysis
patients is associated with increased expression of adhesion molecules, which are key
mediators for renal damage, as mentioned before in this chapter [76]. The excessive
production of ROS also promotes alterations in the endothelium, which is known to be the
first step toward atherosclerosis (see atherosclerosis chapter).
Then, although the generation of ROS due to hemodialysis might be intermittent, the
consequences of their action are beyond to be transitory. Indeed, it has been found that after
hemodialysis there is a decrease of plasma ROS scavenging capacity [77]. This effect is
thought to be related to the loss of antioxidants due to the dialysis process. In consequence,
antioxidant supplementation could be an important therapeutic approach in the prevention of
dialysis induced oxidative stress.
2.3.4. Diabetic Nephropathy

Diabetic nephropathy is a chronic renal disease model, and it represents the most
common form chronic renal failure. Thus, it is pertinent to make a brief mention of ROS
involvement in its pathophysiology even though it does not constitute an ARF cause.
There is increasing amount of evidence supporting a key role of oxidative stress in the
development and progression of diabetic nephropathy. High glucose induces intracellular
ROS production, either directly via glucose metabolism and auto-oxidation, or indirectly
through the formation of advanced glycation end products (AGE) and their receptor binding.
Reactive oxygen species mimic the stimulatory effects of elevated glycemia and upregulate
TGF-β, PAI-1, and other extracellular matrix proteins in glomerular mesangial cells, thus
leading to mesangial expansion and subsequent renal damage [78].
Indeed, it has been suggested that the trigger for hyperglycemia induced damage in the
diabetic kidney is the excessive generation of mitochondrial O2•–. Superoxide leads to the
activation of four major biochemical pathways, including increased AGE formation,
activation of protein kinase C isoforms, and increased flux through the polyol and
hexosamine pathways. In addition, each of these pathways can contribute to ROS generation
[79]
Then, maintenance of oxidative phosphorylation and normalization of mitochondrial
function could be key strategies to reduce the progression of diabetic nephropathy.
Additionally, further investigation regarding other cellular pathways, such as NADPH
oxidase and uncoupling of eNOS, is required to assess their relevance in diabetic
nephropathy and in other models of progressive human renal disease [80].
2.3.5. Nephrotoxic Damage

Aminoglycoside antibiotics are probably the most recognized potentially nephrotoxic
drugs. Unfortunately, their use is limited not only for causing nephrotoxicity (which occurs in
10–15% of cases), but also for irreversible ototoxicity (in approximately 3–25% of patients)
[81].
While gentamicin and other aminoglycosides have been studied extensively, the
biochemical and cellular basis of their nephrotoxicity are not completely understood.
However, it is evident that gentamicin leads to the disruption of the proximal convoluted
tubule and interferes with critical cellular processes through several mechanisms, including
oxidant injury. Gentamicin enhances the generation of ROS by altering mitochondrial
respiration, leading to the generation of H2O2. Also, it induces the release of iron from renal
cortical mitochondria, causing lipid peroxidation in vitro, with iron serving as a potent
catalyst for free radical formation [82].
3. Effects of Antioxidants in Acute Renal Failure

Several investigations have suggested a role of endogenous and/or exogenous
antioxidants in renal protection. However, evidence is not concluding and thereby the role of
antioxidants in acute renal failure still has to be established. It is remarkable to notice that the
beneficial role of antioxidants is out of discussion in chronic renal disease.
The major endogenous mechanism for the removal of ROS is the antioxidant enzyme
system. This system includes two superoxide dismutases that convert O2•– to H2O2 (Cu/Zn-
SOD and Mn-SOD), and two more enzymes, catalase (CAT) and glutathione peroxidase
(GSH-Px), that degrade H2O2 to H2O (for more details see chapter 1).
For example, it has been observed that SOD inhibits ROS generation, decreases lipid
peroxidation in cortical mitochondria and protects the kidney from injury after blood reflow.
In this study CAT activity did not protect against ischemia-reperfusion injury [83].
Nevertheless, a few years later the same authors demonstrated that the inhibition of CAT
before ischemia leads to an exacerbation of the ischemic injury [84]. Accordingly, Baker et
al. showed that kidney tissue taken from animals after ischemia alone was extensively
damaged compared with tissue from SOD-treated animals [53]. Also, it has been confirmed
that an elevated intracellular GSH concentration protects rat renal proximal tubules against in
vitro simulated reperfusion injury [85]. In contrast, it has been reported a non significant fall
in the activity of SOD with no changes in the activity of CAT in erythrocytes of renal
transplant patients [68].
Disparities among results can be attributed to the diverse time gaps used in ischemia-
reperfusion experimental models. Indeed, it has been found that 30 minutes of ischemia
followed by reperfusion has little effect on enzyme activity, whereas longer duration of
ischemia (60 or 90 minutes) results in significant loss of the expression and activity of
catalase, GSH-Px and Cu/Zn-SOD. In the same experimental model, Mn-SOD expression
and activity experienced an induction [86]. Moreover, it has been suggested that both
instability of mRNA for catalase, GSH-Px and Cu/Zn-SOD, and higher transcriptional
activity of Mn-SOD genes are associated with the modulation of antioxidant response to
ischemia-reperfusion injury in kidney [87]
During reperfusion following ischemia, superoxide anion is thought to be mainly
produced by XO. This is supported by several studies showing an increased activity of XO
during reperfusion and an attenuation of ROS generation under these conditions with the use
of allopurinol, a XO inhibitor [83, 88]. Moreover, it has been suggested that allopurinol could
play a role in the prevention of kidney damage during ischemia-reperfusion cycle.
The protective effect of the modulation of antioxidant enzymes against ischemia-
reperfusion induced oxidative stress provide further evidence of the impact of these enzymes
on the degree of tissue damage [89-92].
Conflicting data has been reported regarding the levels of antioxidants in dialysis
patients. Endogenous antioxidant scavengers may be low in dialysis patients due to
diminished oral intake, dietary restrictions, dialytic clearance, or as a result of increased
degradation. For instance, vitamin C deficiency may be secondary to dietary restriction of
fresh fruits and vegetables to avoid hyperkalemia, but also to loss of the vitamin during
dialysis. Plasma vitamin E concentrations are typically normal, whereas erythrocyte and
mononuclear cell concentrations appear to be decreased [93].
Vitamin E appears to be important in the protection against oxidation of low-density
lipoproteins (LDLs) and biological membranes. It has been reported that both oral and
parenteral administration of vitamin E improves renal anemia and erythropoietin
requirements in dialysis patients [94]. More recent reports indicate that vitamin E given
orally also attenuates oxidative stress induced by intravenous iron administration and
significantly decreases the oxidative susceptibility of LDL [95, 96]. However, in a

randomized double-blind placebo-controlled trial, intravenous supplementation with vitamin
E in elective cardiac surgery failed to show a decrease in biochemical markers of oxidative
stress and in the rate of ARF [97].
It has been observed that postdialysis intravenous administration of vitamin C, compared
to ferric saccharate, significantly increases the hematocrit and diminishes erythropoietin
requirements after 8 weeks of therapy [96]. Vitamin C acts by promoting iron release from
storage sites, by elevating its delivery to hematopoietic tissues, and by increasing iron
utilization in erythroid progenitor cells.
While these studies showed beneficial effects of antioxidant supplementation strategies,
it should be mentioned that both vitamin E and vitamin C can be pro-oxidant under certain
adverse circumstances [99,100]
Supplementation with N-acetylcysteine (NAC), a glutathione precursor, is another
pharmacological antioxidant approach. N-acetylcysteine has been demonstrated to exert
beneficial effects in the prevention of oxidant mediated renal injury [101]. Accordingly,
Feldman et al. [102] conducted a prospective randomized controlled open label trial
investigating the role of N-acetylcysteine in the prevention of gentamicin-induced hearing
loss in the setting of end stage renal disease. While the exact mechanism of ototoxicity is
different from that of nephrotoxicity, it should be noted that their common pathogenesis
includes oxidative stress and ROS. This trial showed that administration of NAC at a dose of
600 mg twice a day was effective in decreasing the rate of ototoxicity at both 1 and 6 weeks.
Similarly, Mazzonet et al. demonstrated a protective effect of NAC on gentamicin-induced
nephrotoxicity in rats [103]. However, there are no studies demonstrating a nephroprotective
effect in humans.
Several animal models have attempted to use antioxidant strategies to attenuate cisplatin
nephrotoxicity. Ajith et al. [104] conducted a comparative study of the effects of different
doses of vitamins C and E on cisplatin-induced nephrotoxicity in mice. High doses of both
vitamins were effective in protecting against oxidative renal damage, as measured by
increased SOD activity and concentration of reduced glutathione (GSH), with vitamin C
outperforming vitamin E. Similarly, Lynch et al. demonstrated in a rodent model that
cisplatin nephrotoxicity could be attenuated by the use of allopurinol and ebselen, a seleno-
organic glutathione mimic and ROS scavenger drug [105]. In this model the combination of
allopurinol and ebselen outperformed the administration of each drug individually, leading to
a significant decrease in post-cisplatin serum creatinine and BUN elevations. Moreover,
amifostine, an FDA approved agent for the reduction of renal toxicity in patients receiving
cisplatin, decreases nephrotoxicity by donating protective thiol groups. Unfortunately,
amifostine use is limited by a variety of factors including its cost, side effect profile, and
concerns that its use could interfere with the antitumor effects of cisplatin [106].
Many prophylactic and therapeutic experimental animal studies have suggested the
possibility of attenuating gentamicin-induced renal failure through antioxidant based
therapeutic approaches. Zurovsky and Haber demonstrated that vitamin E and
dimethylthiourea, a potent scavenger of hydroxyl radicals, were effective in preserving renal
function and arresting progressive renal damage associated with gentamicin administration
[107].
Much of the evidence regarding rhabdomyolysis pathophysiology has been achieved in

animal models of myoglobinuric ARF. The most common in vivo model uses intramuscular
injection of hypertonic glycerol. In these conditions it has been reported an increased
generation of H2O2, lipid peroxidation and depletion of GSH stores [108]. Red wine
polyphenols lead to a decreased vulnerability of the rat kidney to ATN caused by
rhabdomyolysis-induced myoglobinuria [109]. Also, resveratrol, a stilbene polyphenol found
in grapes and red wine, has been found to reduce the mortality due to renal ischemia-
reperfusion injury in rats, as well as to improve renal function and decrease histological tissue
damage. This effect appears to be mediated by an increased production of NO in resveratrol
treated rats [110]. In addition, it has been demonstrated that the rat kidney responds to
glycerol-induced rhabdomyolysis with an induction of heme oxygenase as well as the
synthesis of ferritin [111]. This constitutes a protective antioxidant response and suggests a
therapeutic strategy for populations at a high risk for rhabdomyolysis. Unfortunately,
evidence regarding a potential protective effect of antioxidant-based therapeutic approaches
in rhabdomyolysis arises mainly from animal models and has not led yet to clinical studies.
Indeed, the relevance of ROS in rhabdomyolysis has to be further investigated.
Recently, it has been reported a potentially protective effect of aminoguanidine, an
inducible nitric oxide synthase inhibitor with antioxidant properties, in ischemia-reperfusion
renal injury. Aminoguanidine has shown to reduce serum urea, creatinine levels and improve
histopathological lesions when administrated after an ischemia-reperfusion injury to the rat
kidney, but not before [112].
4. Conclusion and Perspectives

Acute renal failure constitutes a major cause of morbidity and mortality, especially in
hospitalized and elderly patients. Oxidative stress is a metabolic derangement that plays a key
role in the development and progression of renal damage. The pathophysiology of ARF is a
wide and highly complex subject, especially due to the multiple mechanisms that can be
involved in its development. Reactive oxygen species appear to play a key role as mediators
in intracellular signaling, leading to deleterious effects such as apoptosis and necrosis.
Consistently, there is an increasing body of evidence supporting a potential role of
antioxidant-based therapy in the treatment and prevention of ARF. However, most of the
available data has been achieved in animal and in vitro models. There are only a few studies
in humans and data are not sufficient to account for the efficacy of antioxidants as preventive
and therapeutic tools.
In consequence, further investigation is required to fully establish the role of oxidative
stress in the pathophysiology of ARF and the potentially protective and therapeutic effects of
antioxidant-based therapies in this setting.
Figure 5-1. Pathophysiology of acute renal failure (ARF). Any of the three classical causes of ARF
leads to two common mechanisms, ischemic damage and nephrotoxic damage. These mechanisms can
activate molecular mediators or directly damage the tubule. Both of these conditions result in a rise in
reactive oxygen species (ROS) concentrations. Therefore, molecular mediators, ROS and direct tubular
injury lead to ARF development and progression.
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Chapter VI
Pre-Eclampsia
Mauro Parra
Fetal Medicine Unit, Obstetrics and Gynecology Department,
University of Chile Clinical Hospital.
Abstract
Pre-eclampsia (PE) is the most important complication of human pregnancy
worldwide and a major contributor to maternal and fetal morbidity and mortality. It is a
disease of two stages. The first stage concerns the relative failure of early trophoblast
invasion and remodeling of the spiral arteries, leading to a poor blood supply to the feto-
placental unit, exposing it to oxidative stress. The second stage is characterized by
maternal endothelial dysfunction, leading to the clinically recognized symptoms of the
syndrome, which include hypertension, proteinuria, thrombocytopenia and impaired liver
function. Furthermore, the modification of spiral arteries occurs during the first and early
second trimester of pregnancy, leading to uteroplacental hypoperfusion and fetal
hypoxia. Despite much work in the last decade, the causes that trigger PE are uncertain
and the predictive value of potential risk factors is poor. Increasing evidence suggests
that placental and systemic oxidative stress plays a crucial role in its development.
Indeed, oxidative stress and disrupting angiogenesis is considered the link bridging the
two stages of the disease. Markers of oxidative stress in women with established PE have
shown both increased lipid peroxidation in placental tissue, along with increased in
maternal plasma biomarkers indicating decreased antioxidant capacity and increased lipid
peroxidation. These findings have contributed to the interest in using antioxidants to
prevent the development of PE. The lack of appropriate early predictors of the disease
has determined that the risk groups for primary prevention of PE should be characterized
on the basis of the clinical history of the patients and from knowing that is possible to
establish some risk factors. A large number of publications suggest a potential role of
antioxidant nutrients in the prevention of PE in women at high increased risk of the
disease. Vitamins C and E have been the main antioxidants agents used for this purpose.
Despite the biological properties of these compounds, exerting ROS scavenging and a
136 Mauro Parra
down-regulation of ROS, the results of clinical trials do not support benefits for routine
supplementation with vitamins C and E during pregnancy to reduce the risk of PE.
This chapter examines the role of oxidative stress in the pathophysiology of PE and
reviews the available data on the use of antioxidant compounds, mainly vitamins C and
E, to prevent the development of this disease.
1. Introduction
1.1. Definition and Classification
For the past hundred years, pre-eclampsia (PE) has been considered a placental
pathology and its clinical management has remained practically unchanged since then [1] PE
is a leading cause of maternal mortality in developed countries [2], and is associated with an
increased rate of perinatal morbidity due to iatrogenic deliveries.
Pre-eclampsia classically defined as a clinical syndrome of still unknown cause that
develops in a previously normotensive woman after the second half of pregnancy, and it is
characterized by an increased blood pressure (140/90 mmHg) and proteinuria greater than
300 mg in a 24 hour urine collection. Both blood pressure and proteinuria are resolved after
delivery of the fetus [3].
The incidence of PE is about 5% of all pregnancies, and in about 20% of cases early
onset PE leads to delivery before 34 weeks [4]. The pathology is more common in conditions
as follows: primagravid women, maternal age above 40 years and multiparous women with
change of partner, increased body mass index and obesity, previous history of pre-eclampsia,
antiphospholipid antibodies, pre-existing diabetes, and multiple pregnancies [5-10].
Hypertension in pregnancy can be classified in four categories : a) pre-existing
hypertension (3-5% of pregnancies), characterized by being present before pregnancy or
diagnosed before 20 weeks of gestation; b) pregnancy-associated hypertension (12% of
pregnancies) as appearance of high blood pressure after 20th weeks of gestation, which in turn
can be sub-classified according to the presence of proteinuria in pre-eclampsia (5-6%) and
gestational hypertension (6-7%); c) superimposed pre-eclampsia (25% of women with pre-
existing hypertension); and d) eclampsia when convulsion is present in a pregnant women
with, or who later develop, hypertension [11].
1.2. Clinical Assessment of Pre-Eclampsia
Normal pregnancy is characterized by a fall in blood pressure due to peripheral

vasodilatation during the second trimester, and increased cardiac output and blood volume by
about 50%. By contrast, severe PE has usually been associated with a low cardiac output and
a high peripheral resistance, although some authors have reported contradictories results [12,
13].
Increased proteinuria observed in PE is characterized by loss of serum proteins and
increase in capillary endothelial permeability. The decrease in blood volume leads to an
increase in tissue edema.
Pre-Eclampsia 137
This change in blood volume is expressed through an increase in maternal hemoglobin

concentration and increase risk of fetal growth restriction [14].Platelet count is reduced in
normal pregnancy (<200x109/L) due to the physiological maternal blood-volume expansion,
however, in severe PE cases there is a further progressive fall in platelet count as a result of
both increased consumption and intravascular destruction [14].
In normal pregnancy, there is a raise in creatinine clearance with a concomitant decrease
in serum creatinine and urea concentrations. In severe PE, serum creatinine tends to increase
and it can be related with poorer outcome [15] The uric acid levels drop in normal pregnancy
as an expression of increased renal excretion. In PE, on the other hand, there can be an
increase in uric acid levels, correlating with poorer maternal and perinatal outcome. It is
thought that this uric acid rise is due to either a decrease in renal function or the occurrence
oxidative stress [16].
Finally, the liver may be damaged in severe cases of PE, associated with upper epigastric
pain, distension and even rupture of the capsule into the peritoneal cavity. Altered liver
enzyme, alanine and aspartate aminotransferase activities can also be seen as part of a
characteristic severe condition called HELLP syndrome (hemolysis, elevated liver enzyme
and low platelet) [16] With regards to the central nervous system, it can be also affected in
PE, especially in severe conditions, such as stroke and eclampsia. Fortunately, in the last ten
years these complications have been reduced due to improvement in the management of these
cases introducing early delivery, antihypertensive drugs and magnesium sulphate [17].
2. Pathophysiology of Pre-Eclampsia
The syndrome of PE involves several organ systems including placenta; kidney, liver,
brain, the vasculature, the hematopoietic and coagulation system. Measurement of specific
markers for each of these systems may not only indicate organ involvement before
manifestation of the full maternal syndrome, but indicate that there are several different
causes and presentations leading to PE.
With certainty, the etiology of PE is still unknown. However, PE is characterized by
certain pathophysiological, hematological and biochemical changes which some of them may
be consequences or causes of this syndrome.
The proposed sequence of events comprises endothelial dysfunction, defective
trophoblast invasion, and consequential impaired placental perfusion, immune maladaptation
and inflammation. The common link between these could be enhanced oxidative stress by
excessive production of reactive oxygen species coupled with inadequate or overwhelmed
antioxidant defense mechanisms. Pre-eclampsia is nowadays considered to be a syndrome
derived from multiple mechanisms that contribute to the pathophysiology of this complex
obstetric condition. There are several hypotheses, although abnormal placentation and
endothelial dysfunction are almost always part of the explanations. The placental disease is
represented by an abnormal extravillous trophoblast invasion of the spiral arteries, leading to
placental hypoperfusion and/or ischemia/reperfusion cycles, which produce an oxidative
stress state and consequently an increase in the reactive oxygen species (ROS) at the
intervillous space. The placental oxidative stress through cytokine and chemokine adhesion
138 Mauro Parra
molecules recruits and activates leukocytes at the intervillous space causing damage to the
endothelial cells. ROS also increase lipid peroxidation and peroxynitration, leading to a
reduction in nitric oxide and prostacyclin bioavailability and DNA oxidation. PE is also
associated with hyperhomocysteinemia, which may increase ROS by reducing nitric oxide
production through increasing asymmetric dimethyl-arginine (ADMA) which competes
directly with L-arginine for eNOS. Together, as outlined in figure 6-1, these changes lead to
the development of all the features of pre-eclampsia such as hypertension, edema, proteinuria
and hypercoagulability.
The linkage between placental hypoxia and maternal vascular dysfunction has been
proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta
or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta
that bind VEGF and PLGF in the maternal circulation. In PE, there is abundant evidence of
altered reactivity of the maternal and placental vasculature and an altered production of
autacoids [18].
Figure 6-1. An schematic diagram illustrating the proposed role of oxidative stress and the main
contributory factors involving endothelial dysfunction, featuring clinical aspects of pre-eclampsia.
ROS, reactive oxygen species; LDL, low density lipoproteins; Hcy, homocysteinemia; OX-LDL,
oxidized LDL, NF-кB, nuclear factor kappa B; iNOS, inducible nitric oxide synthase; ADMA,
asymmetric dimethyl-arginine; eNOS, endothelial nitric oxide synthase; VEGF, vascular endothelial
growth factor; VWF, von Willebrand factor; TM, thrombomodulin; PGI2, prostacyclin; TXA2,
thromoboxane A2, BH4, tetrahydrobiopterin.
Pre-Eclampsia 139
2.1. Placental Insufficiency
Pre-eclampsia occurs only in the presence of the placenta and its resolution begins with
the removal of the placenta. More than 70 years ago, E. W. Page suggested that the feature
that characterized the preeclamptic placenta was its exposure to decreased perfusion [19].
It is accepted today that during early pregnancy, normal placentation occurs in a
relatively hypoxic environment (<2% oxygen) that is essential for acceptable development,
which in turn prevents trophoblast differentiation to an invasive phenotype [20-22]
Intervillous blood flow increases at around 10-12 weeks of gestation and results in exposure
of the trophoblast to increased oxygen tension (pO2) and increases in the activity and levels
of the major antioxidant enzymes in the villous tissues, superoxide dismutase, catalase, and
glutathione peroxidase [23].
Hypoxia inducible factor-1 (HIF-1), and specifically its 1α subunit expression, and
transforming growth factor β3 (TGFβ3), an inhibitor of early trophoblast differentiation,
mediates the effects of low oxygen tension during the first trimester of pregnancy. The
expression of both molecules is high in early pregnancy and falls at around 10-12 weeks of
gestation when placental pO2 levels are believed to increase due to unplug uterine spiral
arteries [20] In addition, there is also evidence that leptin, TGFβ1 and PAI-2 are involved in
the process of physiological trophoblast invasion [24- 26].
In normal pregnancy, progressive endovascular trophoblast invasion occurs progressively
from the decidua into the inner third of the myometrium between 12 to 20 weeks of gestation
[27- 29]. These changes are characterized by spiral artery remodeling including the
disintegration of the tunica media along with the internal elastic lamina, and replacement of
the endothelium with extravillous trophoblast cells expressing an endothelial phenotype. The
changes involve conversion of the narrow muscular arterial tubes into flaccid and wider tubes
that facilitate unimpeded placental perfusion with maternal blood flow required for adequate
exchange of key molecules between the maternal and fetal circulations. As a consequence of
previous physiological changes, the spiral arteries remodeling facilitates the ten-fold increase
in uteroplacental blood flow that occurs between conception and term.
Histological studies have shown that the process of spiral artery vascular remodeling is
partial in pregnancies affected by PE or fetal growth restriction (FGR) [30]. Extravillous
trophoblast invasion and spiral artery remodeling occur either very superficially or not at all
in PE. This dysfunctional process of invasion and replacement of endothelial function results
in both high resistance to flow in the maternal uterine arteries and relative placental
hypoperfusion at a time period in which PE first symptoms may appear.
One of the molecular explanations for an abnormal placentation was made by Cannigia et
al. [31] who found that TGFβ3 expression was increased in human PE placentae when
compared to age-matched controls and that inhibition of TGFβ3 by antibodies restored the
invasive ability to the trophoblast cells in PE explants. The authors speculate that if oxygen
tension fails to increase, or trophoblast does not detect this increase, expression of the two
factors remains high, resulting in shallow trophoblast invasion and predisposing the
pregnancy to PE.
However, it is well known that abnormal placentation does not mean necessary PE and it
is therefore necessary to find a link between the placental and maternal disease. Firstly,
140 Mauro Parra
Roberts et al. [32] proposed a hypothesis about the pathophysiology of PE called the “two
stage model of pre-eclampsia” which intend to resolve this dilemma. The first stage of this
model, whose histological and molecular changes have been described above, is the reduction
of maternal blood flow to the intervillous space. The second step of this model is the
transference of the reduction of placental perfusion to systemic maternal pathophysiology.
This abnormal perfusion of the intervillous space may lead to the production of different
molecules which finally affect endothelial function and reduce organ perfusion. The search
for this factor has led to the identification of numerous substances as candidates to be the
“Factor X”. More recently, there is also an integrated model proposed by Redman et al,
which assumes that the placenta plays a central role in the pathogenesis of PE, but that
abnormal placentation is unlikely to be the exclusive cause of this syndrome [33]. The
authors explain that a normal pregnancy is associated with an apoptotic physiological export
of syncytiotrophoblast microparticles into the maternal circulation, leading to a systemic
inflammatory response [34]. The hypothesis suggests that PE may be explained by either the
presence of a larger or oxidatively stressed placenta [35]. A larger placenta is also seen in
gestational diabetes, twin pregnancy, term or molar pregnancies. An oxidatively stressed
placenta exacerbates the already established inflammatory state in normal pregnancies,
leading therefore to PE.
2.2. Oxidative Stress in Pre-Eclampsia
Although the cause of PE still remains unknown, it has been proposed that enhanced
oxidative stress is a basic component of this condition that could provide the connection
between abnormal placentation and the maternal syndrome [36, 37].
The main source of reactive oxygen species (ROS) initiating the pathophysiological
events appears to be the placenta [38] but maternal leukocytes and maternal endothelium are
also likely contributors. The failure of placental perfusion, explained previously, is likely to
result in hypoxia-reoxygenation cycles which lead to oxidative stress due to changes in the
vascular vasomotor activity by maternal humoral and neural influences [39]. It has been
recently suggested that the cause of PE is the abnormal oxidative insult associated with pre-
eclampsia and not the mRNA expression of antioxidant proteins that may be responsible for
reduced antioxidant enzyme activity in preeclamptic placenta [40].
Other mechanisms of ROS production in PE pregnancies should be the activation of
maternal neutrophils by syncytotrophoblast microvesicles following deportation due to
increased apoptotic or aponecrotic mechanisms locally activated during the passage of
maternal blood through the placenta [41]. Thus, isolated neutrophils from women with PE
synthesize more superoxide than those of normotensive pregnant women [42]. Consecutively,
activated neutrophils may contribute to the activation of the vascular endothelium,
contributing therefore to the pathophysiology of PE [43]. It was found that oxidative stress
early in pregnancy influenced pregnancy outcome, as assessed by the finding that urinary F2-
isoprostanes, biomarkers of lipid peroxidation, are associated with an increased risk of pre-
eclampsia and a decreased proportion of female births [44]. Recent studies have suggested
Pre-Eclampsia 141
that this imbalance between oxidant and antioxidant is the effect of disease and not the
causative factor [45].
2.2.1. Placental Ischemia-Reperfusion and Reactive Oxygen Species

Despite the possibility that the generation of the placental oxidative stress may be related
to hypoxia due to reduced uteroplacental perfusion, hypoxia itself is not sufficient to account
for all the morphological findings [46]. In fact, an ischemia-reperfusion cycle at the
uteroplacental blood flow may be a more important factor for establishing placental oxidative
stress [47]. As a consequence, an increased capacity of placental, as well as of other cells, to
generate ROS has been found in PE [38, 48]. Oxidative stress in this context is a consequence
of an imbalance between excessive generation of ROS and reduced capacity of the
antioxidant defenses, causing placental damage, which in turn could account for the increased
rates of infarction and syncytial necrosis observed in this condition.
The targets and extent of nitration of enzymes, receptors, transporters and structural
proteins may markedly influence placental cellular function in both physiologic and
pathologic conditions [49].
2.2.2. Pro-Oxidant Enzymes

The most common ROS is the superoxide anion. Xanthine and NADPH oxidases have
been identified as major vascular superoxide-forming enzyme systems, but the contribution
of xanthine oxidase (XO) is generally minor [50]. However, XO is abundantly expressed in
cytotrophoblast and syncytiotrophoblast cells due to the fact that ischemia-reperfusion is a
potent stimulus to the conversion from xanthine dehydrogenase [48, 51]. In addition,
NADPH oxidase activity, constitutively observed in the trophoblast of the human placenta, is
highly stimulated in PE [52]. This enzyme consists mainly of 5 subunits and it has been
observed that placentas from preeclamptic patients show increased expression of NADPH
oxidase components p22, p47, and p67 [53].
Other forms of NADPH oxidase are also implicated in the pathophysiology of PE, such
as those present in phagocytes (neutrophilic and eosinophilic granulocytes, monocytes, and
macrophages) and vascular cells. NADPH oxidase mediates increased isolated neutrophils
production of superoxide observed in women with PE [54]. It been well established that
vascular NADPH oxidase plays a major role in the development of hypertension [55] and is a
target for a down-regulation exerted by antioxidant vitamins.
2.2.3. Biomarkers of Oxidative Stress

As we have reviewed, PE is associated with an exaggerated production of superoxide
anion via pro-oxidant enzyme generated due to ischemia-reoxygenation cycle in placenta and
activated leukocytes and endothelial cells. Oxidative stress state can produce lipid
peroxidation, protein carbonylation and DNA alteration in different feto-maternal tissues.
The main biomarkers of oxidative stress are summarized below.
Peroxynitrite: the increased generation of superoxide anion by the placenta, activated
leukocytes, or endothelial cells is accompanied by an up-regulation of inducible nitric oxide
synthase (iNOS), an isoform mainly located in macrophages and neutrophils, leading to
increased formation of peroxynitrite. In fact, the rate constant for the formation of
142 Mauro Parra
peroxynitrite is 3 times faster than that of the interaction of superoxide with superoxide
dismutase (SOD) [56].
Malondialdehyde (MDA): peroxynitrite interacts with lipids leading to peroxidation and
MDA and conjugated diene formation [57]. In PE pregnancies, MDA levels are reported to
be elevated in maternal plasma and placental tissue [58, 59] as well as in erythrocytes, and
the severity of the disease correlates with the MDA concentration in both the serum [38] and
in erythrocytes [60]. Our own data also showed that there was significantly increased MDA
placental production from PE patients compared to normal control group [61]. Additionally,
there was a direct correlation between the lipid peroxidation level and the PE severity [62].
Therefore, PE can be associated with increased lipid peroxidation. It is also remarkable that
MDA products may behave as toxic bifunctional electrophiles, due to reactivity with
proteins, phospholipids, and DNA, generating stable products at the end of a series of
reactions to form propane adducts [63].
F2-isoprostane: this molecule is a product of lipid peroxidation which is formed by a
nonenzymatic peroxidation of arachidonic acid. This compound serves as an index of lipid
peroxidation in several diseases, including PE [64, 65]. The production and secretion rates of
F2-isoprostanes by placentas obtained from women with PE were significantly higher than
those of controls [66], providing convincing evidence that oxidative stress and lipid
peroxidation are abnormally increased in preeclamptic placenta.
Carbonyls: the direct damage of proteins during oxidative stress can give rise to the
formation of protein carbonyls, which may serve as biomarkers for general oxidative stress.
Higher levels of protein carbonyls in both the placenta and decidua were found in women
with mild to severe PE either alone [67] or with concurrent HELLP syndrome [68]. In
addition, our data showed that PE women were characterized by alteration of all oxidative
stress parameters, i.e. there was a significant reduction in total antioxidant capacity of plasma
(FRAP, ferric reducing ability of plasma), increased uric acid, F2- isoprostane, and carbonyl
plasma levels [61].
2.2.4. Antioxidant Defense System in Pre-Eclampsia

Antioxidant enzymes: physiologically by 10 to 12 weeks of gestation, the onset of blood
flow throughout the modified spiral arteries results in an increased oxygen tension and
simultaneous elevation in the expression and activity of the antioxidant enzymes [21]. On the
contrary, the increased concentration of superoxide in the PE placental tissue [69] was found
to be associated with decreased SOD activity and mRNA expression for CuZn-SOD in
trophoblast cells isolated from PE placentas [70]. Also, it was found that these women show a
decrease in plasma levels of SOD [59]. Another study reports reduced catalase and SOD
activities, with a concomitant elevation of lipid peroxidation products [71]. With regards to
total glutathione peroxidase in placenta from PE women, there are contradictory data, since
one study has found increased levels [71] and other reported a decreased activity in placenta
from PE women [72]. It was found that SOD and catalase activity in PE placental tissues
lower than in the control group [61].
Nonenzymatic antioxidants: natural or endogenous antioxidants measured in maternal
blood, placenta, and deciduas are reduced in PE women [67, 73-76] however, they do no
differ between mild or severe conditions [77]. A large number of studies agree that PE is
Pre-Eclampsia 143
associated with a decrease in serum levels of ascorbic acid (vitamin C) and α-tocopherol
(vitamin E) [78, 79] as well as a decrease of other lipid soluble antioxidants such as
coenzyme Q10 [80], carotenoids, and retinol [81]. Other studies have reported that there is no
significant difference in plasma vitamin E concentration, when PE pregnancies are compared
to controls [82].
2.2.5. Oxidative Stress Biomarkers as Predictors of Pre-Eclampsia

In general terms screening tests are available to let a pregnant woman know about
prevention for PE at any one of three different stages: primary, secondary, or tertiary. The
concept of primary prevention is conceived to avoid the occurrence of a disease. Secondary
prevention, in the context of PE, implies inhibiting the disease process before appearance of
clinically recognizable disease. Finally, tertiary prevention, or treatment, means prevention of
complications caused by the disease process.
For the purpose of this chapter, we will only review data available on screening test for
primary and secondary prevention.
Primary prevention of PE should be determined by the clinical history of the patients and
from knowing that it is possible to establish some risk factors. The most significant factors to
be considered in the patient medical files are: the history of PE, antiphospholipid antibodies,
pre-existing diabetes and body mass index above 35 [8-10]. However, all these variables are
only able to predict just 30% of appearance of PE [83].
Several demographic factors have been associated with the risk of developing PE during
pregnancy. Those mentioned above are the most relevant to be used as screening for this
condition.
Secondary prevention of a disease is only possible if the following three requirements are
met: knowledge of pathophysiological mechanisms, availability of methods of early
detection, and resources for intervention and correction of the pathophysiological changes.
Although the pathogenesis of PE is poorly understood, the high resistance placental
circulation shown on uterine artery Doppler study during the second trimester is often used as
a predictor of PE. It has been reported recently that the very high likelihood ratio of PE in the
abnormal uterine artery Doppler screen-positive group confirms the reliability of this second-
trimester screening test in diagnosing impaired placentation [61] and the findings are
compatible with those of other previous screening studies during the first [84] and second
trimester [85-87].
On the other hand, PE pregnancies have been associated with increased PAI-1/PAI-2
ratio, sFlt-1, F2-isoprostane and reduced free PlGF at 23 weeks of gestation, suggesting that
impaired placentation, anti-angiogenic mediators, endothelial dysfunction, and oxidative
stress are the factors that predate the development of PE by several weeks. The findings that
PE women show increased plasma concentration of 8-epi-prostaglandin F2α supports the
hypothesis that poor uteroplacental perfusion predisposes to an increase in placental free
radical synthesis and, thereby, to maternal oxidative stress. Our results are in agreement with
those of Chappell et al who showed that this marker of lipid peroxidation was increased in
women who later developed PE [88].
Other biochemical markers that have been significantly associated with the appearance of
PE are endothelial dysfunction and anti-vasculogenesis biochemical markers. It has been
144 Mauro Parra
found that PAI-1/PAI-2 ratio is significantly increased in women who later develop PE. This
is in agreement with other authors [88] and with the hypothesis that endothelial dysfunction
is the main feature of this condition and predates its onset [1, 89]. It has been observed
increased circulating anti-angiogenic factor (sFlt1) and reduced free PlGF in women who
subsequently developed PE, results in agreement with previous publications [90, 91]. As our
data also show, Levine et al. [91] have demonstrated that alterations of the sFlt1 appeared to
be greater in women who had early-onset PE and in women with PE who delivered a small
for gestational age infant, suggesting that defective angiogenesis may be especially important
in these cases. However, these biochemical markers appear to be a consequence rather than a
cause of this disease, because they do not improve second trimester uterine artery Doppler
detection rate
Although second trimester screening test, either combined markers or uterine artery
Doppler alone, has demonstrated to reach a good detection rate for early-onset PE, all
prevention trial performed till today had failed to demonstrate any benefit in this high-risk
group. Consequently, recent publications have shown that a combined test between clinical,
biochemical and uterine artery Doppler during the first trimester of pregnancy can predict
about 80% of early-onset PE (<34 weeks of gestation) with only 10% false positive rate [83].
These publications are in agreement with our own data, showing that around 70% of
early-onset PE can be predicted correctly at 12 weeks using a combined model of uterine
artery Doppler, placental growth factor and body mass index.
Oxidative stress markers, such as F2- isoprostane, MDA, FRAP and uric acid plasma
levels were not significantly different to the control group [61]. In addition, the urinary
excretion of F2-isoprostanes was associated with an increased risk of PE [ 44 ].
However, a recent gene expression study performed in chorionic villous samples
obtained at 11 weeks of pregnancy from 5 women who later develop PE, have demonstrated
that mRNA expressions of SOD and other markers involved in the development of normal
placentation were significantly altered compared to matched-control group [92].
Other biochemical markers testing during the first trimester of pregnancy with high rate
of detection rate for early-onset PE are placental protein 13 [93] and pregnancy-associated
plasma protein [94]. Plasma levels of these molecules are lower than controls at 11-14 weeks
of gestation. Both markers might play a role in the process of extravillous trophoblast
invasion and spiral artery remodeling [95, 96].
3. Prevention with Antioxidants

To date, many strategies aimed at secondary prevention of PE have been studied,
although none of them has proved to prevent the clinical appearance of PE [11, 97, 98 ].
It is accepted that free radicals are promoting maternal vascular dysfunction and PE in
women are associated with an oxidative stress state, which is characterized by increased
markers of lipid peroxidation, such as MDA [ 64 ] and F2-isoprostane [99] and reduced
plasma and placental levels of antioxidants [100, 101]. Therefore, the possibility of
implementing an antioxidant therapy with vitamins C and E, based on the multiple biological
properties of these agents in addition to prevention of lipid peroxidation, appears to be quite
Pre-Eclampsia 145
well supported [102]. These defense mechanisms, involving antioxidant vitamins and enzyme
systems, may restrain the extent of damage caused by oxidative stress [103]. On this line,
vitamins C and E down-regulates NADPH oxidase, which is the major source of superoxide
anion, at the vascular wall level. Regulation on this enzyme, as reviewed previously, could
play a key role as mediator in the development of systemic pathological processes such as
impairment of endothelium-dependent vasodilatation [104], inflammation and increased
platelet aggregation. In addition, there are also data in animal models, such as pig coronary
artery and aorta from spontaneously hypertensive rats, demonstrating that vitamins C and E
may cause down-regulation of NADPH oxidase and up-regulation of eNOS [105].
Although the exact mechanisms whereby antioxidant vitamins act on those enzymes are
unclear, five possible mechanisms have been suggested: a) regulating protein expression of
the NADPH oxidase at the transcriptional or post-translational levels [106]; b) inhibiting or
interrupting the complex formation of the NADPH oxidase subunit at cell membrane [107];
c) preventing p47phox NADPH oxidase subunit membrane translocation and phosphorylation
[108]; d) stimulating eNOS activity at endothelial cells by increasing the intracellular
availability of the eNOS cofactor tetrahydrobiopterin (BH4) that would further increase NO
synthesis [109, 110]; e) inhibiting the up-regulation of ICAM-1 [111] and increased
production of IL-6 [112] which might be mediated by NF-κB activation in PE.
It has been suggested that the deleterious effect of ROS may be counteracted by an
antioxidant therapy, and that more studies are necessary to determine the optimum dosing and
timing of antioxidant administration, since an inappropriate antioxidant treatment in pregnant
women may have deleterious consequences, reducing placental cells proliferation until to cell
death [113]. Although vitamins C and E inhibit apoptosis of cultured human term placenta
trophoblast [114], it was reported that exposure of these cells to high levels of antioxidant
vitamins C and E may affect placental function, in terms of decreasing secretion of hCG,
placental immunity and increasing production of TNF-α. Such alterations are known to lead
to endothelial dysfunction and adverse pregnancy outcomes, such as fetal growth restriction
[115].
Chappell et al. [87] have carried out a randomized clinical trial using supplementation
with vitamins C and E in a high risk group of pregnant women during the second half of
pregnancy. They have shown that antioxidant vitamins had beneficial effects on biochemical
markers of the disease, and may be beneficial in the prevention of clinical PE, although the
latter was not statistically significant. Recently, there have been published four randomized
trials regarding the effect of antioxidant vitamins in preventing PE in high and low risk
women [115-118]. Beazley et al. [115] planned to randomize 220 high-risk women, but they
stop this study due to lack of funding. Finally, they reported their result base on a group of
only 100 women who were given antioxidant vitamins from 14 weeks onwards. They did not
find any significant difference in the rate of PE between both groups. Poston et al. [117]
randomized 2410 high-risk women who were recruited between 14 to 22 weeks of gestation
according clinical history and current pregnancy. They found that there was no reduction in
the incidence of PE or fetal growth restriction in women who received vitamin E and C. They
also raised a concern about using antioxidant vitamins in pregnant women because it was
associated with increased severity of hypertension. The next study was performed in 1877
nulliparous women who were recruited between 14 and 22 weeks of gestation. They found
146 Mauro Parra
that there were no significant differences between the vitamin and placebo groups in the risk
of PE and other serious maternal or newborn outcomes [118]. Furthermore, a clinical trial
conducting in 707 high-risk Brazilian women recruited between 12 to 20 weeks of gestation
were in agreement with the three previous publications that incidence of PE was not modified
with antioxidant vitamins.
Finally, a recent meta-analysis involving 6533 pregnant women corroborates the
previous data that antioxidant vitamins did not reduce the relative risk of PE and other
adverse outcomes [119]. However, antioxidant vitamins did significantly increase
antihypertensive therapy (77%) and antenatal hospital admission for hypertension (54%).
They conclude that evidence does not support routine antioxidant supplementation during
pregnancy to reduce the risk of pre-eclampsia and called attention on possible serious
complication in pregnancy.
At present, the results of these studies have been mainly disappointing and controversial,
but they should be interpreted with caution. In fact, most studies were done in high-risk
population with extremely diverse pathophysiological backgrounds and also supplementation
was started mostly after 14 weeks of gestation, a time at which physiological placentation has
been already established.
As it has been explained in other section of this reviewed, it could be hypothesized that
the derangement of placentation is a consequence of earlier events during gestation, based on
the normal trophoblast invasion occurring in a way temporally and spatially unique that
involves both degradative and adhesive interactions [1]. Many of these adaptive changes
appear to be disrupted in PE due to decreased degradative ability secondary to lower levels or
cytokine inactivation of MMP-9, [120, 121] or improper expression of adhesion molecules
[29], all processes triggered by oxidative stress. Recently, placental oxidative stress has been
attributed to increased expression of NADPH oxidase [122] and ET-1 [123] in the placenta, a
process occurring very early during placental development, likely accounting for the
impaired trophoblast migration and its consequences (figure 6-2). This view could explain the
failure of the clinical trials to prevent the development of PE through later antioxidant
therapy. Therefore, it is necessary to improve our capacity to develop earlier predictive test
based on uterine artery Doppler in combination with clinical history and biochemical
markers, to detect a high-risk group destine to develop early-onset or severe PE [124].
Endothelial cell apoptosis of the spiral arteries is postulated as the mechanism by which
trophoblast influences vessel remodeling. There are several mechanisms which could activate
apoptosis in vascular cells, one of them being the interaction between Fas/FasL. Furthermore,
it has recently been shown that first trimester trophoblast cells can secrete FasL [125, 126].
Candidates that should be studied for their involvement in the regulation of endothelial
apoptosis by the trophoblast cells include intervillous space levels of oxygen during the first
trimester (hypoperfusion/reperfusion state associated with oxidative stress state), activation
of HIF-1, and the role of L-arginine/nitric oxide synthesis in trophoblast invasion.
Pre-Eclampsia 147
Figure 6-2. Hypothesis to explain the contribution of oxidative stress in the pathogenesis of the
syndrome of pre-eclampsia. The counteracting effect of antioxidant vitamins C and E in two crucial
stages of the process of placentation is indicated by the symbol ( ). ROS, reactive oxygen species;
MMP-9, matrix metaloproteinase-9; VEGF, vascular endothelial growth factor. (Adapted from Rodrigo
et al. Fundam Clin Pharmacol 2007; 21:111-127).
It is also accepted that cytotrophoblast undertakes a program of pseudo-vasculogenesis

during normal pregnancy and it has been postulated that cell-surface endoglin (Eng) may play
a role in the regulation of this process. Cell-surface Eng inhibits trophoblast invasion and
therefore it is speculated that increased soluble endoglin (sEng) observed in PE may be a
compensatory response by the placenta to limit the effects of cell-surface Eng [127]. Thus,
factors associated with vasculogenesis, such as sFlt, PlGF and sEng, have been suggested to
148 Mauro Parra
be causally involved in the manifestation of PE. First of all, the authors showed that sEng and
sFlt-1 can induce endothelial dysfunction in vitro, through inhibiting VEGF and TGF-β
stimulation of endothelial-dependent NO activation. Secondly, treatment of pregnant rats
with sFlt-1 and sEng induced signs of severe PE, including development of HELLP and fetal
growth restriction. Interestingly, they showed that s-Flt-1, through decreasing activity of
eNOS, is associated with increased vascular permeability in the maternal kidneys (increased
proteinuria) and vasoconstriction, while, sEng, through inhibiting the antithrombotic factor
prostacyclin, is associated with the procoagulant state and thrombocytopenia observed in this
condition.
Recent data suggest that in a pathophysiological condition, such as PE, the deleterious
effect of ROS may be counteracted by an antioxidant therapy, and that there is an urgent need
to investigate the optimum dosing and timing of antioxidants administration, since an
inappropriate antioxidant treatment in pregnant women may have deleterious consequences
by reducing placental cells proliferation to cell death.

From the data reviewed we conclude that PE in women is characterized by oxidative
stress, abnormal vasculogenesis and endothelial dysfunction. Furthermore, uterine artery
Doppler characteristics proved to be the best predictor of PE during the second trimester, and
were associated with markers of abnormal vasculogenesis.
The findings observed in this chapter add support strength to the evidence implicating
placental insufficiency and oxidative stress as critical events in the pathogenesis of PE, and
add knowledge about the best way to predict these conditions using a combination between
clinical, biochemical and uterine artery Doppler assessment during the first and second
trimester of pregnancy. However, there is still lacking of data to promote the use of
antioxidant vitamins as early prevention of this condition.
This chapter strengthens the hypothesis that severe cases of PE, with or without fetal
growth restriction, are explained by abnormal mechanisms of early placentation and
vasculogenesis which involve oxidative stress and endothelial dysfunction as constitutive
parts of the PE syndrome. Further investigation of the factors associated with the intriguing
process of extravillous trophoblast invasion to spiral arteries during the first part of
pregnancy is therefore needed. The alteration of this physiological process occurs during the
first 20 weeks of pregnancy and is characterized by a transient coexistence between
trophoblast, endothelial and vascular smooth muscle cells in the invaded spiral
arteriesAlthough it is highly likely that the regulation of this fundamental physiological
process is more complex than it is hypothesized, studies on the regulation of the extravillous
trophoblast induction of endothelial apoptosis and abnormal vasculogenesis would be
important for determining the failure of these processes to occur in PE.
On the other hand, and following our hypothesis, it would be also interesting to study the
interaction between the described poor placentation and its expression at the maternal level,
such as endothelial dysfunction and haematological alterations.
Pre-Eclampsia 149
In summary, further studies on the biochemical alteration of the abnormal placentation

observed in PE and its correlation with the systemic expression of pregnancy-induced
hypertension are necessary for a better understanding of the pathophysiology of the PE
syndrome, and associated conditions. It is also important to establish rational methods of PE
screening, prevention and treatment.
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Chapter VII
Metabolic Syndrome
Rodrigo Castillo
University of Chile
Abstract
The biochemical steps linking insulin resistance with the metabolic syndrome have
not been completely clarified. Mounted experimental and clinical evidence indicates that
oxidative stress is an attractive candidate for a central pathogenic role since it potentially
explains the appearance of all risk factors and supports the clinical manifestations.
Indeed, metabolic syndrome patients exhibit activation of biochemical pathways leading
to increased delivery of ROS, decreased antioxidant protection and increased lipid
peroxidation. The described associations between increased abdominal fat storage, liver
steatosis and systemic oxidative stress, the diminished concentration of nitric oxide
derivatives and antioxidant vitamins, and the endothelial oxidative damages observed in
subjects with the metabolic syndrome support oxidative stress as the common second-
level event in an unifying pathogenic view. Moreover, it has been observed that oxidative
stress regulates the expression of genes governing lipid and glucose metabolism through
activation or inhibition of intracellular sensors. Diet constituents can modulate redox
reactions and the oxidative stress extent, thus also acting on nuclear gene expression. As
a consequence of the food–gene interaction, metabolic syndrome patients may express
different disease features and extents according to the different pathways activated by
oxidative stress-modulated effectors. This view could also explain family differences and
interethnic variations in determining risk factor appearance.
160 Rodrigo Castillo
1. Introduction
The metabolic syndrome is a multifactorial condition leading to accelerated
atherosclerosis and increased risk for diabetes. It is associated with major cardiovascular
events and a high mortality rate [1]. The metabolic syndrome is characterized by different
combinations of three or more of the following features: abdominal obesity, blood
hypertension, hyperglycemia and serum dyslipidemia as defined by the criteria of the Third
Report of the National Cholesterol Education Program Adult Treatment Panel III [2] or by
the updated criteria of the International Diabetes Federation [3].
Epidemiological surveys show that the metabolic syndrome is extremely common. The
1999–2002 National Health and Nutrition Examination Survey estimated the age-adjusted
prevalence of the metabolic syndrome in U.S. adults over 20 years to be between 34.6% and
39.1% and to be even higher if considering adults over 60 years. It is a little more common in
men, and also there exist ethnic differences. Overall, the prevalence of the metabolic
syndrome parallels the increasing aging population and “epidemic” obesity [4].
The link between individual metabolic syndrome components is unknown. Recent
studies support the view that these metabolic abnormalities [5] have a single factor may
underlie the association, the insulin resistance [6]. The fact that insulin resistance and
abdominal obesity are also associated with perturbations in plasma adipokine levels, altered
fatty acid metabolism, endothelial dysfunction, procoagulant state and systemic inflammation
underscores the breadth and complexity of the pathophysiology of this clustering [7].
Therefore, the identification of common basic mechanisms driving to a unifying pathogenic
hypothesis for the metabolic syndrome would be helpful in explaining the clinical
manifestations. In this point, oxidative stress could explain most of the second-level events
resulting in risk factor appearance and may lead to a unitary pathogenic view of this chronic
disorder.
Oxidative stress has been associated with all the individual components and with the
onset of cardiovascular complications in subjects with the metabolic syndrome [8, 9]. In a
recent study [10, 11], the role of oxidative stress in the pathophysiologic interactions among
the constituent factors of the metabolic syndrome has been remarked. Although some of the
constituent characteristics of the metabolic syndrome are known to share common pathogenic
mechanisms of damage, the impact of hereditary predisposition and the regulation of gene
expression as well as the role of environment and dietary habit in determining inflammatory
process-triggered oxidation are still unclear. These aspects of the problem deserve special
attention since it is hypothesized that in patients with the metabolic syndrome, oxidative
stress may be amplified by a concomitant antioxidant deficiency that may favour the
propagation of oxidative alterations from intra- to extracellular spaces and from confined to
distant sites, thus realizing a systemic oxidative stress state [12, 13].
Altogether, these considerations would suggest a unifying hypothesis to explain the
mechanisms underlying the onset and development of metabolic syndrome-associated risk
factors. As the following subsections report, excessive free radical production and oxidative
damages are supported by several experimental demonstrations and human observations.
Therefore, oxidative stress appears to possess the credentials to mechanistically explain the
Metabolic Syndrome 161
perpetuation of insulin resistance, the altered energy production, the endothelial dysfunction,
and the appearance of vascular complications in this condition.
2. Pathophysiology of Metabolic Syndrome

Metabolic syndrome is associated with insulin resistance. It is not a consequence of
insulin resistance alone, but a direct consequence of the lack of insulin action. This is most
evident in patients with insulin receptor mutations or autoimmune antibodies to the insulin
receptor; they may have 100-fold or greater elevations of circulating insulin or require
similarly high doses of exogenous insulin to control diabetes [14]. These patients exhibit a
distinct syndrome with acanthosis nigricans and a high risk of diabetes, but typically have no
obesity, hypertension, or atherogenic dyslipidemia [15]. Moreover, patients with type 1
diabetes mellitus, who lack insulin, do not exhibit the same atherogenic lipoprotein
phenotype typical of patients with metabolic syndrome or type 2 diabetes mellitus. Lean type
1 diabetic mellitus patients do not characteristically have insulin resistance.
If metabolic syndrome does not result purely from a lack of insulin effect, then how
might insulin resistance generate other features of the syndrome? Proposed mechanisms
center around 3 themes: effects of mild to moderate hyperglycemia, effects of compensatory
hyperinsulinemia, and effects of unbalanced pathways of insulin action [16]. Hyperglycemia,
largely postprandial and below diabetic levels, may lead to a variety of effects usually
associated with diabetes. For example, moderate hyperglycemia might be postulated to cause
accelerated atherogenesis via advanced glycosylated end products or via enhanced collagen
formation [17]. (for more details see chapter 8).
Another important mechanism may be the compensatory hyperinsulinemia. The
maintenance of normal post-prandial glucose homeostasis requires that pancreatic beta cells
secrete a normal amount of insulin in response to the hyperglycemic challenge, with resultant
hyperinsulinemia [18]. Insulin stimulates the glucose uptake by muscle, which is the tissue
responsible for the disposal of 80% to 90% of the ingested glucose load, and [2] suppresses
endogenous glucose production, which is generated mainly in the liver. In insulin-resistant
conditions, the ability of insulin to augment glucose uptake and inhibit hepatic glucose
production is impaired. The resultant hyperglycemia presents a stimulus to the beta cells,
which secrete large amounts of insulin after meals. Initially, attention was directed to the
concept that certain organs and tissues can have lesser degrees of insulin resistance than
skeletal muscle and liver [19]. For example, the high insulin concentration required to
produce normal glucose uptake in skeletal muscle may over stimulate cells of the arterial wall
and accelerate atherosclerotic process [20]. In recent years, this concept has been expanded to
include the idea that not only different cell types, but also different metabolic pathways
within the same cell, may differ in their responsiveness to insulin [21].
2.1. Dysfunctional Energy Storage and Obesity
Some investigators regard insulin resistance as a mediating factor in metabolic syndrome,

but not as the primary cause [22]. Dysfunctional energy storage seems to be the fundamental
issue, being insulin resistance the relevant factor leading to abnormalities in the processing
and storage of fatty acids and triglyceride, molecules that account for most of the body’s
energy utilization and storage. In most patients, the key abnormality is simply the presence of
too much triglyceride, or body fat (i.e obesity). The purpose of adipose tissue throughout the
body is energy storage: taking in food calories during and after meals, storing the calories as
triglyceride, and then releasing calories in the form of fatty acids when energy is needed [23].
It is safest for the body to store triglyceride in small peripheral adipocytes. If the capacity of
these adipocytes to store triglyceride is exceeded, triglyceride accumulates in hepatocytes,
skeletal myocytes, and visceral adipocytes. The abnormal triglyceride accumulation may lead
to the development of hepatic and muscular resistance to insulin. This is referred to as the
“overflow hypothesis” [24]. Visceral adiposity can be measured by waist circumference,
waist-hip ratio, or radiographic scans, and it correlates well with insulin resistance and other
features of metabolic syndrome. Excess triglyceride in myocytes and in abnormally large
peripheral adipocytes appears to engender insulin resistance in these cells [25]. Triglyceride
in hepatocytes is recognized as fatty liver and may drive the formation and secretion of
excessive VLDL.
This theory does not postulate an exclusive role for visceral adiposity, because abnormal
peripheral fat cells and triglyceride-laden muscle cells participate in the dysfunctional state
[26]. Body mass index and waist circumference tend to load equally in factor analysis, if
gender is taken into account. Body mass index may be a sufficient measure for many studies,
particularly retrospective analyses of prior data sets. Lipodystrophy syndromes offer striking
examples of the effects of the inability to store triglyceride in the physiologically preferred
small peripheral adipocytes [27, 28]. The mutations underlying many cases of lipodystrophy
are known. These disorders can present in childhood or in adult life with dramatic loss of
subcutaneous fat below the shoulder girdle. These patients can develop severe
hypertriglyceridemia, insulin resistance, fatty liver, and eventually diabetes mellitus [29].
Patients infected with the human immunodeficiency virus, especially those treated with
protease inhibitors; also develop partial lipodystrophy with similarly exaggerated features of
metabolic syndrome [30]. Despite their loss of most subcutaneous fat, these patients develop
abdominal obesity and fat pads around the base of the neck.
In addition to visceral fat, liver, and muscle, excess triglyceride can accumulate in
abnormally large peripheral adipocytes. Cross-sectional studies indicate that large
subcutaneous adipocyte size is associated with insulin resistance [31, 32]. In a study of Pima
Indians, subcutaneous abdominal adipocyte size strongly correlated with risk for developing
type 2 diabetes mellitus. This effect of adipocyte size on the risk of developing diabetes was
independent of and additive to the effect of insulin resistance [33]. Danforth et al. [27] have
proposed that this correlation of adipocyte size and type 2 diabetes mellitus suggests a
difficulty in differentiating new adipocytes. A failure of adipocyte differentiation limits the
pool of adipocytes available for energy storage, and excess triglyceride overflows to other
sites, leading to insulin resistance [34].
Clinical studies in morbidly obese patients showed that the reduction in body mass index
45 to 35 with surgery is associated with a normalization of insulin sensitivity [35, 36]. When
the fat content of muscle was examined in these individuals, it had been reduced to zero,
demonstrating that intramyocellular fat content is an important determinant of insulin
sensitivity. Fat is found in muscle and liver when it overflows from overwhelmed adipocytes;
once fat moves back from muscle and liver to adipocytes, it seems to be stored safely without
causing metabolic derangement [37].
Liver along with adipose tissue participate in maintaining glucose and lipid homeostasis
through the secretion of various humoral factors and/or neural networks [38, 39, 40]. Various
studies have validated the presence of molecular signatures typical of the liver and adipose
tissue in mouse models of obesity [41] and in mice fed with a high-fat diet (HFD) [42]. It is
believed that perturbations in these “intertissue communications” may be involved in the
development of insulin resistance, obesity, and other features of metabolic syndrome [43].
However, it remains unclear which factors alter the communication among tissues and impair
the ability of tissues to adapt to changing metabolic states.
2.2. Oxidative Stress and Insulin Resistance
Reactive oxygen species production is one of many factors that have been suggested to
play a role in the development of insulin resistance, based on the following evidence: i) high
doses of hydrogen peroxide [8] and reagents that accumulate ROS [44] can induce insulin
resistance in adipocytes, ii) increased markers of oxidative stress were observed in obese
humans [45] and rodents [46]. Nevertheless, it remains unclear, whether increased ROS
production causes insulin resistance in vivo. It has been demonstrated that the up-regulation
of genes responsible for ROS production occurs in both the liver and adipose tissue before
the onset of insulin resistance and obesity in mice fed an HFD [47].
It is striking that increased ROS production precedes the elevated levels of TNF-α and
FFAs in the plasma and liver in diabetic patients [48]. Reactive oxygen species triggers the
development of insulin resistance resulting in abdominal obesity, thereby raising the levels of
TNF-α and FFAs. In summary, the HFD induces oxidative stress, potentially through the up-
regulated expression of genes for ROS production and down-regulation of antioxidant genes,
in the liver and adipose tissue [49]. In addition, these changes occur before the onset of
insulin resistance and obesity. Sources of ROS induced by an HFD may differ between the
liver and adipose tissue. These findings suggest that ROS production may be the initial event
triggering HFD-induced insulin resistance and therefore may be an attractive therapeutic
target for preventing insulin resistance and obesity caused by an HFD [50].
2.3. Oxidative Alterations, Visceral Obesity and Liver Steatosis
A number of clinical studies have reported the importance of visceral fat accumulation in
the development of metabolic disorders, including reduced glucose tolerance, hyperlipidemia
and cardiovascular diseases [51].
Visceral fat accumulation causes dysregulation of adipocyte functions, including

oversecretion of leptin and TNF-α, plus a diminished secretion of adiponectin. This results in
the development of a variety of metabolic and circulatory disorders, including quantitative
and qualitative changes in serum lipids and lipoproteins such as small dense LDL [52].
Visceral adiposity represents an independent determinant of all the metabolic syndrome
components. In humans, mesenteric fat has been independently associated with body mass
index and metabolic risk factors better as compared with measured waist circumference [53,
54]. Abdominal adiposity is also crucial as a source of free fatty acids and inflammatory
factors. Indeed, the the International Diabetes Federation gives it a central role in the
diagnosis of the metabolic syndrome [55].
Visceral obesity represents per se a low permanent systemic inflammation, as reflected
by elevated serum markers, such as C-reactive protein and TNF-α [56]. Genetic manipulation
and overnutrition studies have convincingly shown that insulin resistance is regulated by
cytokines and mediators released from mesenteric adipocytes [57].
It is generally accepted that the sequence of events leading to hepatocyte fatty
degeneration begins with insulin resistance, which precedes fat accumulation [58]. Excess
intracellular fatty acids, oxidative stress, energy depletion and mitochondrial dysfunction
then cause cellular injury [59, 60]. NASH, the inflammatory form of NAFLD, is thus viewed
as the result of “two hits,” in which the first hit is fat accumulation [61]. Lipid retention
within hepatocytes triggers oxidative stress (the “second hit”) generating ROS at different
intracellular levels and cytokine release. In particular, the alteration of intracellular fatty acid
trafficking and mitochondrial β-oxidation, consequent to differential expressions of perilipin
and adipophilin [62] and hepatic refractoriness to adipokines [63], contributes to the
impairment of hepatic lipid turnover and leads to lipid accumulation. Lipid accumulation and
insulin resistance activate different sources of ROS: (i) the cytochrome P450 2E1, which
generates ROS during the metabolism of endogenous ketones and dietary constituents [64,
65]; (ii) mitochondria, which continuously generate ROS, being damaged them themselves if
the production of ROS is increased [66, 67]; and (iii) peroxisomes, which generate H2O2 and
are activated when mitochondrial β-oxidation is saturated or impaired [68]. Enhanced hepatic
lipid peroxidation causes changes in physical and chemical membrane properties, with
fluidity and permeability alteration [69] affecting signal transduction and ion exchange
properties [70]. Changes in lipid composition and characteristics induce membrane
remodeling [71]. Most lipid peroxides are volatile molecules that may reach sites distant from
those of generation and cause damages and fibroblastic cell activation in the presence of
inflammation [72]. In this respect, it has been observed that subjects with NASH show high
hepatic and systemic levels of lipid peroxidation products. This phenomenon is associated
with an increased risk for cardiovascular disease [73].
Recently, impaired serum redox balance with decreased antioxidant capacity and
increased lipid peroxidation has been observed in patients with fatty liver, visceral obesity
and metabolic syndrome. In the study by Pou et al. [74], the amount of visceral fat and
systemic oxidative alterations were significantly related, thus indicating that excess visceral
fat is an important and independent determining factor of the observed serum oxidative
changes. Moreover, in these patients, the presence of the metabolic syndrome was predicted
from a linear combination of variables, including liver steatosis, visceral fat and serum
oxidative changes. The participation of the liver both as a damaged organ and a contributory
source for systemic oxidative alterations in patients with the metabolic syndrome and visceral
adiposity is therefore unequivocally suggested [75]. This role is further supported by the
coexistence of fatty liver with blood hypertension and metabolic syndrome in non obese
patients [76] and by the observation that NAFLD is associated with the metabolic syndrome
to a higher extent than excess adipose tissue in obese subjects [77].
Another clinically relevant aspect is the increased vulnerability of fatty livers toward
stress events [78] especially as they occur in transplantation surgery. These mainly depend on
the fact that hepatic steatosis sensitizes hepatocytes to injury and inflammation through
enhanced fatty acid synthase expression and increased fatty acid synthase-mediated apoptosis
[79].
Another potentially damaging factor in NAFLD is intestinal bacteria. The contribution of
small bowel bacteria overgrowth to liver inflammatory processes may in fact be realized
through an increased intestinal permeability that allows entry of gut-derived toxins with
consequent portal inflammation, Kupffer cell activation and liver injury [80, 81]. In this
point, NAFLD patients sowed elevated plasma levels of LPS-binding protein (LBP), a
biomarker of endotoxemia, and they are further increased in patients with NASH. This
increase is related to a rise in TNF-alpha gene expression in the hepatic tissue which supports
a role for endotoxemia in the development of steatohepatitis in obese patients [82]. Indeed,
ROS are generated in the liver by prooxidant inflammatory pathways that are initiated by gut-
derived endotoxin [83, 84]. Excess endotoxin can reach the liver through the portal
circulation as a result of a higher concentration of endotoxin in the gut or through increased
absorption of endotoxin from the gut, i.e.gut leakiness.
Wigg et al. [85] compared a group of 22 healthy controls with a group of 23 patients with
biopsy-proven NAFLD for the prevalence of small intestinal bacterial overgrowth, increased
intestinal permeability and serum endotoxin levels. They found a higher prevalence of small
intestinal bacterial overgrowth (assessed by C14-D-xylose breath test) in patients with
NAFLD, but found no difference in intestinal permeability (as measured by a lactulose–
rhamnose sugar test) or endotoxaemia. Their finding of normal serum endotoxin in the
systemic circulation does not exclude endotoxaemia in the portal circulation. They concluded
that patients with NAFLD do not have a leaky gut, but bacterial overgrowth may contribute
to an endotoxin-initiated hepatic necroinflammatory cascade. However, they did not
distinguish between simple steatosis and steatohepatitis. Furthermore, they only studied small
bowel permeability. Indeed, loss of colonic barrier integrity in patients with NASH could
have a more deleterious effect than loss of permeability of the small bowel, which has
relatively low levels of luminal bacteria. Finally, gut leakiness could still be an important
pathogenic factor in patients with NASH and ‘normal’ intestinal permeability because these
patients may have increased susceptibility to gut leakiness when gut barrier integrity is
challenged and results in the intermittent gut leakiness and endotoxaemia necessary to initiate
a hepatic necroinflammatory cascade and liver cell injury.
In according to this, it have been demonstrated [86] that NASH obese subjects showed a
susceptibility to gut leakiness, rather than overt gut leakiness, in with. This susceptibility to
leakiness may be the cause of the endotoxaemia and may explain why only a subgroup of
patients with NAFLD progresses to steatohepatitis and advanced fibrosis. This finding may
also help us to find the contributing factors in the pathogenesis of NASH that act disruption
of colonic barrier integrity, factors such as NSAIDs, which in turn can lead to endotoxaemia
and provide the ‘second hit’ for development of NASH. Based on our results, it is reasonable
to recommend to those patients with altered fatty acid metabolism and metabolic syndrome
(obesity, diabetes, insulin resistance) and who are more susceptible to oxidative stress to
avoid agents that increase permeability such as NSAIDs and alcohol. Larger, interventional
or longitudinal prospective studies are needed to assess directly the contribution of
susceptibility to gut leakiness in the course of NAFLD.
2.4. Endothelial Oxidative Dysfunction and Blood Hypertension
The participation of arterial hypertension in the generation of systemic oxidative stress

associated with the metabolic syndrome is suggested by a number of observations on the role
of insulin resistance and the sympathoadrenal system [87], NO metabolism changes and the
low circulating levels of vitamin C in patients with high-grade hypertension [88] and the
improvement of systemic oxidative stress with antihypertensive treatment [89, 90] These
considerations have an even higher impact when associated with endothelium activation and
dysfunction as characterized by increased levels of circulating oxidized LDL, intercellular
and vascular adhesion molecules and C-reactive protein [91] and with the evidence that
vascular complications are also associated with oxidative stress events.
In particular, small dense LDL particles are able to filtrate through the endothelium of
blood vessels. Oxidized LDL activates endothelial cells with the promotion of an immune
response leading to the formation of lipid-laden macrophages. Also, in response to
inflammatory stimuli, endothelial cells produce adhesion molecules that will further facilitate
macrophage migration from the blood into the intimae, thus generating an endothelial
damage. These events are favored by insulin resistance and obesity [92]. Other factors have a
role in the modulation of these pathophysiologic mechanisms. Among them, vitamin E is a
fat-soluble vitamin that is sequestered in the hydrophobic interior of membranes where it acts
as an antioxidant, quenching lipid peroxidation. Under normal conditions, the reduced state
of LDL is maintained by vitamin E [93, 94], which also acts by regulating inflammatory
reactions and metabolic pathways, including platelet aggregation [95]. In combination with
tocopherols, vitamin C counters free radicals and regulates vitamin E metabolism by
recycling oxidized tocopherols. The synergic action of these two vitamins is also modulated
by the intervention of glutathione, which maintains vitamin C in the reduced form [96].
Relatively new and interesting pathways of oxidative stress-induced vascular damages
include enzymes such as Nox and homocysteine [97, 98]. Membrane-bound Nox are major
sources of ROS in preatherosclerotic conditions and have been found in human peripheral
and coronary arteries [99]. A direct spatial relationship between Nox-generated ROS and
LDL oxidation was demonstrated in carotid plaques and in lesions associated with unstable
angina [100, 101]. By increasing oxidative stress, activation of Nox in vascular cells has been
reported to be an important mechanism in the pathogenesis of hypertension and
atherosclerosis [102] . Angiotensin II is one of the most potent stimuli activating vascular
Nox. This property clearly links ROS production with activation of the renin–angiotensin
system in hypertension [103]. As a consequence, drugs acting on the renin–angiotensin

system reduce Nox activity, thus rendering this enzyme a specific drug target (figure 7-1).
Figure 7-1. ROS generation and consequent oxidative stress as a “second-level” event causing
metabolic syndrome-associated. FFA: Free fatty acids; NOX: NADPH oxidase; LDL: Low-density
lipoprotein; AOX: Antioxidant defenses; NO: nitric oxide.
Disorders of the folate-dependent methionine metabolism have been described in

experimental models and human conditions associated with a high cardiovascular risk [104].
These metabolic abnormalities result in high levels of homocysteine, a molecule belonging to
the group of thiols. Differently from glutathione and cysteine, which exert protective effects
against ROS, homocysteine is considered to be a “bad thiol” because of its association with a
variety of chronic disease conditions [105]. Homocysteine is formed in the transsulfuration
and remethylation pathways that convert homocysteine to methionine with folate and betaine
intervention. As a consequence of the impairment of the methionine metabolism, the

increased level of circulating homocysteine has been associated with endothelial dysfunction,
both directly and via NO interaction [106]. Although it is known that homocysteine can be
toxic per se by acting as an N-methyl-D-aspartate agonist, thus decreasing the availability of
NO and impairing arterial vasodilation capacity [107], the specific molecular mechanisms of
damage involved are still unclear. Three mechanisms have been proposed, namely, oxidative
stress, endoplasmic reticulum stress and activation of pro-inflammatory factors [108].
Hyperhomocysteinemia has been associated with oxidative stress in liver steatosis, a
hypothesis favored by recent observations [109, 110]. An additional mechanism that may be
involved in homocysteine-mediated vascular alteration in patients with the metabolic
syndrome is the indirect connection between high homocysteine levels and low nitrosothiol
levels. At this concern, it is known that circulating nitrosothiols act as free NO donors for
vascular tone modulation [111]. In addition, by representing a storage form of thiols and
glutathione in particular, extracellular nitrosothiols exert antioxidant functions and favor
removal of toxic products [112, 113], contributing to the role of the extracellular
microenvironment in the regulation of the redox status and function of cell surface proteins
[114]. The above-reported interference of homocysteine with circulating NO availability
could explain, at least in part, the low levels of nitrosothiols and glutathione found in
conditions associated with hyperhomocysteinemia [115]. Therefore, the equilibrium between
“good” and “bad” thiols may determine outcomes in studies of tissue degeneration and
inflammation (i.e., NASH and the metabolic syndrome).
2.5. Experimental Studies of the Metabolic Syndrome
Animal models can be helpful in further understanding the potential pathophysiology of

the metabolic syndrome. Murine models in particular have become quite useful tools in
recent years because the entire mouse genome is now sequenced, and a large number of
transgenic and knockout models are readily available. There are a number of limitations with
these models, however, that must be considered. Rodent lipid physiology, for example, is
significantly different compared with humans. Rodents carry most of their cholesterol in
HDL, not LDL; thus, a low level of HDL is an unusual finding. Blood pressure is usually not
measured in these models, again limiting the use of the “human” clinical definition of the
metabolic syndrome. Nevertheless, there remains much to be learned from animal models that
may be applicable to mechanisms of the metabolic syndrome in humans.
2.5.1. Mouse Models

Various murine models exhibited many of the components of the metabolic syndrome,
i.e., leptin-deficient ob/ob and leptin-resistant db/db mice [116]. More recently, when ob/ob
mice were crossed with the LDL-receptor-deficient mouse, the features of the metabolic
syndrome including obesity, dyslipidemia, hypertension, insulin resistance and impaired
glucose tolerance, and/or diabetes plus hypercholesterolemia resulted in more oxidative stress
and atherosclerosis [117, 118]. A number of less-well known polygenic mouse models have a
mixture of components of the MetS and its associated diseases. It is worth noting that mice
with different genetic backgrounds have a variable propensity to develop the MetS in
response to changes in diet composition [119, 120]. For instance, when C57Bl/6 (B6) and
129S6/SvEvTac [121] mice were placed on a low-fat or high-fat diet for 18 wk, the 129 strain
developed features of the metabolic syndrome, notably obesity, hyperinsulinemia, and
glucose intolerance only on the high-fat diet, whereas the B6 strain developed these features
on both diets [122].
The Jackson Laboratory has carried out a comprehensive assessment of genetic
susceptibility to the metabolic syndrome in inbred mice when challenged with a high-fat,
high-cholesterol diet [123]. A standard protocol was set up to evaluate female and male mice
from 43 inbred strains for 10 traits including all the major criteria of metabolic syndrome
while mice consumed the diet for 18 wk. A few strains of mice developed a phenotype with a
plethora of metabolic abnormalities remarkably similar to the human metabolic syndrome
(strains CAST/EiJ, CBA/J, and MSM/Ms). Other strains had a more limited phenotype, i.e.,
severe obesity (AKR/J and KK/HIJ) vs. protection from obesity (WSB/EiJ); severe
dyslipidemia (MOLF/EiJ) vs. no dyslipidemia (CZECHII/EiJ for males and D2 for females);
and severe insulin resistance (KK/HIJ) vs. being spared from insulin resistance (A/J).
Overall, the discrepant phenotypes within the same environmental exposures may prove
useful in dissecting the genetic and related molecular mechanisms underlying the metabolic
syndrome and its components [124].
2.5.2. Rat Models

A number of models of the metabolic syndrome have been identified in rats. The Zucker
fatty rat was among the first identified [125]. Subsequently, a number of studies have been
published to examine the impact of diet on the phenotypic development of the metabolic
syndrome [126-128]. Wistar Ottawa Karlsburg W rats (WOKW) develop all components of
this syndrome. Genetic analysis of this rat model has identified potential major quantitative
trait loci (QTL) for glucose metabolism on chromosome 3, dyslipidemia on chromosomes 4
and 17, and obesity on chromosomes 1 and 5 [129]. Moreover, the severe insulin resistance
predominant in epididymal adipose tissue of these rats was associated with a 10-fold decrease
in adipocyte adiponectin gene expression and decreased peroxisome proliferator-activated
receptor gene expression, but increased FOXO1 gene expression compared with control rats
[130]. Moreover, the metabolic in WOKW rats was associated with impaired coronary
vasodilatation due to altered adrenoceptor sensitivity [131].
Another example in rats is the corpulent (JCR: LA-cp) rat that like db/db in mice is a
homozygous mutation in the leptin receptor [132]. These rats are obese, insulin resistant, and
hypertriglyceridemic. JCR:LA-cp rats, however, are prone to atherosclerosis [133, 134] and
also appear to be a good model to study the contribution of postprandial lipemia to the
atherosclerotic process. In these rats, lymphatic chylomicron apoB48, fasting and
postprandial plasma apo B48 area under the curve are all elevated [135].
The Prague hereditary hypertriglyceridemic (hHTG) rat was developed as a model of
hypertriglyceridemia. Although these rats are not obese, they are hypertensive, insulin
resistant, and glucose intolerant [136]. Using F2 hybrids, several QTL have been identified
for hypertension and hypertriglyceridemia [137]. Another model of the metabolic syndrome
in rats that includes hypertension is the Lyon hypertensive rat (LH). These rats also have
obesity, dyslipidemia, and an increased insulin/glucose ratio. This rat strain has been used to
identify linkage of body weight, blood pressure, and renal, metabolic, and endocrine
phenotypes [138]. This is a rennin-dependent model of hypertension in which low-dose
(nonantihypertensive) ACE inhibitor therapy affords significant and durable renal protection.
A total genome scan in the offspring of an F2 intercross between the hypertensive and
normotensive Lyon strains has identified a series of QTL for the metabolic syndrome, body
weight, blood pressure, lipid metabolism, and renal function [139]. Other hypertensive rat
models of the metabolic syndrome include SHR/NDmccp(cp/cp) [140] and SHROB
(spontaneously hypertensive, obese rat) [141].
2.5.3. Other Animal Models

Of interest to pet owners and veterinarians alike is the fact that obesity in dogs and cats
has increased in recent years [142], and dogs in particular are models of the metabolic
syndrome. The canine obesity model closely recapitulates the relationship between human
visceral adiposity and insulin resistance. The work of Bergman et al., [143] supports the
portal theory of insulin resistance, in which FFA from visceral adipose tissue directly enter
the liver and unfavorably modify insulin action. Sympathetic nervous system hyperactivity in
this model of obesity may also contribute to excessive free fatty acids (FFA) release,
hypertension, and insulin resistance. As noted previously, a nocturnal increase in plasma FFA
levels may account for both insulin resistance and compensatory hyperinsulinemia.
Obesity is common in cats and is a risk factor for diabetes. The prevalence of diabetes
has increased concomitantly with the increase in obesity, and diabetes is now seen in
approximately 0.5–1% of cats [144]. Cats develop a form of diabetes similarly to type 2
diabetes in humans, characterized by islet amyloid accumulation and loss of β-cell mass
[145]. From more recent studies in felines, it appears that glucose metabolism in cats is
similar to that in humans; however, lipid metabolism is quite different [146].
3. Role of Antioxidants in Attenuation of

Metabolic Syndrome Progression
3.1. Experimental Studies
3.1.1. Antioxidant Vitamins

There is direct evidence that micronutrients have a beneficial effect on insulin sensitivity
and some components of the antioxidant defense system in an animal model of insulin
resistance [147]. In this point, the beneficial effects of antioxidant vitamins supplementation
are attributed to their ability to scavenge free radicals, control nitric oxide synthesis or
release, inhibit reactive oxygen species generation and upregulate antioxidant enzyme
activities that metabolize these molecules [148]. Low levels of vitamin C, a potent dietary
antioxidant molecule, have been associated not only with obesity [149] but also with a
variety of conditions including hypertension, gallbladder disease, stroke, some cancers and
atherosclerosis [150]. Moreover, vitamin C administration ameliorates hyperglycemia and
glycosylation in diabetic-obese rodents [151] and inhibits the activation of inflammatory
response mediated by nuclear factor-kappa B [152]. In addition, to the health effects of

ascorbic acid as antioxidant, this vitamin could be involved in obesity-related mechanisms,
for example, regulation of behavioural activity [153], lipolysis [154] and glucocorticoid
release from the adrenal glands [155]. In hypertensive rats long term Vit C administration
significantly reduced systolic blodd pressure and simultaneously reduced oxidative stress
mediated by NAD(P)H oxidase activation [156]. Vitamin C has beneficial effects not only on
blood pressure but also on endothelial function in hypertensive and diabetic patients [157].
Vitamin C is a soluble compound and it prevents protein and lipid oxidation in the
extracellular environment [158]. In vivo studies confirmed that vitamin C administration
improves arterial vasodilatation by increasing NO production [159].
Demonstration of free radical damage and its prevention by vitamin E in vivo have
lagged because of a lack of sensitive analytical techniques. This, however, has recently
changed; quantification of F2-isoprostanes, isomers of prostaglandin F2, has been suggested
by a number of investigators as a reliable index of in vivo free radical generation and
oxidative lipid damage. F2-isoprostanes are formed in membranes from arachidonyl-
containing lipids by cyclooxygenase enzymes, as well as during free radical-catalyzed lipid
peroxidation [160]. In studies using experimental animals, F2-isoprostanes increased in
plasma and tissues as a result of vitamin E deficiency [161]. Furthermore, in an animal
atherosclerosis model (the apoE-deficient mouse), vitamin E supplementation not only
suppressed F2-isoprostane production but also decreased atherosclerotic lesion formation
[162].
In other animals models in which that estrogen deficiency or ovariectomy results in a
reduction of sexual steroids and increased prevalence of cardiovascular diseases, it was
assessed the benefits of antioxidant vitamins (E and C) for the protection against
cardiovascular disease and oxidative stress. The adjunct antioxidant treatment potentiated the
hormone replace treatment and showed a significant correction of homocysteine and GSH
levels [163]. Further studies are warranted to elucidate the beneficial role of antioxidant
treatment of cardiovascular protection of estrogen deficiency models, relevant to elucidate
clinical complications that present of menopause women.
3.1.2. Flavonoids
Many observational and experimental studies have considered that caloric restriction may
be associated with life prolongation [164]. possibly through an improvement of the cell redox
balance [165]. Also, increased generation of mitochondrial ROS and oxidative damages seem
to be differently induced by nutritional perturbation and state [166]. In animal experiments
hypocaloric diet and antioxidant supplementation were associated with improvement of some
tissue functions and redox states that, conversely, were oxidatively depressed in aged control
animals [167]. A key event associated with diet restriction is the activation of a class of genes
belonging to the Sirt family, which is involved in cell maturation and apoptotic processes.
Recently, Howitz et al. [168] showed that resveratrol, an antioxidant poliphenol of red wine,
was able to activate these genes by mimicking the effect of diet restriction. Successively,
Baur et al. [169] showed that high dose resveratrol was able to contrast the development of
cardiovascular diseases and diabetes in mice that underwent a hyperlipidic diet, suggesting a
role for oxidative stress in systemic inflammation and damages in conditions simulating the
metabolic syndrome. In this point, grape extracts enriched in different polyphenolic families
have been utilized to prevent reactive oxygen species (ROS) production, although having
differential effects on various features of metabolic syndrome when administered to the
fructose (60%)-fed rat (a model of metabolic syndrome) [170]. The effect of pure
polyphenolic molecules (catechin, resveratrol, delphinidin, and gallic acid) prevented insulin
resistance, the elevation of blood pressure and cardiac ROS overproduction and NADPH
overexpression. Indeed, fructose feeding is associated with cardiac fibrosis (accumulation of
collagen I) and expression of osteopontin, a factor induced by ROS and a collagen I
expression inducer. In this model, collagen I and osteopontin expressions could be prevented
by the administration of polyphenolic molecules [171]. The potential use of polyphenols in
the prevention of cardiac complications associated to metabolic syndrome should be further
explored.
3.1.3. Other Antioxidants

Systemic oxidative stress and nitrative stress as well a inflammation increase with the
development of metabolic syndrome-like components in SHR/ cp rats, which display
abdominal obesity, hypertension, hyperglycemia, insulin-resistance, and hyperlipidemia
[172]. Long-term CoQ10 administration can prevent increased oxidative and nitrative stress
[173], as indicated by higher levels of Ox-LDL and 8- OHdG in the serum and of 3-
nitrotyrosine in serum proteins, respectively, and the increased inflammation with activation
of myeloperoxidase, as indicated by higher serum levels of C-reactive protein and 3-
chlorotyrosine in the SHR/ cp rats displaying metabolic-like components. In addition, the
elevated serum insulin levels and high blood pressure were suppressed by CoQ10 intake for
10 weeks. In diabetic rats, CoQ10 treatment also reduced lipid peroxidation and increased
antioxidant parameters like superoxide dismutase, catalase, and glutathione in the liver
homogenates of diabetic rats. CoQ10 also lowered the elevated blood pressure in diabetic
rats, explained to mechanism based on induction of antioxidant defense system [174].
CoQ10 prevents vascular endothelial dysfunction seem to be linked to its hypotensive
effect in SHR/ cp rats. Furthermore, insulin resistance and the consequent hyperinsulinemia,
important components of metabolic syndrome, are associated with endothelial dysfunction,
probably due to increasing oxidative stress [175]. The physiological properties of insulin that
cause enhancement of renal sodium reabsorption and stimulate sympathetic nervous system
activity are believed to play a major role in the development of hypertension [176], although
the underlying mechanisms in the setting of insulin resistance remain obscure [177].
Therefore, the hypotensive effect of CoQ10 observed in SHR/cp rats may be associated with
its alleviation of hyperinsulinemia together with endothelial dysfunction. These findings
suggest that the antioxidant properties of CoQ10 can be effective for ameliorating
cardiovascular risk in metabolic syndrome.
3.2. Clinical Studies
3.2.1. Antioxidants Vitamins

Both vitamins, E and C, appear to be important for the prevention of cardiovascular
events. In fact, consumption of vitamin E has been associated with a lower risk for coronary
heart disease [178] and with reduced LDL oxidation [179]. Also, the connection between
serum concentrations of vitamin E and lipid peroxidation products in relation to cholesterol
level and abdominal obesity has been recently studied in patients with the metabolic
syndrome. An inverse relation between the serum cholesterol-adjusted vitamin E
concentrations and the grade of hepatic steatosis and a linear relation between the extent of
visceral fat and the lipid peroxide/ cholesterol ratio have been observed [61]. In other studies,
supplementation of vitamin E was able to prevent the onset of type 2 diabetes [180] and
improve NAFLD in obese children [181]. All of the studies discussed were carried out with
alpha-tocopherol supplements. Alpha-tocopherol decreases plasma gamma-tocopherol
concentrations, as a result of the function of the hepatic alpha-tocopherol transfer protein,
which preferentially incorporates alpha-tocopherol into the plasma [182], as well as
increasing gamma-tocopherol metabolism. This observation has often been suggested as an
explanation for the null results observed with alpha-tocopherol supplementation in the
majority of prospective clinical trials, especially since gamma-tocopherol concentrations are
inversely associated with increased morbidity and mortality due to cardiovascular disease
[183]. Recently studies showed [184, 185]. that in metabolic syndrome-subjects the
combination of alpha-tocopherol y gamma-tocopherol therapy results in significant
reductions in C-reactive protein, urinary nitrotyrosine, and lipid peroxides. Future studies will
be directed at examining mechanisms for these changes and testing the effect of combined
supplementation on cardiovascular events in high risk populations such as chronic kidney
disease and metabolic syndrome.
Concerning the specific role of vitamin C in oxidative stress-associated arterial
hypertension, mounting evidence suggest the importance of this vitamin in regulating
endothelial function and vasodilation. In fact, vitamin C is known to improve elastic artery
[186], by contrasting endothelial cell oxidation and by stimulating both endothelium-
dependent and endothelium-independent arterial vasodilation [187]. In addition, vitamin C
administration was able to restore endothelium-dependent vasodilation in hyperglycemic
patients [188].
3.2.2. Flavonoids
The Mediterranean diet contains a high rate of olive oil, fish, vegetable and low
consumption of alcohol, thus spreading a wide antioxidant capacity. The Mediterranean diet
has also been associated with a reduced incidence of blood hypertension, suggesting that a
diet regimen well balanced in carbohydrates and fats could be indicated to correct metabolic
abnormalities in metabolic syndrome patients. In a recent controlled crossover trial [189],
lower plasma oxidized LDL and lipid peroxide levels and higher glutathione peroxidase
activity were observed after an olive oil intervention, suggesting that consumption of olive
oil, rich in phenolic antioxidant compounds, could provide beneficial effects in patients with
cardiovascular risk factors. In this respect, it is also known that dietary fats can accomplish
regulation of hepatic lipid metabolism through modification of gene transcription [190]. This
is achieved by long-chain polyunsaturated fatty acids that are able to direct (i) fatty acids
away from triglyceride storage by enhancing their oxidation and; (ii) glucose away from fatty
acid synthesis by increasing its flux to glycogen [191].
Increased consumption of fruits and vegetables has also been shown to be associated
with a reduced risk for stroke in most epidemiological studies [192, 193]. In a recent meta-
analysis of prospective cohort studies, He et al. [194] demonstrated that intake of fruits and
vegetables higher than the average of three servings per day was associated with a lower risk
for stroke, thus providing strong support for the use of antioxidant vitamin-rich food in the
diet of patients with cardiovascular risk factors. However, experimental and human studies of
the addition of antioxidants to diets and other treatments in patients with NASH and
metabolic syndrome yielded controversial results. In particular, although a vitamin E-
deficient diet elevated the lipid peroxidation levels in the rat liver, both ubiquinol and
glutathione seem to protect mitochondria from lipid peroxidation more than vitamin E [195].
In humans, whereas addition of vitamin E to ursodeoxycholate in the treatment of NASH
patients improved laboratory test and hepatic histology findings in a small number of
metabolic syndrome patients [196], a combined vitamin E and vitamin C treatment did not
improve necro-inflammatory activity or alanine aminotransferase and was not superior to
weight loss in reducing biochemical indexes in two different studies of NASH patients [197].
In this point, is relevant mentioned some studies that demonstrate scavenger properties that
polyphenolic compounds in chronic consumtion in patients with NASH, however these
benefits are not expressed in functional parameters [198].
3.2.3. Other Antioxidants

Another natural food compound with protective properties is betaine. Betaine is
distributed widely in plants (wheat germ, bran and spinach), and rich dietary sources include
seafood, especially marine invertebrates [199]. The principal physiologic role of betaine is
the methyl donor (transmethylation) in the methionine cycle. Inadequate dietary intake of
betaine leads to disturbed hepatic methionine metabolism resulting in elevated plasma
homocysteine concentrations and to inadequate hepatic fat metabolism leading to steatosis
and subsequent increased serum lipid levels. These metabolic alterations may contribute to
coronary, cerebral, hepatic and vascular diseases. Betaine has been shown to protect internal
organs, improve vascular risk factors and enhance performance [200].
Coenzyme Q10 (CoQ10) is an endogenously synthesized compound that acts as an
electron carrier in the mitochondrial respiratory chain [201]. In addition to its unique role in
mitochondria, CoQ10 functions as an antioxidant, scavenging free radicals and inhibiting
lipid peroxidation [202]. Recent studies have provided evidence of the potential value of
CoQ10 in prophylaxis and therapy of various disorders related to oxidative stress. There is
promising evidence of the beneficial effect of CoQ10 in hypertension and heart failure [203].
It has been reported that CoQ10 concentrations and redox status are associated with
components of metabolic syndrome [204]. The administration of CoQ10 notably suppresses
oxidative and nitrative stress, inflammation, hypertension, and hyperinsulinemia [205]. These
findings suggest that the antioxidant properties of CoQ10 can be effective for ameliorating
cardiovascular risk in metabolic syndrome. Table 1 shows a summary of some clinical trials
that support interventions with antioxidant in metabolic syndrome.
Finally, natural elements appear to have a role in the regulation of serum glycemia and
associated metabolic dysfunctions. In particular, some observations have suggested that
excess intake of refined carbohydrates is associated with decreased levels of serum chromium
[206] and that this element has potential benefits on hyperglycemia, diabetes and elevated
serum lipids [207]. It has been suggested that chromium explicates its action by improving
some insulin effects, including the glucose transport within mitochondria, and improving the
energetic demand.
Table 7-1. Clinical trial that support the antioxidant

interventions in metabolic syndrome
Compound Mechanism Effects in Human
Vitamin C ↓ ROS production Improve endothelial function

Schneider et al., 2005
Moreau et al., 2005
Scavenger ROS ↓ Endothelial oxidation

May & Qu, 2005
Vitamin E Scavenger ROS ↓ LDL oxidation

Lapointe et al., 2006
Antiinflammatory ↓ C-reactive protein

Skalicky et al., 2008
Flavonoids ↓ ROS production ↓ LDL oxidation

Fito et al., 2004
Feillet-Coudray et al., 2008
Scavenger ROS ↓ Liver steatosis

Kaviarasan et al., 2007
Betaine ↓ Homocysteinemia Improve vascular function

↓ Liver steatosis
Craig , 2004
Coenzyme Q10 Scavenger ROS ↓ Hyperinsulinemia

↓ Blood pressure
Rosenfeldt et al., 2007
Pepe et al., 2007
ROS: Reactive oxygen species; LDL: Low density lipoprotein.

The metabolic syndrome is common, and the associated risk burdens of diabetes and
cardiovascular disease are a major public health problem. The hypothesis that the main
constituent parameters of the metabolic syndrome share common pathophysiologic

mechanisms of damage provides a new conceptual framework for future research, although
clinical trials will be necessary to confirm that the results from animal studies are applicable
to humans.
Actually is the well-documented beneficial effects of exercise and body weight reduction
in the prevention of insulin resistance and in the amelioration of diseases associated with the
metabolic syndrome, the paradigm discussed in this review suggests that interrupting
intracellular and extracellular ROS overproduction would contribute to normalizing the
activation of metabolic pathways leading to the onset of diabetes and its complications and
contrast the appearance of endothelial dysfunction leading to cardiovascular complications.
This view supports the hypothesis that oxidative stress, mechanistically explaining the
perpetuation of insulin resistance and endothelial dysfunction, may contribute to the
appearance of cardiovascular complications in patients with the metabolic syndrome.
Under conditions of elevated metabolism, many tissue-specific cells are continuously
subject to insult from ROS, such as the superoxide radical and H2O2. This is probably a
common feature for elements of the metabolic syndrome such as hypertension,
hypertriglyceridaemia, diabetes and obesity. Moreover, an increase in ROS production is one
of the earliest events in cases of glucose intolerance, and it may be the cause of pancreatic β-
cell dysfunction as well as hepatic pathologies. Interestingly, β-cells produce ROS in
response to increased glucose concentrations, but express relatively low levels of free-
radical-detoxifying enzymes. This combination might make β-cells particularly sensitive to
oxidative stress. It is also becoming more appreciated that hepatic steatosis and
steatohepatitis are closely related to the generation of ROS.
Nonetheless, drugs currently approved for use in clinical practice are highly effective for
the treatment of modifiable risk factors, and notably hypertension and dyslipidaemia. At
present, however, physicians tend to target cardiovascular risk factors in isolation, and as a
direct consequence of treating individual risk factors. In order to obtain maximal reductions
in cardiovascular disease events and to optimise clinical benefit, therapeutic strategies which
target multiple cardiovascular risk factors for the management of global cardiovascular risk
should be used. An integrated approach to the control of blood pressure and dyslipidaemia,
alongside interventions to improve insulin sensitivity, weight loss, and reduce smoking, may
therefore represent an effective therapeutic strategy for the attenuation of atherogenesis and
the prevention of cardiovascular disease in high-risk patients.
Future research should help further define the potential role of antioxidant
supplementation to diet and exercise. Indeed, for many years, interest has focused on
strategies that enhance removal of ROS using either antioxidants or drugs that enhance
endogenous antioxidant defense. Although those strategies have been effective in
experimental models, several trials have shown that they do not reduce cardiovascular events
and in some cases have actually worsened the outcome. An intriguing alternative approach to
reduce oxidative stress is inhibiting ROS production by blocking enzymes involved in its
synthesis. This hypothesis opens testing novel molecules that could interfere with the
production of free radicals and may result in reversing, or even retarding, diseases caused by
oxidative and inflammatory processes, such as the metabolic syndrome.
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Chapter VIII
Diabetes Mellitus
Rodrigo Castillo
University of Chile.
Abstract
Elevation of glycemia in diabetic patients may lead to the autooxidation of glucose,
glycation of proteins, and the activation of polyol metabolism. These changes accelerate
the generation of reactive oxygen species (ROS) and increase oxidative modification of
lipids, DNA, and proteins in various tissues. Thus, oxidative stress occurring in this
setting may play an important role in the development of the chronic complications of
diabetes, such as nephropathy, neuropathy, and lens cataracts. Langerhans islets are more
vulnerable to the occurrence of oxidative stress, since they contain low levels of
antioxidant enzyme activities compared to other tissues. High glucose concentrations are
known to give rise to a manifestation named glucose toxicity. Major manifestations of
glucose toxicity in the pancreatic β-cells are defective insulin gene expression,
diminished insulin content, and defective insulin secretion. The link between the clinical
complications and oxidative stress-related parameters has been established by the study
of advanced glycation end products (AGEs). Among the latter, heterocyclic amines,
acrylamide, and AGEs are well-known compounds hypothesized to cause harmful health
effects. First, AGEs act directly to induce cross-linking of long-lived proteins, such as
collagen, to promote vascular stiffness, thus altering the structure and function of
vasculature. Second, AGEs can interact with their receptors to induce intracellular
signaling leading to enhanced oxidative stress and elaboration of key proinflammatory
and prosclerotic cytokines. Over the last decade, a large number of preclinical studies
have been performed, targeting the formation and degradation of AGEs, as well as their
interaction with specific receptors. Translational research with humans is now under way
to ascertain whether this protection can be provided to patients experiencing inadequate
glycemic control.
1. Introduction
Diabetes mellitus is a major cause of morbidity in the western world, and of the most
common severe chronic illnesses, affecting over 230 million people worldwide with an
estimated global prevalence of 5.1% [1]. The associated complications, mainly coronary
disease, poses enormous public health and economic burdens, novel preventive and
regenerative therapies have emerged in the past decade with the aim to preserve pancreatic β-
cell mass and delay the onset of diabetes.
This illness is characterized by a chronic metabolic disorder caused by defects in both
insulin secretion and action. An elevated rate of basal hepatic glucose production in the
presence of hyperinsulinemia is the primary cause of fasting hyperglycemia. In this setting,
after a meal, impaired suppression of hepatic glucose production by insulin, and decreased
insulin-mediated glucose uptake by muscle, contribute almost equally to postprandial
hyperglycemia. The reason for the injury related to hyperglycemia is the formation of
advanced glycation end products (AGE) such as glycated proteins, glucose oxidation-derived
metabolites, and increased free fatty acids [2]. These effects result in oxidative stress in the
mitochondria, as well as in the activation of oxidative and inflammatory signaling pathways.
The latter is continued with damage to the insulin-producing cells, resulting in various
complications of diabetes. The majority of these complications, including retinopathy,
nephropathy, atherosclerosis and subsequent coronary artery disease, cerebral vascular
disease, and peripheral artery disease, can be related to microangiopathy or endothelial injury
[3].
It is clear that glucose toxicity can result in abnormal fatty acid metabolism, namely
autooxidation of glyceraldehyde, which generates hydrogen peroxide and ketoaldehydes.
This can lead to chronic oxidative damage. In the presence of reactive metals, hydrogen
peroxide could form the hydroxyl radical leading to toxicity. In addition, glucose toxicity
results in protein kinase C activation and its downstream effects on transforming growth
factor β, vascular endothelium growth factor, endothelin 1, and nuclear factor κB, among
others [4]. Therefore, the formation of glycation products and sorbitol is important in the
pathogenesis of the complications of diabetes. These same products are involved in the
process of aging, resulting in DNA strand breaks and production of reactive dicarbonyls [5].
The most concerning aspect of the disease is the functional impairment in β-cell caused by
oxidative stress, resulting in a loss of insulin gene expression in the islet cells. Furthermore,
the hyperlipidemia that often accompanies diabetes mellitus can result in fatty acid-mediated
oxidative damage and metabolic disturbances in the β-cells [6]. Although the use of
antioxidants has been proposed to prevent some of the complications of diabetes [7, 8], this
intervention may provide only a partial solution. Nonetheless, it is important to understand
the role of oxidative stress in the disease process of diabetes.
This chapter is aimed to show the state of the art about the insights that will foster further
investigation into the mechanisms by which oxidative stress influences the onset and
progression of diabetes. In addition, the rationale suggesting potential therapeutic and
preventative measures for this frequent condition, based on the use of antioxidants, will be
discussed.
Diabetes Mellitus 195
2. Pathophysiology of Diabetes Mellitus

2.1. Type 1 Diabetes
Type 1 diabetes is presented as a chronic immune-mediated disease with a subclinical

prodromal period characterized by selective loss of insulin-producing β-cells in the
pancreatic islets in genetically susceptible subjects. Autoreactive T cells, both CD4 and CD8,
have been implicated as active players in the β-cell destruction. The most important genes
contributing to disease susceptibility are located in the HLA class II locus on chromosome 6.
In addition, ten other genes or genetic regions have also been associated with this disease [9].
A series of autoantigens have been identified, including insulin, glutamic acid decarboxylase,
the protein tyrosine phosphatase-related islet antigen 2, and most recently the zinc transporter
Slc30A8 residing in the insulin secretory granule of the β-cell [10]. Nevertheless, only a
relatively small proportion, less than 10%, of individuals with HLA-conferred diabetes
susceptibility progress to clinical disease. This implies that additional factors are needed to
trigger and drive β-cell destruction in genetically predisposed subjects.
Clinical type 1 diabetes represents end-stage insulitis, a histologic change in the islets of
Langerhans characterized by edema and lymphocyte infiltration, and it has been estimated
that at the time of diagnosis only 10–20% of the β-cells are still functioning.
It is generally accepted that the destruction of the β-cells is mediated by cellular immune
responses. This is supported by the following facts: (i) T cells are present in insulitis; (ii)
disease progression is delayed by immunosuppressive drugs directed specifically against T
cells; and (iii) circulating autoreactive T cells can be detected in patients at clinical
presentation of type 1 diabetes [11]. It has remained open where potentially islet-autoreactive
T lymphocytes are activated initially. The activation of T cells requires the presentation of
autoantigenic determinants to self-reactive T cells by major histhocompatibility complex
(MHC). The MHC is a set of molecules displayed on cell surfaces that are responsible for
lymphocyte recognition and antigen presentation. Indeed, the MHC molecules control the
immune response through recognition of our own cells, and reacting against foreigner
antigens. However, MHC II molecules may not be expressed normally on β-cells in vivo. It
has been shown by in vitro experiments that the expression of MHC II molecules can be
induced on the surface of the β-cells by the combined effect of interferon-γ and TNF-α [12],
making possible the locally activation of naïve autoreactive T cells in the islets.
Alternatively, and even more likely, autoantigens are presented to naïve autoreactive T cells
by antigen-presenting cells (APC) which primarily express MHC II molecules. It has been
postulated that the initial encounter of APC and naïve self-reactive T cells takes places in the
pancreatic lymph nodes. The activated T cells are capable of invading the islets, where they
become reactivated by encountering cognate β-cell autoantigens and thereby initiating
insulitis. It seems that the autoimmune response is antigen-driven in type 1 diabetes. This is
supported by the fact that the strongest genetic susceptibility is associated with MHC class II
alleles.
Recent studies have shown that type 1 diabetes is a proinflammatory state [13, 14].
Schalkwijk et al. [15] reported elevated C-reactive protein (CRP) levels in Type 1 diabetes,
compared with controls patients without macrovascular disease. In the EURODIAB study
[16], levels of CRP, plasma interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were
found high in type 1 diabetic subjects and, in a cross-sectional study, correlated with the
severity of diabetic vascular disease. In this point, animal models of type 1 diabetes provide
strong support to the hypothesis that Toll-like receptor-induced innate signaling pathways are
involved in the proinflammatory process leading to autoimmune diabetes [17]. Studies
performed in peripheral blood cells and sera from patients with type 1 diabetes indicate that
aberrant innate functions might exist in such patients [18], but the relevance of these
alterations to the mechanism leading to type 1 diabetes is currently unclear.
These observations indicate that any intervention manipulating the autoimmune response
would have the highest likelihood of success, if initiated as soon as possible after the
appearance of the first signs of β-cell autoimmunity. There may be a critical window of 1
year for immune intervention after the emergence of the first signs of the disease process in
type 1 human diabetes.
2.2. Type 2 Diabetes
In patients with type 2 diabetes and established fasting hyperglycemia, the rate of basal
hepatic glucose production is excessive, despite plasma insulin concentration that are
increased two-fold to four-fold [19]. These findings provide unequivocal evidence for hepatic
resistance to insulin, and this evidence is substantiated by an impaired ability of insulin to
suppress hepatic glucose production. Accelerated gluconeogenesis is the major abnormality
responsible for the increased rate of basal hepatic glucose production. The increased rate of
basal hepatic glucose production is closely correlated with the increase in fasting plasma
glucose level [20].
Muscle tissue in patients with type 2 diabetes is resistant to insulin [21. Defects in insulin
receptor function, insulin receptor-signal transduction pathway, glucose transport and
phosphorylation, glycogen synthesis, and glucose oxidation contribute to muscle insulin
resistance. In response to a meal, the ability of endogenously secreted insulin to augment
muscle glucose uptake is markedly impaired [22]. Muscle insulin resistance and impaired
suppression of hepatic glucose production contribute almost equally to the excessive
postprandial increase in the plasma glucose level [23].
Under diabetic conditions, ROS [24] are produced in various tissues such as nerve cells
and vascular cells, and are involved in the development of diabetic complications [25, 26].
Recently, pancreatic β-cells emerged as a target of oxidative stress–mediated tissue damage
[27, 28]. Pancreatic β-cells express the high Km glucose transporter GLUT-2 abundantly and
thereby display highly efficient glucose uptake when exposed to high glucose concentration.
Also, because of the relatively low expression of antioxidant enzymes, such as catalase and
glutathione peroxidase [29], pancreatic β-cells may be rather sensitive to ROS attack when
they are exposed to oxidative stress. Thus, it is likely that oxidative stress plays a major role
in β-cell deterioration in type 2 diabetes.
There are several sources of ROS production in cells: the nonenzymatic glycosylation
reaction [30, 31], the electron transport chain in mitochondria [32], and the hexosamine
pathway [33]. Among these, the glycation reaction seems to have broad pathological
significance in diabetic complications, because under hyperglycemia the production of

various reducing sugars, such as glucose, glucose-6-phosphate, and fructose, increases
through glycolysis and the polyol pathway. All of these reducing sugars are known to
promote glycation reactions of various proteins. In diabetic animals, glycation is observed
extensively in various tissues and organs, and various kinds of glycated proteins. The latter
might include glycosylated hemoglobin, albumin, and lens crystalline produced in a
nonenzymatical manner through the Maillard reaction. During this reaction, which in turn
produces Schiff bases, Amadori product and AGE, ROS are also produced [34]. Also, the
electron transport chain in mitochondria is likely to be an important pathway to produce
ROS. Indeed, it was suggested that mitochondrial overwork, which causes induction of ROS,
is a potential mechanism leading to impaired first-phase of glucose stimulated insulin
secretion found in the early stage of diabetes. In addition, ROS are produced also through the
hexosamine pathway and that activation of the pathway leads to deterioration of β-cell
function by provoking oxidative stress [35] (figure 8-1).
Figure 8-1. Sources of cellular ROS in chronic hyperglycemia TCA, Tricarboxylic acid.
2.3. Advanced Glycation End Products and Oxidative Stress
The increasing body of evidence targeting accumulation of AGEs and/or their receptors
(RAGE) that mediate the biological actions could potentially confer benefits on diabetes-
related end-organ injury [36]. Advanced glycation end products are a complex group of
compounds formed via a non enzymatic reaction between reducing sugars and amine residues
on proteins, lipids, or nucleic acids. The major AGEs in vivo appear to be formed from highly
reactive intermediate carbonyl groups, known as α-dicarbonyls or oxoaldehydes, including 3-
deoxyglucosone, glyoxal, and methylglyoxal [37, 38]. Some of the best chemically
characterized AGEs in humans include pentosidine and N-carboxymethyl lysine. Apart from
endogenously formed products, AGEs can also originate from exogenous sources such as
tobacco smoke and diet [39, 40]. Food processing, especially prolonged heating, has an
accelerating effect in the generation of glycooxidation and lipid oxidation products, and a
significant proportion of ingested AGEs is absorbed with food. Tissue and circulating AGE
levels are higher in smokers and in patients on high AGE diets, with concurrent increases in
inflammatory markers [41]. Furthermore, there is evidence from animal studies that exposure
to high levels of exogenous AGEs contributing to renal and vascular complications [42].
Nevertheless, it remains to be determined the relative importance of these exogenous sources
of AGEs in the pathogenesis of diabetic complications.
Advanced glycation end products accumulate within the various organs that are damaged
in diabetes, what is accelerated by hyperglycemia. The intermolecular collagen cross-linking
caused by AGEs leads to diminished arterial and myocardial compliance and increased
vascular stiffness, phenomena that are considered to partly explain the increase in diastolic
dysfunction and systolic hypertension seen in diabetic subjects [43]. Advanced glycation end
products accumulate in most sites of diabetes complications, including the kidney, retina, and
atherosclerotic plaques [44, 45]. Advanced glycation end products have been measured and
reported to be linked to the sustained effects of prior glycemic control on the subsequent
development of vascular complications.
Oxidative stress may play an important role in the development of complications in
diabetes such as lens cataracts, nephropathy, and neuropathy. glycation reactions, especially
Maillard reactions, occurring in vivo as well as in vitro and are associated with the chronic
complications of diabetes mellitus, aging, and age-related diseases by increases in oxidative
chemical modification of lipids, DNA, and proteins [46]. In particular, long-lived proteins
such as lens crystallines, collagens, and hemoglobin may react with reducing sugars to form
AGEs. Recently, we found a novel type of AGE, named MRX, and we found that MRX is a
good biomarker for detecting oxidative stress produced during Maillard reaction [47]. Lipid
peroxidation reaction in hyperglycemia and hexanoyl modification formed by the reaction of
oxidized lipids and proteins might be important for oxidative stress development. Indeed, the
hexanoyl lysine (HEL) moiety in proteins, the earlier and stable markers for lipid
peroxidation–derived protein [48], has been identified in oxidized LDL and erythrocytes of
patients with type 1 diabetes [49]. On the other hand, macrophages and neutrophils play an
important role in oxidative stress during hyperglycemia, and it has been determined that
oxidatively modified tyrosines are a good biomarker for the occurrence of oxidative stress at
an early stage. In this point, the interaction of AGEs with RAGE in endothelial and
inflammatory cells induces intracellular generation of ROS, mainly mitochondrial electron
transport chain, NADPH oxidase, xanthine oxidase, and arachidonic acid metabolism [50].
Omori et al [51] describe the kinetics of p47phox activation comparing neutrophils from
diabetic and healthy subjects, suggesting that hyperglycemia increases AGE prime
neutrophils. In turn, this increases the oxidative stress through the induction of the p47phox
translocation to the cell membrane, and preassembly with p22phox by stimulating a RAGE-
ERK1/2 pathway. Several reports have linked ROS with intracellular and extracellular
inflammatory signals, which induce the signal transduction from RAGE to NF-κB [52, 53].
Each of these pathways is closely linked to AGE binding to RAGE, because blockade of the
receptor with either anti–RAGE IgG or excess soluble RAGE prevents their activation [54].
Therefore, the inhibition of AGE formation, blockade of the AGE-RAGE interaction, and
suppression of RAGE expression or its downstream pathways may be a novel therapeutic
strategy for the treatment of vascular complications in diabetes.
2.4. Role of Oxidative Stress in Diabetes
2.4.1. Clinical Evidence for Glucose Toxicity
The term glucotoxicity (or glucose toxicity) refers to a phenomenon responsible for the
pathologic changes on cellular function and structure in tissues throughout the body, due to
the adverse effects of elevated blood glucose levels.
The dimension of time is essential to the toxic effects of glucose; they are best
understood in the context of chronic, time-related elevations of blood glucose over many
months and years rather than days. Because blood glucose levels in nondiabetic people rise
postprandially, it seems unlikely that short periods of elevated blood glucose are significantly
toxic to cells. The concept of glucose toxicity at the level of the pancreatic islet β-cell is more
relevant to type 2 diabetes than to type 1 diabetes because, as mentioned above, patients with
type 2 diabetes typically retain functional β-cells for many years after the onset of the
disease. Even though optimal medical management for type 2 diabetes regulates fasting
glucose levels, most patients continue having abnormally elevated glucose levels
postprandially. Such patients are continually in double risk because they have a disease that
both decreases β-cell function and has an outcome (hyperglycemia) that continually damages
the remaining β-cells.
For many years it has been suggested that patients with diabetes undergo chronic
oxidative stress. This can be appreciated by measurements of biomarkers for oxidative stress
in patients with type 2 diabetes using various laboratory techniques, including high-
performance liquid chromatography, gas chromatography/mass spectrometry, and
immunostaining of pancreatic biopsies. Elevated oxidants and markers for oxidative tissue
damage, such as hydroperoxides, oxidation of DNA bases, 8-epi-prostaglandin F2α, and 8-
hydroxy-2’-deoxyguanosine, have been reported in patients with diabetes [55, 56, 57].
Moreover, therapy with sulfonylureas, which low blood glucose levels in diabetic patients,
has been associated with an increase in red blood cell glutathione [58], which enhances
intracellular antioxidant defense mechanisms. Coincidentally, several conventional
antihyperglycemic drugs can also show antioxidant activities [59, 60]. Activity of the rate-
limiting enzyme for glutathione synthesis, γ-glutamylcysteine ligase, was reported to increase
with improved glycemic control [61]. Using human isolated islets, it has been reported that
exposure to high glucose concentrations increases intra islet levels of peroxide [62]. This
increase was blocked by mannoheptulose, which prevents glucose metabolism by the β-cell.
Recent studies suggested that subclinical cardiovascular disease, including complications
in diabetes, is associated with oxidative damage and precedes future cardiovascular disease
[63, 64]. Blood levels of glucose, D-dimer, glutathione and total cholesterol contribute
significantly to a diabetic oxidative damage, constituting a panel of biomarkers that may be

helpful in evidence-based pharmacological intervention with anti-aggregation and/or
antioxidant agents against cardiovascular disease in diabetes. Actually, some clinical trials
have suggested that non-insulin-dependent diabetes mellitus patients present increased lipid
peroxidation, changes in antioxidative defense (decreased CuZnSOD activity in
erythrocytes), and alterations in erythrocyte morphology compared with insulin-dependent
diabetic patients [65, 66]. Diabetic patients with microvascular complications show,
increased oxidative stress related-parameters such as malondialdehyde levels, together with
decreased glutathione peroxidase and superoxide dismutase activities. These factors may
contribute to the occurrence of micro vascular complications and mortality in non-insulin-
dependent diabetes mellitus patients.
2.4.2. Experimental Studies in Animal Models of Diabetes
Observations using clinical material obtained from humans that suggested a link between
glucose toxicity and oxidative stress have stimulated a great deal of research in animal
models of diabetes. The advantages of studying the streptozotocin-treated diabetic mouse and
the manipulation of apoE as the preferred model to the streptozotocin-induced diabetic rat
was first reported by Yamamoto et al. [67] . Nonetheless, there is a more extensive literature
concerning the development of vascular disease in the streptozotocin-diabetic rat. For
instance, in the aorta from this model it was reported a triphasic change in endothelial
function enhanced at 1-week post-streptozotocin, unaltered at 1–2 weeks, and impaired at
8 weeks [68]. Kobayashi and Kamata [69], reported a decrease in aortic endothelial function
9 weeks after streptozotocin treatment and this was accompanied by an increase in oxidative
stress, but no change in mRNA eNOS expression. In contrast, in rats treated for 2 weeks with
streptozotocin, aortic eNOS mRNA and NADPH oxidase were increased and endothelial
dysfunction was associated with a reduction in the bioavailability of NO as measured by
electron spin resonance [70]. In the current study [71], it was also shown that biomarkers of
oxidative stress were elevated in the aorta from the streptozotocin-treated groups at 16 weeks.
Similar changes occur in the conduit vessels, what has also been reported for the small
mesenteric vessels from the apoE−/− streptozotocin diabetic mouse [72]. Recent studies
followed in ApoE-deficient (ApoE-/-) and glutathione peroxidase (GSH-Px) double-knockout
(ApoE-/- GSH-Px-/-) mice diabetic with streptozotocin, showed that lack of functional GSH-
Px accelerates diabetes-associated atherosclerosis via up-regulation of proinflammatory and
profibrotic pathways. These data establishe GSH-Px as an important antiatherogenic
therapeutic target in patients with or at risk of diabetic macrovascular disease [73].
A model of type 2 diabetes are Zucker diabetic fatty (ZDF) rats, showing an inadequate
control to maintain normoglycemia. These animals are leptin receptor deficient and are
characterized by postweaning development of marked obesity, hyperglycemia, and
hypertriglyceridemia [74]. The chronic exposure of this animal type to high glucose levels
over many months caused diminished insulin gene expression, insulin content, and glucose-
induced insulin secretion [75, 76]. These abnormalities were associated with decreased levels
of two critical insulin promoter transcription factors [77, 78]. The ZDF rats became more
hyperglycemic and they developed defects in these factors, as well as decreased insulin
mRNA levels, insulin content, and glucose-induced insulin secretion in isolated islets [79].
When the animals were given troglitizone, a glucose- and triglyceride-lowering drug that is
also an antioxidant, during the first 6–16 weeks of age, the development of hyperglycemia
was substantially prevented. There exists in vivo evidence that the relentless progression of
hyperglycemia in the animal had secondary glucotoxic effects on the remaining β-cells [80].
To ascertain whether these glucotoxic effects were due to oxidative stress, the animals were
treated with two antioxidants, N-acetylcysteine (NAC) or aminoguanine (AG). These drugs
were given to ZDF rats from the 6th to the 12th week of age. Both drugs prevented the rise in
blood oxidative stress markers, including malondialdehyde and 4-hydroxy-2-nonenal. The
drugs ameliorated the development of hyperglycemia, glucose intolerance, defective insulin
secretion, decrements in β-cell insulin content, and insulin gene expression [81]. These
studies strengthened the hypothesis that chronic oxidative stress is a major mechanism for the
clinical phenomenon termed glucotoxicity of the pancreatic β-cell. Similar observations were
made using another type 2 diabetics models, the db/db mouse, where antioxidant treatment
NAC, vitamins C plus E, or both for 6 weeks revealed that the β-cell mass was significantly
larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a
possible cause, the antioxidant treatment suppressed apoptosis in β-cell without changing its
rate of proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis
induced by oxidative stress causes reduction of β-cell mass [82].
Recent studies confirm the importance of oxidative stress in diabetic vascular
dysfunction [83, 84, 85]. These data reveal a different origin of ROS under basal conditions
and during stimulation with contracting agents. The increased basal superoxide radical of the
smooth muscle of diabetic rats appears to be derived from NADPH oxidase. The free radicals
produced by NADPH oxidase cause the up-regulation of both constitutive and inducible
COXs in the vascular smooth muscle cells [86]. These enzymes generate oxygen-derived free
radicals and/or metabolites of arachidonic acid, which in turn cause depression of the
contractile process but hyper-responsiveness of the cell membrane receptors of the vascular
smooth muscle cells [87]. Therefore, the mechanisms determining an endothelial dysfunction
are implicated in macro- and microvascular complications in diabetic patients, derived in part
from an imbalance of vasoconstrictor prostanoids.
3. Prevention with Antioxidants

3.1. Clinical Trials
In the last time, much attention has been focused on attenuation of oxidative stress by
dietary antioxidants to assist in the prevention of diabetes mellitus. An increase in oxidizing
response above a certain threshold produces, in the absence of a concomitant rise in
antioxidant/reducing response, oxidative stress that is associated with complications in
diabetes. In relation to this concept, the absence of complications in type 1 diabetes patients
up to 5 years after onset of the disease may be associated with the oxidizing and reducing
balance which needs to be maintained in order to prevent or delay the onset of oxidative
stress [88]. The effective diabetic control involves evaluation of the oxidizing/antioxidant
balance besides glycemic control. A simple technique involving reduction of 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in
order to determine quantitatively the antioxidant status of plasma [89]. Human plasma
contains several antioxidant effectors including ascorbate, urate, α-tocopherol, albumin and
protein sulphydryl groups [90]. Previously it has been found that oxidized human serum
albumin, in contrast to normal albumin, decreases antioxidant activity and is able to trigger
the oxidative burst of human neutrophils [91]. In this point, various studies show that plasma
from healthy subjects exhibited a higher antioxidant status compared with plasma from
diabetic patients, indicating that the latter may contain high levels of protein and oxidized
lipids, possibly occasioned by oxidative stress present in diabetes [92, 93]. In several of its
roles, selenium functions as a dietary antioxidant and thus has been studied for its possible
role in chronic diseases [94]. Indeed, it could play an important role in diabetic
complications, performing as a cofactor for antioxidant GSH-Px activity [95]. Kornhauser et
al., [96] found that in type 2 diabetes mellitus patients with microalbuminuria showed a
positive correlation with glucose, and a negative correlation with serum selenium and
glutathione peroxidase, suggesting that this may be implicated in the diabetic nephropathy. In
other recent study that supports the importance of impaired antioxidant status for the
development of insulin resistance and type 2 diabetes [97], serum concentrations and dietary
intakes of beta-carotene and alpha-tocopherol, independently, predicted insulin resistance and
type 2 diabetes incidence during 27 years of follow-up in a community-based study of men.
On the other hand, dietary supplementation with micronutrients may be a complement to
classical therapies for preventing and treating diabetic complications. Supplementation is
expected to be more effective when a deficiency in these micronutrients exists. Nevertheless,
many clinical studies have reported beneficial effects in individuals without deficiencies,
although several of these studies were short-term and had small sample sizes [98]. Recently,
it was found that strong antioxidative activity in curcuminoids, the main yellow pigments in
Curcuma longa (turmeric), have been used widely and for a long time in the treatment of
sprain and inflammation in indigenous medicine. Curcumin (U1) is the main component of
turmeric, and two minor components are also present as the curcuminoids. Curcuminoids
were reported to possess antioxidant activity [99, 100] and inhibit the microsome-mediated
mutagenicity of benzo(a)pyrene and 7,12-dimethyl-benz(a)anthracene, and it was also
reported that U1 acts as a strong inhibitor of tumor promotion. This effect can be explained as
roughly parallel to the relative antioxidant activity [101, 102].
In the case of the antioxidant supplement, various studies support the preventive effects
on metabolic and clinical complications of diabetes, mainly in type 1 diabetes [103, 104,
105]. This clinical trial identifies positive effects of these nutrients on various outcome
measures relating insulin resistance and cardiovascular factors [106]. However, the potential
role of these agents, such as alpha-lipoic acid, chromium, folic acid, isoflavones, magnesium,
pycnogenol, selenium, vitamin C, vitamin E, and zinc in the treatment of type 2 diabetes are
not well characterized. This can be mainly due to the lack of rigor in applying the inclusion
criteria and in excluding those concomitant pathologies that would be interfering with
oxidative stress-related parameters. In the case of vitamin E, it is reported to reduce oxidative
stress at levels of 200 mg day or more, especially in patients with renal disease [107] and
coronary artery disease [108]. Accordingly, Engelen et al., [109] developed a clinical trial
where the combination of micronized fenofibrate 200 mg/day and vitamin E 400 IU/day
tended to increase the resistance of non-HDL lipoproteins to copper-mediated oxidation.
Vitamin E alone administration, decreased the oxidation of non-HDL lipoproteins as shown
by a reduction of thiobarbituric acid reactant substances formation. This protective effect of
vitamin E tended to be enhanced by micronized fenofibrate.
The supplement of vitamin C also showed an attenuation of oxidative damage in diabetes
mellitus patients [110, 111]. It is known that vitamin C at pharmacologic doses decreases
sorbitol accumulation, product that contributes to the progression of chronic diabetic
complications. One the first trials reported of vitamin C supplements intake of 100 or 600 mg
daily for 58 days in young adults with insulin-dependent diabetes mellitus and nondiabetic
adults [112]. This supplement diminished significantly the sorbitol accumulation in the
erythrocytes of diabetics, displaying low toxicity. This accounted for the efficacy of vitamin
C over pharmaceutical aldose reductase inhibitors. Other study, a placebo-controlled trial,
tested the hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-
induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus [113]. Venous
blood samples were obtained at rest, after a maximal exercise challenge and before and 2h
after oral ingestion of 1g ascorbate or placebo. Diabetic patients exhibited an elevated
concentration of lipid peroxidation associated with a depression of retinol and lycopene.
Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in
both groups and attenuated the exercise-induced oxidative stress response compared with
healthy individuals.
Finally, flavonoids are described to exert a large array of biological activities, which are
mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability.
Most of these evidences have been obtained by in vitro studies on individual compounds and
at doses largely exceeding dietary doses [114]. Various authors have found that flavonoid
consumption has a positive effect on insulin resistance and cardiovascular outcome measures,
but only when combined with soy proteins [115, 116]. Indeed, antioxidant effects have been
measured on serum and macrophages, which could contribute to attenuation of
atherosclerosis development in non-insulin dependent diabetes mellitus patients [117].
3.2. Experimental Studies of Antioxidants in Diabetes
There exists evidence that the over expression of antioxidants such as Cu/Zn superoxide
dismutase [118], catalase [119], thioredoxin [120], and metallothionein [121] targeted at β-
cells counteracts the development of type 1 diabetes. Since the pancreas has been shown to
have low levels of the antioxidant enzymes superoxide dismutase and catalase [122], these
findings suggest that oxidative stress plays a key role in the pathogenesis of diabetes [123].
Indeed, nitric oxide (NO) and ROS induced by inflammatory cytokines such as interleukin-
1β, TNF-α, and interferon-γ are considered to be crucial mediators of β-cell death [124, 125].
This is supported by several studies showing that pretreatment with antioxidants before the
injection of streptozotocin attenuates the development of hyperglycemia and insulitis in the
multiple low-dose streptozotocin models [126, 127, 128]. In the case of vitamin E, chronic
effect of supplementation in addition to insulin can have additive protective effects against
deterioration of renal function in rat model of diabetic nephropathy [129]. In addition, kidney
tissue levels of malondialdehyde and inducible NO synthase (iNOS), and reduced serum
glutathione peroxidase improved towards control levels with vitamin E administration. These
effects could be associated with protection of vessel structure and function in the damage of
microvascular complications [130]. Similar experimental models in STZ-treated rats have
been used to assess the effect of short-term antioxidant therapy based on oral vitamins C and
E administration, with similar antioxidant response [131, 132]. In the case of vitamin C, the
use for attenuating oxidative injury might become a useful adjunct to prevent albuminuria
and renal sclerosis in rat diabetic nephropathy [133]. It was demonstrated that ascorbic acid
directly modulates contractile responses of diabetic rat aortas, likely through mechanisms in
part dependent of preservation of endothelium-derived NO [134]. In this point, vitamin C
may act as a direct ROS scavenger in endothelium; resulting in increased availability of the
eNOS cofactor tetrahydrobiopterin, thereby enhancing eNOS activity [135].
With respect to ROS sources, Yamamoto et al., [67] described for the fisrt time increased
oxidative stress linked to altered expression of NADPH oxidase and eNOS at an early stage
of the induction of diabetes with streptozotocin in the apoE−/− mouse. In addition,
expression of both mitochondrial SOD initially (at 4-week post-streptozotocin) and then the
cytosolic SOD (at 8-week post-streptozotocin) increased in the mesenteric arteries perhaps
reflecting a compensatory response to the increase in oxidative stress. However, SOD1 and
SOD2 levels were not different when the streptozotocin-treated group were compared to the
control group at 16 weeks again, suggesting that the reported changes in SOD are linked to
the initiation of type 1 diabetes following treatment with streptozotocin and the onset of
diabetes-related vascular dysfunction. Nevertheless, expression of eNOS mRNA and protein
remained significantly elevated at 16 weeks. An enhanced expression of eNOS may
contribute to vascular dysfunction and accelerated atherogenesis as reported in the apoE−/−
mouse [136].
On the other hand, Ding et al. [137] have reported that in the small mesenteric arteries
from the streptozotocin-diabetic apoE−/− mouse the component of acetylcholine-mediated
relaxation attributed to NO from streptozotocin-treated diabetic apoE−/− mice was enhanced
in comparison to the non-diabetic control apoE−/− mouse. This suggested that the ability of
eNOS to generate NO is not compromised in this type 1 diabetic model, possibly reflecting
an enhanced synthesis of tetrahydrobiopterin that has also been reported in the vasculature of
the apoE−/− mouse [138]. The eNOS expression and endothelial function in the
streptozotocin-type 1 apoE−/− mouse differs from that reported for the db/db mouse model of
type 2 diabetes. In small mesenteric arteries from these mice there was, a complete absence of
the contribution from NO to endothelium-dependent relaxation, elevated oxidative stress, and
no change in eNOS mRNA or protein expression [139]. In the case of the NADPH oxidase,
an increase in gp91phox expression has also been linked to neovascularization following
tissue ischemia [140]. In the aorta from the streptozotocin-rat model of type 1 diabetes [141],
reported that 8 weeks after treatment with streptozotocin Nox1 protein expression was
doubled, but the expression of nox4 was unchanged and acetylcholine-mediated
vasodilatation and NO generation was decreased despite an increase in eNOS protein. In this
view, there are conflicting reports on the effects of NOS inhibitors on type 1 diabetes in
animal models [142, 143]. Papaccio et al., [144] reported that multiple low-dose
streptozotocin treatment did not stimulate NO production at the islet level, although
superoxide dismutase activity was decreased. These data suggest that NO itself may have no
major role in multiple low-dose streptozotocin-induced diabetes. However, Lenzen [145] and
Mabley et al. [146] have demonstrated that peroxynitrite plays a role in the pathogenesis of
islet cell dysfunction and the destruction associated with the multiple low-dose streptozotocin
model of type 1 diabetes.
Recently, it has been demonstrated that edaravone, a potent scavenger of hydroxyl and
peroxyl radicals, protects against NO-induced cytotoxicity in cultured astrocytes, although
edaravone does not affect the release of NO or its metabolism [147]. In addition, edaravone
protects against S-nitroso-N-acetyl-DL-penicillamine, a NO donor that induces cytotoxicity
in the rat pancreatic β-cell line INS-1. These findings suggest that NO plays a role in the
pathogenesis of diabetes and that edaravone may attenuate multiple low-dose streptozotocin-
induced diabetes by inhibiting a NO-mediated mechanism.
Considering the NADPH oxidase as a potential pharmacological target in experimental
diabetic model, some evidences support that the experimental inhibition of this enzyme is
associated to a potential benefit in diminishing the complications associated with the
microvascular [148] and macrovascular damage [149]. In this point, some drugs with
antioxidant properties have been used as coadjutant therapy in vascular disorders associated
with diabetes progression such as β-blockers [150], ACE inhibitors [151], and statins. These
drugs normalizes endothelial function and reduce oxidative stress in diabetes by inhibiting
the activation of the vascular NADPH oxidase, thereby preventing eNOS uncoupling due to
an up-regulation of the key enzyme of tetrahydrobiopterin synthesis [152]. Other of their
properties involves an improvement of the endothelium-dependent vasodilatation [153],
lipid-independent antioxidant [154] and anti-inflammatory effects [155]. The proposed
mechanism for these pleiotropic effects, even at low-dose, involves the inhibition of
inflammatory intracellular pathway via ERK1/2/NF-kappaB-pathway [156], and inactivation
sources of ROS such as NADPH oxidase [157]. Both of these effects have been demonstrated
sufficiently to improve endothelial function under experimental diabetic conditions.
3.3. Role of Antioxidants in Reducing the Advanced Glycation End Product

Formation
Aminoguanidine was one of the first inhibitors of AGE formation studied [158, 159], and
is thought to act as a nucleophilic trap for carbonyl intermediates. In animal studies,
aminoguanidine has prevented a wide range of diabetic vascular complications [160, 161,
162]. In clinical trials, it has been found a reduction in AGE-hemoglobin independent of
HbA1c lowering [163, 164].
Placebo-controlled clinical trials have been assayed with aminoguanidine in types 1 and
2 diabetes examining renal outcomes [165, 166]. Reductions in proteinuria and decreased
progression of retinopathy were observed, although the study did not demonstrate a
statistically significant beneficial effect on the progression of nephropathy. Further clinical
evaluation of this agent has been limited due to long-term toxicity. Indeed, some patients
have developed antimyeloperoxidase and antineutrophil antibodies [167], and also

glomerulonephritis [166].
Pyridoxamine is a derivative of vitamin B6. It prevents the degradation of protein-
amadori intermediates to protein-AGE products. In murine models it has reduced
hyperlipidemia and prevented AGE formation [168, 169]. Thomas et al. [170] showed that
pyridoxamine antagonizes angiotensin II-induced elevation in serum and renal AGEs,
prevents renal hypertrophy, and decreases salt retention in experimental models.
Pyridoxamine also prevents diabetes-induced retinal vascular lesions. Some preliminary
clinical trials with this agent have been performed [171].
Benfotiamine, a lipid-soluble thiamine (vitamin B1) derivative, prevents activation of
three major pathways of hyperglycaemic damage (hexosamine pathway, intracellular AGE
formation, and the diacylglycerol-protein kinase C pathway) by increasing the activity of
transketolase, the rate-limiting enzyme of the nonoxidative branch of the pentose phosphate
pathway [172]. In animal studies, high-dose thiamine and benfotiamine therapy increased
transketolase expression in renal glomeruli, and inhibited the development of
microalbuminuria and diabetes-induced hyperfiltration [173]. Benfotiamine has improved
nerve conduction velocity in the peroneal nerve [174] in diabetic patients. Accordingly, a
short 3-week clinical study showed alleviation of painful neuropathy [175]. Nevertheless,
long-term human data are still lacking. Recent studies also show that benfotiamine prevents
macrovascular and microvascular endothelial dysfunction and oxidative stress after a high-
AGE meal [176].
Methylene bis 4,4-(2 chlorophenylureido phenoxyisobutyric acid) (LR-90) has been
investigated in a number of animal studies by Figarola et al., [177] The compound LR-90
inhibited albuminuria, reduced serum creatinine concentration, and circulating AGE levels in
diabetic rats without any effect on glycemic control. Also, LR-90 prevented
glomerulosclerosis and collagen deposition in association with reduced glomerular AGE
accumulation. The effect of LR-90 is also currently being tested on macrovascular
complications in a range of animal studies. Interestingly, LR-90 has recently inhibited the
expression of proinflammatory molecules stimulated by RAGE pathway in human
monocytes[178], suggesting that this agent has novel antiinflammatory properties with
protective effects against diabetic vascular complications. There are plans for this agent to be
further evaluated in the clinical context.
Another therapeutic approach that is being considered involves soluble RAGE (sRAGE)
that blocks AGEs from binding to RAGE. It remains to be fully elucidated if sRAGE acts
whether as an antagonist inhibiting RAGE dependent signaling pathways or through binding
various RAGE ligands such as AGEs, thus preventing these putative proinflammatory
molecules from acting on other receptors (e.g scavenger receptors) to promote end-organ
injury [179]. Studies with RAGE knockout mice that do not express sRAGE or full-length
RAGE suggest that the key mechanism of sRAGE action is via inhibition of RAGE
dependent phenomena. In the next few years, either sRAGE or possibly a nonpeptide RAGE
antagonist will be examined clinically, although it is possible that nondiabetic diseases may
be the focus of the RAGE clinical development program.
The pathophysiological crosstalk between the AGE-RAGE system and angiotensin II has
also been associated with diabetic microangiopathy in various diabetics’ models [180].
Angiotensin convertase enzyme inhibitors as well as angiotensin II antagonists appear to

decrease the formation of AGE, as assessed in a series of in vitro studies as well as in animal
models of diabetes [181, 182]. Furthermore, ACE inhibitors, based on in vitro, preclinical,
and clinical studies, appear to promote surface receptor AGE expression, thus providing an
additional mechanism to inhibit AGE induced organ injury [183]. The explanation of these
effects may be supported by a down-regulation of RAGE mRNA levels in a dose-dependent
manner, demonstrated in some in vitro models. Indeed, ACE inhibitors may act as an anti-
inflammatory agent against AGE by suppressing RAGE expression via PPAR-gamma
activation in the liver and may play a protective role in vascular injury in diabetes [184]
(table 8-1).
Table 8-1. The antioxidant interventions targeting

the advanced glycation end products pathway
Compound Mechanism Effects in humans

↓nephropathy, retinopathy
↓ Angiotensin II
ACE inhibitors Yoshida et al., 2006
NADPH inactivation
Coughlan et al., 2007
↓nephropathy, retinopathy
Aminoguanidine Inhibition AGE formation
Bolton et al., 2004
Inhibition AGE formation ↓retinopathy
Pyridoxamine
↓ Cholesterol Degenhardt et al., 2002
↓ Oxidative stress ↓nephropathy
LR-90
ECM fibrosis Figarola et al., 2007
Anti-RAGE RAGE blockade ↓Nephropathy
sRAGE Kunt et al., 2004
Thallas-Bonkeet al., 2004
ACE: Angiotensin convertase-enzyme; NADPH: nicotinamide adenine dinucleotide phosphate-oxidase;

RAGE: Receptor advanced glycosilation end products; LR-90: Methylene bis 4,4--(-2
chlorophenylureido phenoxyisobutyric acid).

Diabetes mellitus is a chronic disease caused by an impairment of β-cells function and
survival, giving rise to the development of hyperglycemia. Consequently, the exposure to
high blood glucose levels over many years leads to adverse structural and functional changes
in tissues, a phenomenon termed glucose toxicity. The finding that diabetic patients have
increased clinical biomarkers for oxidative stress and tissue damage could suggest a possible
linkage between glucose toxicity and oxidative stress, what has been supported by
experimental studies.
Under diabetic conditions, ROS are produced through several processes such as the
nonenzymatic glycosylation reaction, alterations in the electron transport chain in
mitochondria, and the hexosamine pathway. Oxidative stress and consequent activation of the
inflammatory and apoptotic pathways are likely involved in progression of pancreatic β-cell
dysfunction found in diabetes type 1. Antioxidant treatment can protect β-cells against
glucose toxicity and thus exert beneficial effects reported from clinical and biochemical
assays. In the case of type 2 diabetes, ROS are accepted as major factors in the onset and
development of their macrovascular complications. The underlying mechanism of this
deleterious effect involves an inactivation of the signaling pathway between the insulin
receptor and the glucose transporter system leading to the onset of insulin resistance in this
setting.
When comparing metabolic pathways of ROS production in the β-cell, it has been
suggested that secretagogues causing increased insulin secretion can also lead to increased
ROS production, via mitochondrial and NADPH oxidase mechanisms. The development of
oxidative stress could be due not solely to the relatively of NADPH oxidase activity, but also
to the low activity of antioxidant enzymes found in the islet β-cell. Therefore, together with
scavenging ROS with antioxidant substances, it is plausible the design of future therapies
based on targeting NADPH oxidase in the islet, thereby causing a diminution of ROS
production. These interventions may be beneficial for maintaining β-cell integrity in the
difficult environment of nutrient oversupply and immune challenge.
The biochemical process of advanced glycation appears to be enhanced in the diabetic
patients as a result of not only hyperglycemia but also due to other stimuli such as oxidative
stress and increased free fatty acids. These might generate a heterogeneous group of chemical
moieties that appear to induce directly and indirectly the development and progression of
vascular complications. A range of pharmacological strategies, predominately being
examined in preclinical contexts, appears to show great promise in reducing AGE induced
injury by interfering with either the accumulation of AGE ligands or interrupting the AGE-
RAGE interaction. It is anticipated that over the next few years, findings from clinical studies
will assist clinicians in determining the relevance of targeting advanced glycation as an
approach to reducing diabetic complications.
Finally, a sufficient supply of dietary antioxidants may prevent or delay diabetes
complications, including renal and neural dysfunction by providing protection against
oxidative stress, although the detailed examination of protective mechanisms is uncertain.
Therefore, it is extremely important that future clinical trials exert strict clinical criteria of
selection, sources and doses of antioxidants.
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Chapter IX
Nonalcoholic Steatohepatitis
Juan Gormaz1 and Ramón Rodrigo2

1
University of Chile
2
Abstract
Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases
characterized mainly by macrovesicular steatosis that occurs in the absence of alcoholic
consumption. NAFLD is closely associated with comorbid conditions, such as obesity,
dyslipidemia, and insulin resistance. It is a medical condition in which the liver is
invaded with fat and excessive amounts of lipids are present within hepatocytes. There is
increasing evidence to consider that fatty liver is the hepatic manifestation of the
metabolic syndrome, a growing problem in the modern western world. NAFLD might
worsen into a more serious condition, known as nonalcoholic steatohepatitis (NASH), in
which fat accumulation is accompanied by an inflammatory process in the liver.
The clinical relevance of these conditions is given by the high prevalence of NAFLD
in the general population and to the possible evolution of NASH towards end-stage liver
disease, including hepatocellular carcinoma, as well as the need for liver transplantation.
The molecular mechanism whereby NASH might eventually lead to fibrosis, and severe
cirrhosis in some patients, is a process associated with increased production and release
of inflammatory mediators, such as nitric oxide (NO), cytokines, and reactive oxygen
species (ROS) by the cells. Oxidative stress caused by increased ROS plays an important
role in the pathogenesis of NASH. These reactive species would derive from
mitochondria, cytochrome P-450 2E1, peroxisome, and iron overload in the liver with
steatosis. Excessive ROS is considered to cause simple steatosis to progress to NASH.
Regardless the origin of hepatic fat, it could produce a rise of hepatic free fatty acids. The
latter, particularly the polyunsaturated ones, are closely linked to ROS generation by
different pathways, including increased oxidation in different cellular organelles,
disruption of mitochondria and endoplasmic reticulum, microsomal cytochrome P450
224 Juan Gormaz and Ramón Rodrigo
activation and ceramide formation. In addition, increased ROS production could derivate
not only in hepatocyte cell death but also in the activation of liver resident cells, such as
Kupffer, stellate and endothelial cells. This might enhance the original oxidative stress,
inflammatory response and subsequent immune infiltration thus aggravating NASH.
Up to date no absolute effective medical treatment is available for NASH patients.
Therapy is predominantly aimed at controlling the comorbid conditions, such as obesity,
insulin resistance, and dyslipidemia. However the major role of oxidative stress in the
pathogenesis of NASH suggests that the antioxidant treatment would be an effective
therapy. Hence, both several substances with different antioxidants mechanism and
effects related with the redox balance have been assayed in small clinical trials. These
agents have shown the ability of improve the outcome of patients, thus opening the door
to new strategies to manage or treat this disease. This chapter provides the clinical and
experimental evidence to support the role of oxidative stress in the pathophysiology of
NAFLD and NASH, as well as the molecular bases promoting the development of
mechanism-based therapeutic interventions, mainly clinical trials aimed to target specific
pathways involved in the pathogenesis of NASH.
1. Introduction
The liver plays an important role in lipid metabolism, including cholesterol, fatty acid
and triglycerides synthesis, together with lipoproteins management [1]. Numerous factors,
such as alcohol abuse, obesity, metabolic syndrome, hepatotoxic drugs, between others, can
lead to metabolic disorders damaging liver structure and function. Nonalcoholic fatty liver
disease (NAFLD) represents a spectrum of histologic findings ranging from simple steatosis
to nonalcoholic steatohepatitis (NASH) and cirrhosis [2]. Nonalcoholic steatohepatitis is
characterized histologically by the presence of hepatocyte ballooning, Mallory’s hyaline,
inflammation, and fibrosis, which occur in the absence of excessive alcohol consumption
[3].The diagnosis of hepatic steatosis and steatohepatitis or nonalcoholic steatohepatitis
(NASH) is not yet possible without liver biopsy [4]. Nonalcoholic steatohepatitis is one of the
most prevalent forms of chronic liver disease and it is estimated to affect nearly 4% of the
United States population [5], approximately 1% of worldwide population [6]. Moreover, up
to 20% of affected patients will develop cirrhosis [7].
In addition, near 70% of the cases of cryptogenic cirrhosis present characteristics of
NASH [8]. This condition may represent the final step of NASH, which constitutes a lost of
the typical necroinflammatory and steatotic characteristics, in up to 80% of patients [9, 10].
The 5- and 10-year survival in NASH has been calculated at 67% and 59%, respectively,
although death often may be from comorbid pathologies.
Currently it is accepted that NASH may lead to serious liver failure or hepatocellular
carcinoma, [11, 12]. Within the North American population, the prevalence of NAFLD is
nearly 20% [13, 14]; Japan and Italy have similar statistics [15, 16], becoming the most
common cause of liver disease in Western countries [17].
Recent researches have demonstrated that NAFLD is closely related with obesity,
dyslipidemia, insulin resistance, and has been described as the liver component of the
metabolic syndrome [18]. For example, in obese population with a body mass index greater
than 30 Kg/m2, the prevalence of NAFLD increases extremely, oscillating between 74% and
Nonalcoholic Steatohepatitis 225
90% [19, 20]. Epidemiological data also suggests a strong relation between insulin resistance
and NAFLD [21].
When considering the role of liver in lipid metabolism, it is not surprising that liver cells
are capable of storing lipids as energy reserves. However, hepatic lipid content is usually
small (less than 5% of fat by wet weight); when the liver lipid reserves exceeds this
percentage, it is assumed a liver steatosis condition [1]. In this regard, primary NAFLD is
now considered as the hepatic manifestation of the metabolic syndrome [22-24], a cluster of
disorders including central obesity, insulin resistance with or without type 2 diabetes
mellitus, dyslipidemia and hypertension (see Chapter 7).
The pathogenesis of NAFLD is attributed to a multi-hit process involving insulin
resistance, oxidative stress, apoptotic pathways, and adipokines. In these days, there is no
established treatment for this pathology except for weight loss and treating each component
of the metabolic syndrome. Nevertheless, a large number of agents are being considered in
clinical trials performed in patients with NASH.
Hepatic steatosis should not be considered a benign feature, but rather a silent killer. This
is supported by several studies demonstrating that this pathology has the potential to progress
to NASH [25]. The pathophysiology of NAFLD is not fully elucidated, but the initial state
involves accumulation of excess fat within the hepatocyte due to metabolic conditions
leading to hepatic steatosis. The next stage involves the development of oxidative stress that
causes structural damage to biomolecules, and activates inflammatory chain resulting in
NASH [26]. Fat droplets accumulate in the cytoplasm of liver cells and their excessive
accumulation may lead to cell damage or cell death, a phenomenon known as lipotoxicity
[27, 28]. Lipotoxicity is associated with oxidative stress and the consequent activation of
proinflammatory pathways, leading to immune infiltration, damage amplification, cell death
and fibrosis [17].
2. Pathophysiology of Nonalcoholic
Fatty Liver Disease and Steatohepatitis
The pathogenesis of NASH is multifactorial. Among others, the causes of hepatocellular
injury in steatotic liver include the following: oxidative stress, lipid peroxidation,
mitochondrial dysfunction, and dysregulation of immune system. Because not all steatotic
livers progress to NASH, some other environmental factors, or a combination of genetic
factors, are thought to be required for progression to NASH and fibrosis.
A number of factors aim to the multifactorial nature for the mechanism of NAFLD,
including derangements in metabolic parameters, endotoxin-induced cytokine release and
oxidative stress. The metabolic parameters include mitochondrial dysfunction, amino acid
imbalance, hyperglycemia and imbalances in antiketogenic hormones in portal blood.
Nonalcoholic fatty liver disease can progress to end stage liver disease; however, the cause of
the NAFLD progression remains elusive. There is a hypothesis of “two hits” [29] postulating
that liver fat accumulation per se is not harmful. The “first hit” is the excessive hepatic fat
deposition. The “second hit”, for presumed transition of simple steatosis to NASH. Despite
the involvement of many other proinflammatory mediators, lipid peroxidation is one
hypothesis as the “second hit” to explain the development of the disorder in humans.
However, the author of this hypothesis, in the light of more recent studies, proposed a
modification of the two-hit model that places more emphasis on the role FFAs [30].
Accordingly, data from animal models suggest that oxidative stress contributes to
steatohepatitis. Indeed, an increase of lipid peroxidation has been documented in humans as
an important mechanism of progression of NAFLD [31]. The predominant metabolic features
of the increased lipid peroxidation further suggest a close association between the oxidative
imbalance and the dyslipidemia, thereby leading to functional deterioration of the steatotic
liver [32].
There are no tested non-invasive diagnostic modalities to distinguish NAFLD and
NASH, but new biomarker panels are approximating the liver biopsy in accuracy [4]. The
diagnosis of hepatic steatosis and steatohepatitis or NASH is not yet possible without liver
biopsy. Liver classical biochemical parameters, like aminotransferases, tend to be
inconclusive and cannot be used reliably to confirm the presence or stage the extent of
fibrosis. Nevertheless, a growing body of evidence strongly suggests that hepatic fatty acid
amount may impact the degree of liver damage and therefore disease evolution [33]. Liver
histology in NASH is usually classified through score systems based on morphological tissue
parameters. Thus, Brunt et al. [34] proposed a classification of NASH as mild, moderate, or
severe in relation to the degree alteration of parameters like steatosis, hepatocyte ballooning,
lobular inflammation, and portal inflammation.
2.1. Hepatic Steatosis
Hepatic steatosis is caused by an imbalance between the delivery of fat in the liver and
its subsequent secretion or metabolism. Thus, fat accumulates when the delivery of fatty
acids to the liver, either from the circulation or by de novo synthesis within the liver exceeds
its capacity to metabolize the fat by β-oxidation or secrete it as very low-density lipoproteins
(VLDL). A derangement in any of these pathways, alone or in combination, causes fat
accumulation in the liver.
Concerning the development of hepatic steatosis in NAFLD, it has been demonstrated
that fat in the hepatocytes comes from multiple different origins including dietary lipids, fatty
acids released from adipose tissue, and from de novo liver lipogenesis [1]. In humans it was
demonstrated that after 2 weeks of high-fat diet (56% of energy as fat) lipid accumulation
increased in hepatic tissue, while an isocaloric low fat diet (16% of total energy as fat)
diminished liver fat content [35]. Moreover, studies in both humans and animal models
indicate that adipose tissue is one of the major sources of the increased lipid movement to the
liver [17]. Finally, together with dietary fat and fatty acids secreted from adipose tissue,
hepatic tissue is also capable of de novo lipogenesis. Although the de novo lipid synthesis is
probably not relevant in healthy lean subjects [36], it has been shown that in NAFLD patients
it can generate up to 24% of liver fat [1].
In addition, fatty acids inside the hepatocytes are metabolized by one of two
mechanisms: oxidation to generate energy or esterification to generate triglycerides, which
are either integrated into VLDL particles for export or store within the hepatic tissue.
Problems in any of these pathways could contribute to hepatic steatosis development [1, 37].
Defective mitochondrial/peroxisomal β-oxidation or microsomal cytochrome P450 4A ω-
oxidation and/or diminished ability of the liver to export lipids may be induced by mutations
of genes encoding enzymes of lipid metabolism, as shown for mice deficient in liver carnitine
acyl transferase-1 and triacylglycerol transferase with consequent impairment of fatty acid
oxidation and triacylglycerol export, respectively. Alternatively, dietary fats can accomplish
regulation of hepatic lipid metabolism through modification of gene transcription, as
achieved with ω-3 and ω-6 long-chain polyunsaturated fatty acids (LCPUFAs) [38]. Under
physiological conditions, LCPUFAs metabolism maintains an adequate ω-3/ω-6 proportion
but any imbalance in this relation can influence metabolic-inflammatory pathologies
including NAFLD [39].
2.2. Obesity and Insulin Resistance in Hepatic Steatosis
Obesity promotion of steatosis is primary linked to the imbalance between the delivery of
fat to the liver and its subsequent secretion or metabolism (positive energy balance). It is well
known that high-fat diets foment an influx of ingested lipids to the liver promoting steatosis.
In addition, high content digestible carbohydrates diets stimulate steatosis, due to the fact that
permanent and excessive ingest of glucose elevates the amount of circulating insulin, the
most lipogenic endocrine mediator.
On the other hand, recent investigations have revealed that obesity plays a major role in
the development of insulin resistance [40], known as a promoter of steatosis. Insulin
resistance leads to steatosis in a similar way as high-digestible carbohydrates diets.
Sustained hyperglycemia and/or hyperinsulinemia, promotes steatosis through the
activation of lipogenic transcription factors (TFs). Nonetheless, de novo liver lipogenesis is
up-regulated independently by glucose and insulin [41, 42]. Glucose acts directly by
generating energy and acetil-CoA, a precursor of all de-novo synthesized lipids, and
indirectly through the activation of TF carbohydrate response element binding protein
(ChREBP). Insulin acts indirectly through the activation of TF sterol regulatory element-
binding protein-1c (SREBP-1c). Together, both TFs induce the synthesis of all the machinery
involved in lipogenesis [43].
2.3. Fatty Acid Accumulation: Lipotoxicity
It should be mentioned that steatosis may be a key step in the development of liver
damage in NAFLD. Indeed, this metabolic derangement may affect the liver structure and
function, but also other organs. These alterations may be caused by a phenomenon known as
lipotoxicity, a condition that accounts for the pathologic changes in non-adipose tissues, such
as the liver and pancreas, due to the adverse effects of excess fatty acid accumulation. In
addition, lipotoxicity is associated to redox imbalance leading to increased formation of ROS
[33]. Increased ROS production in presence of an excess of FFAs has been demonstrated in
several models of NASH. For example, a research performed in CHO cells showed that cell
death induced by saturated fatty acids depend on ROS production [44]. In addition, other
studies have found that ROS plays a primary role in the activation stage of programmed cell
death induced by FFAs [33]. Increased generation of ROS in the presence of large amounts of
FFAs has been validated in many animal models of NASH [45, 46]. Livers of patients with
NASH have augmented amounts of lipid peroxidation by-products, providing more evidence
of increased ROS in this condition [37]. Oxidative stress may cause various kinds of
functional and structural damage in non-immunological liver cells, like hepatocytes or
endothelial cells [1]. In parallel, ROS stimulate the activation of other hepatic immune related
cells, including Kupffer and stellate cells.
2.4. Free Fatty Acids as a Source of ROS in Hepatocytes
Several experimental models have shown that excess of FFAs are a major source of ROS
in NASH [45, 47, 48]. The most important ROS associated to this pathology are superoxide
anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen molecules, establishing that
oxidative stress exerts a central role in the development of NASH [6]. Increased hepatocyte
FFAs triggers multiple processes capable of generating ROS [17]. First, FFAs could cause
mitochondrial dysfunction, a process associated with enhanced ROS production and ATP
depletion. Second, FFAs could generate an endoplasmic reticulum (ER) stress response, a
phenomenon that stimulates ROS production. Third, increase in FFAs determines
hyperactivation of some isofoms of microsomal cytochrome P450, leading to an increase in
ROS formation. Fourth, a rise in cytoplasmic FFAs levels would increase ceramide
formation, a well known pathway of ROS generation (figure 9-1).
2.4.1. Mitochondrial Dysfunction

Mitochondria play a central role in cellular metabolism. It is the site of free fatty acid β-
oxidation and the citric acid cycle, which generates NADH and FADH2. The latter
compounds transfer electrons to the respiratory chain, and finally to molecular oxygen, a
process that generates ATP, the primary energy molecule of the cell. It has long been
recognized that the mitochondrial electron transport chain is a main site of ROS generation
[49]. Several lines of evidence show that NASH patients are characterized by abnormal
mitochondria from both morphological and functional point of view [17, 37].
As previously mentioned, liver steatosis is at least in part, derived from an excessive
hepatocyte lipogenesis, process that physiologically inhibit mitchondrial β-oxidation in order
to avoid ATP depletion in a futile cycle of synthesis-oxidation of fatty acids. However, under
steatosis conditions this mechanism could overload mitochondria with FFAs. Currently, it is
well known that excessive accumulation of FFAs in liver cells directly induces mitochondrial
dysfunction and oxidative stress. This process is associated with a sequence of events that
includes mitochondrial depolarization, increased ROS production, release of cytochrome c,
an electron carrier of the respiration chain between mitochondrial complexes II and III, and
organelle dysfunction. FFAs-induced mitochondrial failure would be dependent on lysosomal
disruption and activation of cathepsin B, peptidase involved in lysosomal metabolism,
because the inhibition of these downstream events protects against FFAs-induced
mitochondrial dysfunction and oxidative stress both in vitro and in vivo [50]. Reactive
oxygen species production occurs because mitochondrial impairment causes an electron flow
interruption that blocks respiratory chain leading to the transfer of electrons from respiratory
intermediates to molecular oxygen to produce superoxide anions and hydrogen peroxide [45],
which in turn starts a self-sustaining loop that worsen original mitochondrial injury [17]. This
mitochondrial degeneration impairs β-oxidation capacity of the organelle, generating more
free fatty acids accumulation, leading to a positive feedback mitochondrial dysfunction [37].
Additionally, mitochondrial injury may be a cause of reduced hepatocellular ATP stores in
NASH [10], hampering the regeneration of antioxidant defenses, the reparation of damaged
organelles and the maintenance of cellular homeostasis, thus encouraging the development of
oxidative stress. Finally, ROS-associated release of mitochondrial cytochrome c is intimately
linked to mitochondrial dependent apoptosis process related to caspase cascades activation.
Figure 9-1. A comprehensive model detailing the different sources of hepatocyte free fatty acids (FFAs)
and their implication on non alcoholic fatty liver disease (NASH) development related to oxidative
stress generation via different pathways. Dual arrows imply bidirectional relation.
2.4.2. Endoplasmic Reticulum Stress

Liver ER is a central organelle for lipid metabolism, biological membranes assembly and
lipoprotein management [51], constituting a major site of FFAs accumulation. Since this
organelle comprises more than half of the total membranes in the hepatocyte and has the
highest cellular calcium concentration, together with a unique oxidative environment to
support disulfide bond formation [33], it becomes in the best cellular place for FFAs
spontaneous precipitation and oxidation. This process is associated with deficiencies in
protein synthesis and folding. This phenomenon is related with the disruption of the
organelle, called ER stress [33]. The RE stress is highly related with ROS generation [52].
Maintenance of integrity of ER membrane structure is essential to physiological oxidation of
proteins, associated to disulfide formation and cellular Ca2+ handling [53]. Hepatic steatosis
and NASH are associated with known triggers of the ER stress.
Accumulation of saturated FFAs and its calcium precipitates in ER decrease the fluidity
of the organelle, affecting their ability to fold and export proteins. Accumulation of unfolded
protein initiates the activation of an adaptive signaling cascade known as unfolded protein
response (UPR) [54]. Unfolded protein response could derivate in ROS generation by
extrinsic and an intrinsic pathways. In the first case, unfolded protein accumulation in the ER
may elicit Ca2+ leak into the cytosol. The very close proximity of ER and mitochondria leads
to accumulation of this cation near mitochondria allowing it to enter to this organelle and
depolarize its inner membrane. This process triggers increased mitochondrial ROS
production, cytochrome c release and mitochondrial dysfunction. High levels of ROS
generation within the mitochondria further increase ER disruption and sensitize the Ca2+
release channels at the ER membrane, by oxidizing a critical thiol in the ryanodine receptor
causing its inactivation, both processes enhancing Ca2+ release from ER create a vicious
cycle that threaten to cell survival [52]. In addition, this vicious cycle could activate both
mitochondria-dependent and mitochondria-independent caspase cascades, the latter
associated to protein kinases Ca+2 dependent (PCKs). The intrinsic pathways of ROS
generation by ER is related with oxidative protein folding, a highly regulated process
catalyzed by a family of ER oxidoreductases, including protein disulfide isomerase. In these
reactions, molecular oxygen serves as the terminal electron acceptor for disulfide bond
formation. The enzymes ER oxidoreductases use a flavin-dependent reaction to pass electrons
directly to molecular oxygen, a reaction that has the potential to generate ROS. It has been
estimated that near 25% of the ROS generated in secretory specialized cells may result from
the formation of disulfide bonds in the ER during oxidative protein folding, ROS that
otherwise would be removed by antioxioxidant defenses [54].
However under ER stress this ROS production is enhanced because the activation of
UPR allows only properly folded proteins to exit the ER. Misfolded proteins are either
retained within the ER lumen in complex with molecular chaperones to repair them.
Catalyzed reparation of these proteins use reduced glutathione (GSH), one of the most
important endogenous antioxidant, to return thiols involved in non-native disulfide bonds to
their reduced form so they may again interact with ER reductase to be reoxidized. This
process generates a futile cycle of disulfide bond formation and breakage, in which each
cycle would generate more ROS and consume GSH. Alternatively, because both protein
folding and unfolding are highly energy-dependent processes, ATP depletion as a
consequence of protein misfolding, could aggravate oxidative stress [52]. Recent studies
show the presence of ER stress in mice with hepatic steatosis. In addition, a new small
clinical trial has demonstrated that there is a progressive degree of activation of the UPR
between steatosis toward steatohepatitis in NAFLD patients [54].
2.4.3. Cytochrome P450 Activation

Cytochromes P450 are an integral ER superfamily of heme-monoxygenases essential to
metabolize an important range of compounds for a proper elimination from the body. While a
limited number of these enzymes are involved in the synthesis of steroid and bile acid
production, most cytochromes P450s metabolize xenobiotics or foreign compounds including
several drugs, toxic compounds and chemical carcinogens. Cytochromes P450 catalyze the
oxidation of carbon and nitrogen functional groups usually resulting in the incorporation of
an alcohol moiety. In some cases, an epoxide is incorporated to an aromatic ring that is
spontaneously converted to an alcohol by non-enzymatic reaction with water, or transformed
to a trans dihydrodiol by epoxide hydrolases [21].
Paradoxically, some of these enzymes also can cause cell toxicity by different pathways,
among other, the generation of ROS through the oxidation of several compounds [17].
Cytochromes P450 that metabolically activate toxic compounds and carcinogens are limited
to a few isoforms including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2E1, and in a lesser
extent the CYP3A subfamily. Cytochromes P4501A1 and CYP1B1 mainly activate
polycyclic aromatic hydrocarbons. Cytochrome P4501A2 carries out the N-oxidation of
heterocyclic amine food mutagens and arylamines and finally CYP2E1 metabolizes
numerous compounds, such as aromatic, aliphatic and halogenated hydrocarbons, many of
which are industrial solvents, and some of which are cancer suspect agents. The increased
availability of FFAs determines the activation of microsomal cytochrome P450 isoforms
CYP4A10/4A14 and especially CYP2E1, both involved primarily in FFAs ω-oxidation,
leading to an increased ROS production [17]. The mechanism of this reaction involves the
flavoprotein-mediated donation of electrons and during the catalytic cycle, P450s use
hydrogen from NADPH to reduce O2 leading to the production of H2O2 and superoxide
radical [55]. Interestingly, even in the absence of substrate, these enzymes can generate ROS.
This phenomenon, known as futile cycling, is due to P450-derived NADPH oxidase activity,
a process independent of exogenous xenobiotic substrates [56]. These ROS generated within
the ER membranes produce peroxidation of LCPUFA, a process that foment oxidative stress
and ER stress. On the other hand, FFAs indirectly could cause induction of P450s ω-
oxidation, because in the presence of lipid excess, increased FFAs oxidation leads to an
increase in ketone bodies, mediators that are inducers of these enzymes [21].
The participation of P450s ω-oxidation in NASH oxidative stress was proven by a study
in a genetic model of GSH synthesis deficiency in mice, NASH spontaneously develops and
is associated with elevated CYP2E1 expression and increased lipid peroxidation that is
decreased by addition of the CYP2E1 inhibitor diallyl sulfide [21]. In animal models of high
fat diet-NASH induced, several studies have demonstrated an elevated expression and
activity of CYP2E1. Obese humans have both elevated CYP2E1 activity and induction,
indicating that CYP2E1 is related to hepatic pathology resulting from morbid obesity.
Moreover, the level of P450 correlated well with the degree of steatosis. Likewise, in rodents
a similar high level of CYP2E1 activity was found in non-diabetic patients with NASH,
indicating that the activity of this P450 isoform is elevated in humans with this pathology.
Also, CYP2E1 protein levels are spontaneously induced in patients with NASH [21].
2.4.4. Ceramide Formation

Ceramide is a sphingolipid, a category of lipids derived from the aliphatic amino alcohol
sphingosine. Sphingolipids are important structural constituents of biological membranes in
animal cells, mostly plasma membranes. During the last years, sphingolipids have been
studied due to their role in signal transduction, proliferation, differentiation and immune
response [49]. Despite its physiological role, a growing body of researches also suggests that
ceramide and/or its derivatives can induce cellular ROS production in different cell models
through the activation of NADPH oxidase [57] and inducible nitric oxide synthase (iNOS)
[58]. Also, ceramide might down-regulate antioxidant enzymes [59], and/or alter the
mitochondrial function [60].
In endothelial cells ceramide was able to induce endothelial dysfunction via superoxide
generation, resulting in peroxynitrite formation. This activation was blocked by NADPH
oxidase specific inhibitors, N-vanillylnonanamide, apocynin, and diphenylene iodonium, but
not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport
chain enzymes [57]. On the other hand, in different cell models ceramide can induce
apoptosis by activating the pro-inflamatory TF nuclear factor kappa B (NF-қB) [61], which
upregulates the expression of iNOS. The increased formation of nitric oxide (NO) induce the
production of reactive nitrogen species (RNS), specially peroxynitrite, a powerful long life
oxidant agent, which is probably an important cause of apoptosis [62]. Different iNOS
inhibitors, such as nicotinamide and aminoguanidine, diminished NO formation under these
conditions in vitro and prevent ceramide β-cell apoptosis and diabetes in vivo [63]. In relation
to antioxidant enzymes, ceramide was reported to participate in the regulation of
mitochondrial manganese dependent superoxide dismutase (Mn-SOD) and catalase in various
cell types. Ceramide mediated the generation of ROS and subsequent activation of redox-
sensitive transcription; these factors may be involved in the up-regulation of the Mn-SOD
gene expression.
The mechanism for ceramide-induced inhibition of catalase is not clear, but the
inhibitory effect of ceramide on phosphatidylinositol-3-kinase has been reported to be
involved in this process [45]. Finally, naturally occurring 16 carbon ceramide was shown to
cause an increase in ROS generation through mitochondria, promoting cytochrome c release
[60].
The de novo ceramide production or its glycosyl derivatives, has been proposed as an
important mediator in the cytotoxicity of saturated FFAs in NASH [64]. For example it was
known that FFAs, induce ceramide synthesis in different cell types, and inhibitors of de novo
ceramide synthesis diminished the induction of apoptosis by FFAs. The role of ceramide
toxicity in liver diseases has been confirmed in several model systems especially in mouse
with truncated ceramide synthesis. These animals were more resistant to alcohol-induced
cirrhosis than normal mice. In addition, hepatocytes from the transgenic mice were less
sensitive to TNF-α induced apoptosis [65].
2.5. Non FFAs Oxidant Species Production
In addition to the FFAs associated generation of ROS, some NASH patients could have
type II diabetes or insulin resistance. Both conditions can expose the liver to exacerbated
levels of glucose, process that could lead to glucotoxicity.
Despite glucotoxicity is poorly studied in liver models, it is a well known source of ROS
in other cellular models, such as β-cell [53] and neurons [66]. The glucose-induced rise in
ROS would be associated to different mechanism, for example mitochondrial dysfunction and
ER stress, induction of lipogenesis, activation of immune system and stimulation of fibrosis
by the route of advanced glycation end-products (see Chapter 8) [17, 53, 67]. On the other
hand, beyond the ROS production by processes directly associated to liver hepatocyte
metabolism, there exists a primary hepatocyte ROS production derived to extrahepatic non-
immunological tissues, especially adipose tissue. This would be a second line of association
between obesity and NASH, because several lines of evidence suggest that adipose tissue
generates and releases a variety of inflammatory molecules, such as TNF-α [1], able to
stimulate ROS production in many non immunological cell types including hepatocytes [40].
Moreover, plasma TNF-α level correlates with body fat mass [10].
2.6. Liver Oxidative Stress
Despite the fact that the presence of elevated levels of ROS could be dangerous for any
kind of cells, the hepatocyte is one of the best equipped for this type of aggression, because
unlike other cells, multiple liver physiological processes are associated with the generation of
large amounts of oxidant species. For example, xenobiotic detoxification, permanent fatty
acids oxidation, protein secretion and peroxisomal function, constitute challenges forcing the
hepatocyte to develop the best antioxidant defenses of mammalian cells, enabling the liver to
survive adequately with ROS concentrations being toxic for other cells. Hence under normal
conditions, hepatic aerobic metabolism involves a steady-state production of pro-oxidants,
such as ROS and RNS, which are balanced by a similar rate of their consumption by
antioxidants. However, as mentioned above, the steatotic liver seems to be particularly
susceptible to oxidative damage. Oxidative stress occurs when there is an imbalance between
the generation of ROS and the antioxidant defense systems in the body so that the latter
become overwhelmed [68]. Oxidative stress is developed in NAFLD at the stage of steatosis
and is exacerbated in steatohepatitis. This metabolic derangement is accompanied by a
decrease in both glutathione content and the activity of antioxidant enzymes, associated with
liver cytochrome P450 CYP2E1 isoform induction. Additionally, the antioxidant capacity of
plasma is inversely correlated with the progression of NAFLD toward NASH.
Recently, it was suggested an impaired glutathione metabolism towards an oxidant status
in NASH patients, correlating this with a higher intake of saturated fat and a lower intake of
carbohydrates [69].
Finally, a recent study in a rat model it was shown that shows that increased activity of
the renin-angiotensin system through the vasoconstrictor Angiotensin II causes development
and progression of NAFLD by increasing hepatic ROS [70]. Oxidative stress generates the
chemical alteration of essential biomolecules causing their inactivation; hence it leads to

direct hepatocyte cell injury. In addition, oxidative stress by-products, such as some toxic
aldehydes, have longer half-lives than ROS and are able to damage the hepatocyte and
diffuse out to extracellular targets [71].
The well established major oxidative stress occurrence in NASH pathogenesis was
demonstrated in human biopsies by several markers [72]. First, there is a significant increase
of lipid peroxidation by-products (e.g. thiobarbituric acid reactive substances, TBARS;
malondialdehyde; 4-hydroxynonenal) [73, 74]. Second, it has been found an increase of nitro-
tyrosine protein modifications [75]. Third, it has been detected the presence of DNA
hydroxylation [73]. Fourth, there are increased plasma oxidative stress parameters such as
thioredoxin, among others [76]. Fifth, reduction of antioxidant defenses, including
diminished levels of GSH and coenzyme Q10 and lower antioxidant enzymes activities such
as glutathione S-transferase, CuZn-superoxide dismutase and catalase [74].
The combination of direct oxidative stress effects and their by-products effects have the
intrinsic ability to generate cell necrosis. However, before this irreversible process occurs,
oxidative stress generates an adaptive response, aimed to avoid damage propagation and
stimulate regeneration: this is the inflammation process. Oxidative stress derivate
inflammation is a finely regulated process that involves several changes in genetic expression
and important modifications in hepatocyte metabolism. Many of these changes would be
target to trigger apoptosis, probably to avoid a malignant transformation associated to a
chronic and moderate oxidative stress or to prevent the explosive inflammation response
associated to cell necrosis.
2.7. Liver Inflammation
Inflammation is a complex, conserved and non-specific biological response of cells and

tissues to injurious stimuli, such as cell necrosis, exposition to pathogens and toxic
substances, among others. It is a protective biological response that involves the local
vascular system, the immune system, and various cells within the injured tissue in order to
remove the harmful as well as initiate the reparation of the affected tissue. Inflammation is
characterized by vasodilation and augment in vascular permeability with the subsequent
increased movement of plasma and immune cells, specially macrophages, neutrophils and
monocytes, from the blood to the affected tissues, process called infiltration. This
phenomenon begins with a cascade of biochemical events related with the activation and
release of inflammatory mediators, including cytokines and prostaglandins, by resident
immune and non-immune cells of the injured tissue. Inflammation mediated by oxidative
stress is originated by two main pathways; the intrinsic and the extrinsic pathways. The
intrinsic pathway relates to the ability of ROS to induce directly in the affected parenchymal
cells the expression of different kinds of proinflammatory mediators, without immune cell
mediation. For example, ROS can promote directly the formation and release of inflammatory
mediators in parenchymal cells by regulated molecular mechanism independent from external
stimulus. The extrinsic pathway is associated with the ability of parenchymal oxidative stress
to induce the release of inflammatory mediators by surrounding resident immune cells. This
process is mediated mainly by the ability of ROS to trigger apoptosis in an affected cell,
through a controlled series of events related with the sequential activation of several proteins
[67]. Apoptotic cell bodies have the ability to induce the activation of different immune cells
and stimulate the differentiation of pro-immune cells to their functional state, both capable to
express large amounts of new proinflammatory mediators. These mediators induce the
relaxation of endothelial cells junctions and stimulate the expression of adhesion molecules
in non-immune cells of the injured tissue. Both of these processes are responsible for the
influx of circulating not resident immune cells into the affected tissue [37]. This infiltration,
carried out by cells of innate immune system, is the first organism defense barrier to contain
an aggression. Indeed, it plays and important role in the development of a later adaptive
immune response, if necessary, and it is fundamental to the regeneration of the damage
tissue.
On the contrary, chronic inflammation is a pathological condition that favors cell death
and threatens to tissue reparation because inflammatory mediators and infiltration stimulate
cell death directly and indirectly. Then, their maintenance in time prevents cellular
regeneration and begins to affect the healthy surrounding tissue.
During inflammation, one of the most important molecular mechanisms to stimulate cell
death is the generation and release of powerful oxidant species such as ROS and RNS to
affected tissue. These species have the ability to destroy pathogens and infected or modified
cells that in most cases are more susceptible to an oxidative threat than healthy cells. Despite
of its recognized efficiency, this mechanism has a limitation; when the oxidant species do not
meet its goal within a reasonable period they begin to affect the healthy surrounding cells
spreading oxidative stress conditions, which in turn induces inflammation, generating a
vicious cycle that propagates and matures the original inflammatory response, being a key
step in chronic inflammation [77].
Chronically inflamed hepatic tissue is the most important classical marker of NASH and
practically defines the disease, being the marker that makes the difference between simple
steatosis and steatohepatitis. Unlike other diseases such as viral hepatitis and or infection, in
NASH the main injurious stimuli that induce the inflammation is the hepatocyte oxidative
stress, phenomenon that starts the vicious circle that in fatty liver conditions is very difficult
to be stopped.
As mentioned below, virtually in all pathologies related with oxidative stress, especially
in NASH, oxidant species induce directly the expression of pro-inflammatory mediators. This
process occurs through the activation of pro-inflammatory TFs such as NF-κB and activating
protein-1 (AP-1), among others, in all cell types of the affected tissue. These TFs are
sensitive to a decrease in intracellular redox potential [43]. Nuclear factor kappa-B
preferentially induces the transcription of pro-inflammatory mediators including paracrine
molecules such as TNF-α and interleukin-1, cell surface molecules such as TNF-α receptor
and intracellular molecules like iNOS. In contrast, AP-1 typically stimulates the expression of
pro-apoptotic proteins (e.g. p53) and adhesion molecules. Hepatocyte released paracrine
molecules react with surrounding endothelial cells stimulating them to express other pro-
inflammatory mediators, especially adhesion molecules. In parallel, these paracrine
molecules activates surrounding immune resident cells that starting to release large amounts
of ROS and RNS and secrete more inflammatory mediators.
2.7.1. Liver Endothelial Cells

Liver sinusoidal endothelial cells constitute the sinusoidal wall, also recognized as the
endothelium, or endothelial lining. The endothelial cells can be regarded as unique capillary
cells which differ from other tissue capillary cells, because of the presence of several open
pores or fenestrae that lacks the classical diaphragm and a basal lamina underneath the
endothelium. Fenestrae filter all the fluids, solutes and particles that are exchanged between
the sinusoidal lumen and parenchymal tissue, allowing only particles smaller than the
fenestrae to move in a bidirectional way [78]. Injured sinusoidal endothelial cells play direct
and indirect roles in the development of inflammation and propagation of the initial
hepatocyte oxidative stress. During these processes, there are changes in the molecular
expression profile of the normal hepatic endothelial cells. These changes are characterized by
expression of high levels of inflammatory molecules such as MIP-1β (Macrophage
inflammatory protein 1β), and ITAC (IFN-gamma-inducible T cell alpha chemoattractant)
and adhesion molecules such as ICAM-1 (inter-cellular adhesion molecule-1) and VCAM-1
(vascular cell adhesion molecule-1). In addition, these cells begin to directly generate ROS
by enzymatic and not enzymatic mechanism [79].
2.7.2. Kupffer Cells

The most important of the liver immune cells are Kupffer cells, the resident liver
macrophages, which play significant roles in immunomodulation, phagocytosis, and
biochemical attack [77]. Any liver insult, including inflammatory substances derivates from
hepatocytes by oxidative stress, immediately triggers Kupffer cells activation [80]. The
presence of an excess of lipids, including neutral ones, such as triacylglycerols (TGs), appear
to sensitize these macrophages to liver insults, for example, adding TGs to
lipopolysaccharide enhanced the ability of the latter to induce the expression of pro-
inflammatory mediators in Kupffer cells. As Kupffer cells account for 70-80% of the whole
body macrophages [80] its chronic activation results in the secretion of large amounts of
RNS, derived from the induction of iNOS, [77]. This process increases tremendously the
original liver oxidative stress, becoming in a key step in the development and progression of
NASH. In addition, activated Kupffer cells are known to generate and secrete important
quantities of inflammatory mediators associated to liver injury, such as TNF-α, interferon α
and β (INF α/β), interleukin-1β, interleukin-6 and granulocyte colony-stimulating factor
(GCSF) [81-83], enhancing the original inflammatory response. This also directly affects
hepatocytes. For example, TNF-α, one of the most powerful pro-inflammatory cytokines that
are the most abundant cytokine in NASH, has the ability to generate oxidative stress in
healthy hepatocytes promoting lipolysis, increasing cytosolic FFAs and interrupting
mitochondrial electron transport chain with similar effects than FFAs, ROS and Ca2+,
fomenting apoptosis and/or necrosis [17].
2.7.3. Infiltration
The joint activation of hepatic resident immune and endothelial cells amplifies the
original inflammation signals, triggering immune infiltration with different types of
circulating inflammatory cells. Immune infiltration is associated with the recruitment of
circulating macrophages and leukocytes, mostly monocytes and neutrophils, into the liver
vasculature [84, 85]. In NASH, the major function of these cells is to remove dead cells and
cell debris in preparation for tissue regeneration. However, because of the nature of these
immune cells, during long-term infiltration non-affected neighbor hepatic tissue may also be
damaged, which can aggravate the original liver injury [86]. Activation of infiltrated immune
cells release more ROS and RNS, thus further enhancing oxidative stress. In addition, if the
rate of removal of dead cells and regeneration of tissue is lesser than the parenchymal
destruction rate, such as in NASH, infiltration cells become more active and begin to secrete
the same inflammatory paracrine molecules secreted by Kupffer cells. This event amplifies
the damage to its highest level fomenting the irreversible loss of the affected parenchyma
which is replaced by non functional tissue in the process called fibrosis.
2.8. Fibrosis
Fibrosis is the replacement of the parenchyma tissue by fibrous connective tissue as a

reparative or reactive process. It is a regenerative pathological response that involves the
synthesis, release and deposit of an special kind of extracellular matrix characterized by their
density and rigidity. This dense connective matrix, also called dense fibrous tissue, has
collagen fibers as main element, composed of type I and type III collagen that forms strong
structures which separates affected tissue from different and/or healthy tissue. Collagen rich
connective tissues are manufactured by fiber-forming cells or fibroblasts in response to a
complex network of signals associated to final stages of pathological states, including heavy
oxidant species, pro-inflammatory mediators and apoptotic bodies, among others. Obesity
and related insulin resistance promotes fibrosis progression by different mechanisms
including steatosis, hyperleptinemia, increased TNF-α production and impaired expression of
peroxisome proliferator-activated receptors-γ receptors (PPAR-γ) [87]. The renin-angiotensin
system, plays a vital role in blood pressure regulation (see Chapter 2), and appears to promote
hepatic fibrogenesis too [70]. Fibrosis is one of the final stages of all chronic liver disease; it
is carried out by activated fibroblast derived from hepatic stellate cells.
2.8.1. Hepatic Stellate Cells

Hepatic stellate cells (HSTs) are pericytes located in the perisinusoidal space of the liver.
As a relatively undifferentiated cell, under physiological conditions it usually remains in a
quiescent state, but still has the ability to differentiate into fibroblast, macrophages or
contractile myofibroblasts as required [88]. In response to chronic inflammation and oxidant
species these cells experience a massive activation and become highly proliferative and
fibrogenic beginning the generation and deposition of extracellular matrix components,
mainly type I collagen [80]. Hepatic injury in NASH related with oxidative stress, and
inflammation leads to cytotoxic events that activate hepatic stellate cells and deposition of
collagen [89]. Activation of stellate cells is the key step in the development of liver fibrosis.
This process is mediated mainly by ROS, RNS and inflammatory cytokines derived from
damaged hepatocytes and activated Kupffer cells, but mainly from activated infiltrating
immune cells [80]. Injured sinusoidal endothelial cells can also play an important role in
hepatic fibrosis participating in both early HSTs proliferation and plasmin activation [90].
These processes are associated with fibrosis [91]. Stellate cells also can secrete ROS and
other inflammatory molecules [17, 80] favoring the perpetuation of the inflammatory
response. Hence, these cells have the ability to directly induce inflammation, hepatocyte
necrosis, and liver fibrosis, all of the classical histological markers of NASH [37].
2.8.2. Fibrogenesis in NASH

Profibrogenic mechanisms operating in NASH are partly in common with those observed
in other chronic liver diseases. The differences are related with the presence of increased
circulating adipokine levels, altered glucose metabolism and the hormonal profile associated
with the metabolic syndrome. These conditions might have a specific role in the induction of
fibrogenesis in NASH. Reactive oxygen species and RNS stimulate the progression of liver
fibrosis and increase the procollagen expression in HSTs. Oxidative stress-derived molecules,
for example reactive aldehydes, induces activation of these cells. Pro-inflammatory mediators
stimulate immune cell release of fibrogenic factors, such as platelet-derived growth factor
and transforming growth factor β1, which stimulate the profibrogenic actions of HSTs [17].
Hepatocytes apoptotic bodies stimulate fibrogenesis in vivo [92] because phagocytosis of
these particles by hepatic stellate cells activates these cells and stimulate NADPH oxidase-
mediated production of ROS. Angiotensin II induces profibrogenic actions in HSTs, which
express all components of the renin–angiotensin system, moreover interference with this
system attenuates fibrosis development in different laboratory models of chronic liver disease
[93]. In relation to comorbidities, insulin resistance is a potent predictor of fibrosis, because
elevated glucose or insulin levels up-regulate transforming growth factor β (TGF-β) and
connective tissue growth factor. Hyperglycemia could also affect hepatic stellate cells
biology because receptors for advanced glycation end-products are expressed in these cells
and have a role in migration of activated hepatic stellate cells [94]. Leptin, the most abundant
adipokine in obesity conditions is a potent profibrogenic factor, acting directly on hepatic
stellate cells [95].
2.8.3. Cirrhosis
The final stage of liver fibrosis is cirrhosis, condition defined as huge and extended
distortion of normal hepatic architecture. Cirrhosis is characterized by replacement of liver
tissue with regenerative nodules surrounded by dense layer of fibrotic tissue. As time goes by
regenerative nodules develop in the midst of scars which may result in advancement and
neoplastic transformation of the cirrhosis. Hepatic cirrhosis is usually considered irreversible
and treatment is only supportive. The evolution rate of early NASH fibrosis to cirrhosis and
the morphology of the last condition vary from person to person, presumably, because the
wide range of comorbid conditions that could accompany NAFLD during their progression
and the different individual’s response to those pathologies.
3. Antioxidant Therapies
Up to date, there is no an effective medical treatment available for NASH patients.
Therapy is predominantly aimed at controlling comorbid pathologies, such as obesity, insulin
resistance, and dyslipidemias [96]. Weight loss is mostly advocated but it is difficult to
achieve in the majority of patients. Indeed, some affected individuals have been reported to
worse their hepatic damage with excessive weight loss [97, 98].
Table 9-1. Antioxidant tested for the treatment of NASH
Agents Proposed mechanisms References

Glycyrrhizin Free radical scavenging Wu et al. 2008 [100]
Inhibition of FFAs accumulation
Anti-inflammatory
Immunomodutation
Vitamin E Free radical scavenging Lavine 2000 [106] *
Regeneration of oxidized glutathione to the Hasegawa et al. 2001[107] *
reduced active form. Kugelmas et al. 2003 [108]*
Vajro et al. 2004 [109]*
Harrison et al. 2003 [110]*
Sanyal et al 2004. [7]*
Dufour et al. 2006 [6]*
Probucol Free radical scavenging Merat et al. 2002 [115]*
Promotes endogenous antioxidants synthesis Merat et al. 2002 [117]*
Merat et al. 2008 [108]*
Tokushige et al. 2007 [118]*
N-acetylcysteine Free radical scavenging Thong-Ngam [119]
Stimulates glutathione synthesis Baumgardner et al. [120]
Gulbahar et al [121] *
Pamuk and Sonsuz [122]*
De Oliveira et al. [123]*
Vitamin C Free radical scavenging Shireen et al. 2008 [127]
Regenerates oxidized vitamin E and glutathione Harrison et al. 2003 [110]*
to the reduced active forms.
S- Glutathione synthesis through formation of Vendemiale et al. 1989 [135] †
adenosylmethionine cysteine Labo and Gasbarrini 1975 [136] †
Frezza et al 1990, [137] †
Mato et al 1999 [138] †
Betaine Increasing S- adenosylmethionine Miglio et al [142]*
Abdelmalek et al. [143]*
Genistein Free radical scavenging Yalniz et al. 2007 [145]
Anti-inflammatory
Antifibrotic
*Clinical Trials
† Tested in no NASH liver disease models.
In relation to liver steatosis, a major concern in treating is to prevent progression to

NASH, but there is not general consensus on the effectiveness of any therapeutic agent for
treating fatty liver [25]. However, the role of oxidant processes in the progression of hepatic
steatosis to NASH and in NASH worsening, opens the door to an intervention pathway, being
logical to think that antioxidant therapies oriented to the neutralization of oxidative stress,
such as inhibition of oxidant species production and scavenging of oxidant species would be
an effective therapy in treating NASH by amelioration of the pathology driving forces and
reduction of the secondary effects [11] (table 9-1).
3.1. Treatment of Conditions that Induces the Liver Oxidative Stress
Stopping or reducing liver metabolic oxidant species production would seem to be the
best strategy to treat NASH. In fact currently most accepted treatments are based on this
strategy, such as weight loss, hypolipemiant and hypoglycemiant therapies. The first two
strategies are focused in lipotoxicity reduction, process highly associated to oxidative stress.
In addition, weight loss would limit oxidant species generation through the reduction of
adipose tissue, an important source of inflammatory molecules associated to NASH
development. Hypoglycemic agents, associated to insulin resistance therapy, would tend to
reduce liver glucose levels, and FFAs lipogenesis, both processes also associated to liver
oxidative stress. Recently, the utilization of substances that directly prevent lipotoxicity by
inhibition of FFAs accumulation, such as vegetable bioactive compounds (e.g. glycyrrhizin)
is beginning to be studied.
3.1.1. Metabolic Treatments

Several clinical trials have studied the effects of weight loss in NASH using different
strategies such as diet, exercise, and gastric surgery. Most of that trials show improvements in
NASH markers [10], including histological parameters. Antidiabetic therapy studies with
drugs (e.g. metformin and glitazones) also have shown damage reduction. On the contrary,
antihyperlipidemic therapy using diverse pharmacological agents, such as fibrates and statins
has not been conclusive.
In spite of the absence of absolute evidence of the benefits of these therapies, metabolic
control of oxidant species production seems to be a good alternative to treat NASH.
Preventing the production of new oxidant species by the original metabolic inductors could
be expected to have benefits in these patients. However, it should also be considered that the
presence of large amounts of already formed oxidant molecules could catalyze their own
propagation, thereby increasing cell damage, what should need a specific pharmacological
therapy. Moreover, this metabolic prevention of oxidant species production probably would
not finish in short term with the heavy immune ROS and RNS generation associated to
immune activation. Hence, the direct elimination of produced oxidant species is still very
important.
3.1.2. Glycyrrhizin
Glycyrrhizin is a triterpene glycoside considered the major bioactive compound of
licorice root extract. Licorice is one of the most traditional medicinal plants; it has an
ancestral use in traditional Chinese medicine for the treatment of several inflammatory
diseases [99]. Glycyrrhizin has a variety of pharmacological properties including anti-
inflammatory, antioxidant, and immune-modulating activities and has been used to reduce
liver inflammation and hepatic injury. The mechanism by which glycyrrhizin improve NASH
has been studied in HepG2 (human liver cell line) and might be associated with the
prevention of FFAs-induced toxicity and subsequent cell apoptosis [100]. These effects are
mediated by 18 beta-glycyrrhetinic acid (GA), the biologically active metabolite of
glycyrrhizin. This metabolite also stabilizes lysosomal membranes, inhibits expression and
activity of the proapoptotic lysosomal peptidase cathepsin B, reduces FFAs-induced
oxidative stress and inhibits mitochondrial cytochrome c release. Animal models of high fat
diet-induced NASH show that GA prevents hepatic lipotoxicity and liver injury in vivo [100].
One of the most remarkable aspects of glycyrrhizin is that in addition to their direct
antioxidant properties, it also has the ability to attack the cause of steatotic hepatocyte
oxidative stress: the lipotoxicity. This feature makes the glycyrrhizin a promising agent to
complement a traditional antioxidant therapy that is focused only in remove, in a directly or
indirectly way, previously generated oxidant species. Moreover, as licorice root extract has
an ancestral use in the treatment of liver diseases, glycyrrhizin is in a privileged position to be
proved in NASH clinical trials.
3.2. Oxidant Species Scavengers
As oxidant species play a major role in NASH pathogenesis, antioxidant supplementation

has been thought as a therapeutic option [25], and many antioxidants have been studied in
NASH [101]. An antioxidant agent is any molecule that favors, through any mechanism, the
loss of reactivity in oxidant molecules. Direct antioxidants avoid oxidative reactions at any
level and indirect antioxidants stimulate the generation and/or activity of direct antioxidants.
In relation to direct antioxidants, vitamins E, probucol and vitamin C appear to be the only
tested in clinical trials, and related to indirect antioxidants only N-acetylcysteine, S-
adenosylmethionine and Betaine have been studied. Recently, other antioxidant substances,
such as genistein, a phytoestrogen found in soybeans, shown promising in animal models of
the pathology; however there are no clinical trials that prove its effectiveness in human
patients.
3.2.1. Vitamin E
The term vitamin E is related to a family of lipid soluble substances called tocopherols
and tocotrienols, each with different suffix; α, β, γ, and δ. The α-tocopherol is the most
abundant natural form and is the major tocopherol in human plasma [71]. This vitamin has
powerful free radical scavenging abilities, breaking free radical chain reactions, that is of
principal importance in lipid peroxidation due to interrupt unsaturated fatty acids degradation
[6]. Animal models of liver disease associated with oxidative damage have shown that
vitamin E reduces lipid peroxidation, corrects oxidative stress and improve fibrosis [102-
105]. For example, in a mouse model of NASH, the intervention group significantly
improved reduced glutathione reserves, with a subsequent decline in levels of TBARS and
improvement in fibrosis histological score [65].
In clinical trials vitamin E has been studied alone or associated with other therapeutic
agents. In the first study, Lavine [106] used increasing doses of vitamin E, up to 1200 IU/day,
in a small group of affected children demonstrating improvement in aminotransferases, but
liver histology was not analyzed. In the second study, Hasegawa et al. [107] in a little
uncontrolled pilot trial, demonstrated fibrosis improvement in 66% of adult NASH patients
treated with vitamin E in doses of 300 mg/day during 12 months. Later, in other pilot study
carried out by Kugelmas et al. [108] with NASH adult patients, vitamin E and aerobic
physical activity supplementation showed no effects after 3 months of 800 IU/day vitamin
supplementation, suggesting that in short-term interventions this vitamin provides no
apparent added benefit or physical activity masked possible vitamin effects. However, Vajro
et al. [109], in a pediatric randomized placebo-controlled trial, showed hepatic enzyme

improvement in 14 children with hepatic steatosis treated during 5 months with low doses of
vitamin E (400 IU daily for the first 2 months and 100 IU daily for the final 3 months).
Interestingly, the subgroup of treated patients that did not show weight loss during the trial,
showed improvement in aminotransferases levels, suggesting that vitamin E therapy may be
applicable in obese children who are unable to adhere to a calorie restriction treatment.
Moreover, the most complete randomized placebo-controlled clinical study of vitamin E
carried out by Harrison et al. [110], in adult patients with biopsy-proven NASH, showed a
significant improvement in fibrosis score of liver biopsies in the intervention group after 6
months. However, in this group patients received 1,000 daily UI of vitamin C, in addition to
1,000 IU daily of vitamin E.
Finally, other currently published vitamin E intervention trials evaluate the effect of
vitamin E in combination with other therapeutic products not necessarily antioxidants agents.
Sanyal et al. [7] showed in a 6 months pilot study that mixed therapy with vitamin E and
pioglitazone (a hypoglycemiant drug) generates a greater improvement in NASH histology
than only vitamin E therapy. Nonalcoholic steatohepatitis patients were randomized with 400
IU daily of vitamin E alone versus vitamin E plus 30 mg daily of pioglitazone. Both
intervention groups show improvement in liver histology and aminotransferases
normalization. However, the mixed therapy group showed greater improvement in several
biopsy parameters such as steatosis, inflammation, hepatocyte ballooning, and Mallory
hyaline at the end of the trial. In other more recent randomized placebo-controlled trial,
Dufour et al. [6] demonstrated NASH improvements with a vitamin E-based therapy plus
ursodeoxycholic acid (UDCA), a secondary bile acid with well known hepatic cytoprotective
properties and immunomodulatory effects. Initial biopsy-proven NASH patients were
randomly assigned to receive doses of UDCA 12–15 mg/(kg/day) with 400 IU of vitamin E
twice a day, UDCA with placebo, or placebo with placebo during 2 years before a second
biopsy. The initial inter-group body mass index did not change significantly during the 2
years. However, the UDCA plus vitamin E group improved laboratory values and hepatic
steatosis of NASH patients. Interestingly, the UDCA alone group had no differences in any
parameter with the placebo-placebo group.
3.2.2. Probucol
Probucol is a synthetic polyphenol drug, with hypolipidemiant effects, widely used in
heart and blood vessel diseases [111]. Probucol have strong antioxidant properties being able
to block oxidative modification of low density lipoproteins (LDL) in vivo [112]. Like other
hydrophobic direct antioxidants, probucol suppresses the propagation of ROS, especially free
radicals [113]. In addition, it appears that this drug can promote endogenous antioxidants
production [114]. Probucol tends to accumulate in fat, therefore it should be expected to
specifically deliver its antioxidant effect in fatty liver diseases [115]. Animal studies with
probucol, show that this drug ameliorates oxidative stress-related parameters in models of
lipid derivate hepatic injury [116].
In clinical trials probucol has been studied alone or associated with other drugs. These
trials have been carried out mostly in Iran by Merat et al. [101, 115, 117] who found that the
treatment with probucol would be effective against NASH. In the first Merat´s pilot study,
biopsy-proven NASH patients were treated during 6 months with 500mg of probucol daily.
Aminotransferase levels improved significantly [115]. The second Merat´s study was a
randomized double-blind controlled study with biopsy-proven NASH patients who were
treated with 500 mg of probucol daily or placebo during 6 month. These results confirmed the
former [117]. Nevertheless, in both studies no final biopsies were performed. The most recent
Merat´s study included biopsies performed before treatment and after twelve month of 500
mg probucol daily, who showed improvements in liver enzymes and necroinflammation
biopsy score but not in fibrosis parameters [101].
Finally, Tokushige et al. [118] evaluated the effect of probucol in combination with
pantethine, the dimeric and more biologically active form of pantothenic acid (vitamin B5)
that among other effects promotes fatty acid oxidation and reduces serum triglyceride. In this
trial, biopsy-proven NASH patients with hyperlipidemia were treated with probucol (500
mg/day) and pantethine (600 mg/day) for six months, showing important improvements in
aminotransferases. In eight patients, liver biopsy was performed before and after the
intervention. Four of these patients showed inflammation improvement, and two showed
fibrosis improvement.
3.2.3. N-Acetylcysteine
N-acetylcysteine is a thiol compound derived from the metabolism of amino acid L-
cysteine. It is an endogenous precursor of glutathione formation with marked direct and
indirect antioxidant properties. Its indirect antioxidant activity is related to its ability to
stimulate glutathione generation. The direct antioxidant effect is derived from the thiol
reductive ability. Several investigations of N-acetylcysteine in NASH animal models show
improvements in different pathology markers. Thus, in diet induced NASH rats Thong-Ngam
et at. [119] found improvement in levels of total glutathione and decrease in
necroinflammation after treating the animals with N-acetylcysteine. In the same animal model
Baumgardner et al. [120] demonstrated that N-acetylcysteine improves several markers of
NASH progression, including TBARS and activation of cytochrome P450 CYP2E1 isoform,
by inhibiting the development of oxidative stress and TNF-α secretion, without blocking the
development of steatosis.
Only three small clinical trials of N-acetylcysteine in NASH are available. The first study
was a controlled trial conducted by Gulbahar et al [121] in NASH patients treated with diet
management followed by 600 mg/day of N-acetylcysteine. In this trial, significant
improvement in aminotransferase levels was found. Nonetheless, no hepatic biopsies were
performed.
Later in a randomized placebo study with biopsy-proven NASH patients, Pamuk and
Sonsuz [122] found significant decrements in aminotransferase levels in the intervention
group after a month treatment period with oral administration of 600 mg/day of N-
acetylcysteine. Unfortunately, this study lacks of final biopsies too.
Other study, based in combined therapy with N-acetylcysteine (1.2 g/day) and the
hypoglycemiant agent metformin (850–1000 mg/day) during a year, was performed by De
Oliveira et al. [123] in obese biopsy-proven NASH patients. This trial excluded individuals
with comorbidities other than obesity such as hyperlipidemias and diabetes. Biopsies were
performed in all patients at the end of the therapy. After the intervention, most biochemical
and histological NASH parameters showed significant improvement, reducing several

markers of NASH, including fibrosis.
3.2.4. Vitamin C
Vitamin C or L-ascorbate is a glucose derivate lactone, which humans cannot synthesize.
This compound is necessary for a range of essential metabolic reactions (e.g. collagen
hydroxylation) [124]. In addition, L-ascorbate is a strong water-soluble reducing agent with
important antioxidant functions demonstrated in in vitro [125]. This vitamin, in addition to its
direct free radical scavenging abilities, has the property to regenerate oxidized vitamin E and
glutathione to the reduced active forms [126]. Some animal studies have shown that vitamin
C is able to improve liver antioxidant defenses [127] and others showed that this vitamin can
prevent liver induced oxidative damage [128]. Thus, it is reasonable to think that vitamin C
therapy could improve NASH.
The sole clinical trial testing the effect of vitamin C in NASH patients was the previously
mentioned Harrison et al. study [104]. However, in this study it is difficult to establish the
contribution of vitamin C alone, as it was assayed in combination with vitamin E.
3.2.5. S-Adenosylmethionine
S-adenosylmethionine is an amino acid derived from folate cycle by the reaction of
methionine and adenosine triphosphate. It is fundamental in a several group of reactions by
its ability to donate a methyl group, these reactions include DNA methylation and
phosphatidylcholine synthesis [129]. The indirect antioxidant ability of S-
adenosylmethionine would be due to the production GSH through the formation of cysteine.
Animal studies of different models of hepatitis show a significant increased of cytosolic and
mitochondrial GSH after the incorporation of S-adenosylmethionine [55, 130, 131].
Clinical studies have demonstrated that patients with different liver diseases have a
deficient conversion of methionine to S-adenosylmethionine, with the subsequent and
decreased levels of plasma and hepatic GSH [132-134]. S-adenosylmethionine has been
shown to reverse the decreased GSH hepatic levels in liver disease patients after 36 weeks of
oral therapy [135]. In addition, other clinical human studies have shown the benefits of S-
adenosylmethionine therapy in different forms of liver disease, including alcoholic
steatohepatitis and intrahepatic cholestasis of pregnancy [136-138]. To our knowledge, no
published clinical trials to date have used S-adenosylmethionine in NASH.
3.2.6. Betaine
Betaine or trimethylglycine is a physiological metabolite of choline that serves as an
alternative methyl donor in the homocysteine derivate synthesis of methionine and in the
phosphatidylcholine synthesis [139].
The hypothetical indirect antioxidant effects of betaine would be associated with its
ability to increase S-adenosylmethionine [71]. Animal models of alcohol-induced fatty liver
demonstrate that betaine oral therapy increases hepatic S-adenosylmethionine levels, and
prevents the progression of steatosis [140]. Betaine also decreases mitochondrial derivate
apoptosis in rat hepatocytes treated with bile acids [141].
There are very few clinical trials of betaine in NASH. Miglio et al. [142] randomized
NAFLD patients to compare the effects of an oral form of betaine glucuronate in combination
with nicotinamide ascorbate and diethanolamine glucuronate versus placebo. The
intervention group shows improvement in ultrasound scoring of liver steatosis and
aminotransferases, compared to the placebo group. However, it was unknown how many
patients had NAFLD versus NASH because no biopsies were performed.
Other study, an uncontrolled small pilot trial of betaine anhydrous (20 gr daily) in
biopsy-proven NASH patients conducted by Abdelmalek et al. [143] for 1 year, demonstrated
some liver improvements. Overall fibrosis, necroinflammatory grade and steatosis showed
improvements after the final biopsy. Nevertheless, at the end of the trial, serum triglycerides
showed a nonsignificantly increase, supporting a possible betaine lipotropic effect.
3.2.7. Genistein
Genistein is a phytoestrogen present in soybeans, which has a variety of pharmacological
features including, antineoplasic, antioxidant and anti-inflammatory actions. Phytoestrogens
are isoflavones, compounds with well known direct antioxidant properties and proven
abilities to improve various diseases associated with oxidative stress [144].
Finally, a hypothesis of the role of the as antioxidants therapeutic tools in NAFLD or

NASH patients is shown in figure 9-2.
Figure 9-2. Diagram representing the different possible strategies to treat NASH in relation to inhibit
direct and indirect mechanisms associated to oxidative injury focusing in antioxidants agents. Dual
arrows imply bidirectional relation, T imply direct inhibition and dotted T imply indirect inhibition.
Animal models show improvement of NASH after genistein treatment, and recent in vitro
studies revealed that genistein affects proliferation of HSCs and consequently displays
antifibrotic effects. For example, Yalniz et al. [145] showed in a recent study that genistein
remarkably prevented the emergence of NASH by improving the biochemical and
histopathological abnormalities via attenuating oxidative stress. Liver lipid peroxidation
levels were significantly higher in the untreated group in comparison to the genistein group,
measured by MDA detection. In addition, treatment with genistein improved inflammation
parameters decreasing significantly TNF-α plasma levels, a known mediator associated to
oxidative stress. Despite the need to conduct clinical trials to demonstrate the therapeutic
effects of genistein in human NASH patients, it is known that the consumption of soy
products rich in phytoestrogens has proved to have numerous favorable effects on various
diseases. Moreover, the underlying mechanism of action, based mainly in antioxidant
properties, and their animal studies, suggest that this compound, associated to traditional
therapy, could be promising in the treatment of NASH patients.
4. Conclusion and Perspectives

In conclusion, it is clear that the overabundance of oxidant species is a key step in both,
the development of liver steatosis and the progression from simple steatosis to NASH.
Hepatic steatosis should not be considered benign because this condition itself plays a major
role in NASH development. Hence, NAFLD patients should be monitored for possible
prevention of NASH, particularly individuals with comorbid conditions strongly linked to
inflammation and oxidative stress, such as obesity, insulin resistance and hyperlipidemia. The
missing link between simple hepatic steatosis and NASH is associated with metabolic
impairment of the liver due to accumulation of FFAs agents that are strongly associated to
oxidant species production by different pathways. The response to these challenges is partly
determined by genetic factors associated with the activity of antioxidant defense systems.
When these defenses become overwhelmed, oxidative stress is triggered. Consequently, this
metabolic derangement causes chronic inflammation, cell death and fibrosis of the liver. Thus
increased oxidative stress has been well documented in humans as an important mechanism
of progression of statosis consecutively to inflammation and fibrosis. Therefore, a great
interest of antioxidant therapies to diminish liver oxidative damage has arisen, since
metabolic therapy has not given promising results in improving biochemical markers and
liver morphology of these patients. Among the studied antioxidant substances, Vitamin E
seems to have one of the best therapeutic effects, likely to be improved in combination with
other antioxidants agents, suggesting that the combination of substances with different
antioxidant mechanism could be more effective than the use of only one substance. Other
potentially therapeutic agents, such as probucol, vitamin C and betaine among others, have
been used on the basis of there direct or indirect antioxidant properties. Moreover it should be
noted that more clinical trials are currently being performed in order to improve the clinical
outcome of these patients.
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Chapter X
Neurodegenerative Disorders
Rodrigo Pizarro
Institute of Biomedical Sciences,
Abstract
Oxidative stress has been related to the pathogenesis of virtually every
neurodegenerative disease. However, two clinical entities stand out for the major
epidemiological burden they provide, as they are intimately related to our increasingly
aging population. Alzheimer’s disease and Parkinson’s disease are the two most
prevalent neurodegenerative diseases affecting roughly over 5.5 million people in the
United States alone. In spite of this, the understanding of their underlying
pathophysiological mechanisms is scarce, and thus, they have remained difficult to treat,
prevent or cure. Early diagnosis is fundamental, as is in early stages of the
neurodegenerative process when therapeutic interventions in both animal models and
clinical trials have proven more beneficial. However, early detection can be a painstaking
procedure due to their subtle, highly unspecific first clinical features and still rather
undeveloped biomarkers. It is in all of these tasks where the understanding of the roles of
oxidative stress in the pathogenesis of such conditions has been proved beneficial.
Biochemical markers of oxidative stress now seem a feasible way of early detection of
such diseases. We could also give possible explanations for the mixed results obtained in
clinical trials using antioxidant supplementation against neurodegenerative diseases. The
task now is to continue looking deeper at oxidative stress in the mechanisms of such
diseases, but also to develop new therapeutic resources to meet the needs of a rapidly
growing population. This chapter deals with the general pathophysiology of oxidative
stress and its role in the pathogenesis and antioxidant supplementation in the detection,
understanding and treatment of Alzheimer’s disease and Parkinson’s disease.
258 Rodrigo Pizarro
1. Introduction
Neurodegenerative diseases (ND) are becoming ever more prevalent with the increasing
amounts of aging population. This new epidemiologic scenario proposes a new endeavor to
both clinicians and the scientific community, as the knowledge of the pathogenesis of these
diseases is scarce, and thus, have remained challenging to treat, prevent, or cure.
Neurodegenerative processes have been revealed in an enormous and heterogeneous set of
inherited and sporadic conditions, characterized by two main events: 1. A progressive
neuronal loss in the affected central or peripheral nervous system structures, leading to
clinically relevant impairment of the cerebral function and; 2. Clinical manifestations of a
progressive decline in cognitive (dementia) and/or motor function that is beyond what might
be expected from normal aging [1].
1.1. Normal Physiologic Cognitive Decline
The normal aging process is generally associated with a gradual loss of neurons, a lower
ability of the brain to create new synapses and various biochemical changes at the membrane
level. The latter influences axonal signal transduction, regulation of membrane bound
enzymes, ion channel structure as well as the maintenance of various receptors [2]. Also,
cerebral blood flow decreases with aging and is associated with a loss of endothelial function
[3]. Endothelial cells form a critical component of the blood-brain barrier (BBB) and are
actively involved in the transport of nutrients from the blood to the brain [4]. Cognitive
deterioration is so frequently observed in the elderly that it is commonly thought of as an
inevitable end-point of normal cerebral decline. However, dementia increases its prevalence
in an exponential manner up to age 80, where its prevalence begins to drop to completely
flatten by age 95, where it stabilizes at 40%. Therefore, neurodegeneration should be
considered an age-related- pathological process, distinct from those implicated in normal
ageing [5].
1.2. The Burden of Neurodegenerative Diseases
Dementia currently affects over 24 million people worldwide, with 4.6 new reported
cases each year. With the increasing aging of the population, it’s to be expected that this
figure will double in the next 20 years, up to an estimate of 42.3m in 2020 and 81.1m by
2040 [6]. Older age has been consistently associated with an increased risk of developing
dementia [7]. The prevalence of dementia in people age 60 to 64 is roughly 1%, while it can
reach well over 30% in people over 85 years old [6]. Dementia represents a leading cause of
death in the elderly. It’s a common cause of morbidity and mortality, with 50% of females
and 40% of males aged over 84 years dying of dementia [8]. As in any age-related pathology,
the shorter life expectancy of men biases lifetime history of the disease and the proportion of
death rates between sexes [7].
Dementia has been systematically underestimated in mortality data, as recent studies
reveal. Death certificates tend to list the immediate cause of death of patients with dementia
as pneumonia, sepsis or trauma while omitting dementia completely. Physicians tend to
Neurodegenerative Disorders 259
overstate other causes of death, such as cardiovascular events and/or hesitate to determine
dementia out of fear of the social pressure, diagnostic limitations or social security issues [8].
Dementia’s associated morbidity and mortality rates can be explained due to its effects
on common physiologic processes such as swallowing. Successful swallowing requires the
input from cortex, sub cortex, brain cortex and cranial nerves [10]. The alterations in the
process of swallowing can thereby predispose the person with dementia to dehydration and/or
malnutrition, and even acquire and die from aspirative pneumonia [9, 10].
Figure 10-1. Sources of reactive oxygen species involved in the pathogenesis of Alzheimer’s disease.
There is sufficient evidence to support the establishment of oxidative stress before the deposition of the
Aβ plaques (primary insult?). Aβ-peptides are generated from APP. Aβ peptides as well as APP have
been found inside mitochondria and included in membranes where they increase ROS production by
complex I uncoupling. However, Aβ peptides are also ROS generating agents per se. Other sources of
ROS are metallic ions (Fenton reaction), and NADPH oxidase. Oxidative stress leads to grave
macromolecular damage and microglia activation, which leads to further oxidative stress and pro-
immflamatory chemokine production. Reactive oxygen species and inflammation promote NF-κB
activating pro-inflammatory genes perpetuating the oxidative stress-inflammation cycle until the cell
sustains irreversible damage to progress into neurodegeneration.
1.3. Diagnostic Procedures and Potential Treatments
Even though the sensitivity of clinical diagnosis can reach 93% in skilled hands, its
reported specificity is much lower, at 55% [11]. This has left dementia as a severely under
diagnosed entity. The recognition of dementia at early stages or its less frequent presentations
can be even more challenging on solely clinical basis. With the introduction of potentially
successful treatment strategies, the need for a reliable biomarker has become even more
pressing. New neurochemical determinations are now being tested to detect the disease at its
260 Rodrigo Pizarro
initial stages, where interventions have been more promising. These procedures should also
aid in the process of making a correct differential diagnosis of the disease in its unusual
presentations [12].
Several relevant alterations have been reported regarding the poorly understood
pathogenesis of neurodegenerative disorders. Among them, the role of oxidative stress has
been well established as one of the primary events in the neurodegenerative process [13-15].
Moreover, the evidence regarding the attack by reactive species of oxygen (ROS) and
nitrogen (RNS) on lipids, proteins and DNA has been found in virtually every type of brain
disease, as well as in the physiologic aging process [1].
1.4. The Role of Oxidative Stress in Neurodegenerative Diseases
Reactive oxygen species are constantly being produced by normal cell metabolism [16].
In normal cell functioning, there is a balance among the ROS production and the antioxidant
defense. If this thin balance is altered, due either to excess in the ROS production or a deficit
in the antioxidant defense, oxidative stress is established. Depending on the magnitude of this
phenomenon, there are several possible cellular responses. Moderate oxidative stress leads to
the activation of cell proliferation and protein induction mechanisms with contained
macromolecular damage. In massive ROS production and liberation there is inevitable
damage of macromolecules such as lipids, proteins, RNA, and DNA, which has been shown
to be involved in the etiology, and/or progression of multiple human diseases [17-19].
Macromolecular damage leads to cellular malfunctioning and if the sustained injury reaches a
point of no return, the cell enters programmed death or necrosis [20, 21].
It has been well established that the mitochondria is a major contributor of ROS
production and thus, tissues with high metabolic rate are in need of high antioxidant
protection, to cope with these exigent requirements [21]. There would only be a ROS
handling deficit under massive pathologic hyper-production as occurs in mitochondrial
dysfunction as well as hypo-metabolism and/or when ROS are supplied from external sources
as occurs in pathologic conditions. Therefore, these entities have been related to the
pathogenesis of neurodegenerative procecess such as AD [17]. In hypoxemia, for example,
there is a raise on the mitochondrial ROS production, by the effect of Ca+2 overload and other
mechanisms, leading to an augment in superoxide (O2•–) that surpasses the antioxidant
defense [20-22](for further general aspects of oxidative stress, refer to Chapter 1).
Cerebral tissue appears particularly susceptible to oxidative stress-mediated injury. The
central nervous system (CNS) has a high metabolic oxidative rate. While accounting for only
2% of the body weight, 20% of the resting body oxygen consumption is destined to cerebral
functioning [1]. At the same time, the brain is composed mostly by post-mitotic neurons, and
has a very high content of substances susceptible to oxidation such as polyunsaturated fatty
acids (PUFA) and catecholamines. It also posses a high content of transition metals and
ascorbate levels, which act together as potent pro-oxidants, coupled to relatively limited
antioxidant capabilities. Thus, the brain is quite exposed to the attack of oxidative stress [1,
19, 23].
For many years researchers have been trying to elucidate the precise role of ROS on the
pathogenesis of neurodegenerative disorders. However, as the pathogenesis of such diseases
has remained elusive, treatment and prevention strategies have not been successful in
modifying their natural history, or considerably diminishing their symptoms in order to
improve the quality of life of their victims [19]. There are several hypotheses to explain the
mechanisms of ND. Among these, the role of oxidative stress has been gaining strength, as it
encompasses the action of many other agents such as redox active trace metallic elements,
mitochondrial dysfunction and oxidative injury through pro-oxidant peptide (β-amyloid)
mechanisms [24]. Over the last decade, free-radical-mediated damage has been associated
with virtually all stages of AD [19, 25]. Data from clinical trials and experimental models
suggests that the pathologic expression of AD’s characteristic proteins is related to the
establishment of oxidative stress per se leading to increased membrane lipid peroxidation
[26-28].
This chapter will focus on the role of oxidative stress in the pathogenesis of ND and
available evidence to sustain the use of redox-state modifying strategies. In order to achieve a
clearer understanding of the matter at hand, Alzheimer’s and Parkinson’s disease will be
analyzed separately from further on.
Alzheimer’s Disease
Alzheimer's disease (AD) is the most frequent neurodegenerative disorder associated
with the onset of dementia in the elderly. It’s the most common form of dementia in adults
over age 65, and the fourth leading cause of death in the United States, currently affecting
over 4.5 million Americans. The mean expected lifespan following diagnose is up to 8.5
years with a range of 1–25 years [29-31]. Alzheimer’s disease is a mainly sporadic disease,
where ageing represents the main risk factor [32]. Studies reveal that up to 5% of AD could
be caused by missense mutations for either Alzheimer β-amyloid (Aβ), precursor protein
(APP) or some of the enzymes involved in its metabolism [12].
Alzheimer’s disease presents a cognitive and memory impairment mostly derived from
hippocampal malfunctioning, leading progressively to the dramatic and socially disabilitating
condition of the well known, late stages of the disease. Clinical features of AD include
anterograde amnesia and loss of language, motor skills, abstract reasoning, concentration and
executive function that have a substantial effect on daily functioning (table 10-1) [33].
Mild cognitive impairment (MCI), as first described by Petersen et al [34] (table 10-2), is
defined as a cognitive decline of the individual which is more pronounced that what should
be expected for his/her age and education level, but does not interfere in a marked manner
with the activities of daily life [35, 36].
There is a well-established augmented risk for patients with MCI, who are up to 10 times
more likely to develop AD than the general population. Progression from MCI to early AD
dementia increases annually at a rate of 10-12% per year in contrast to the 1-2% of general
population. Conversion to dementia is finally 80% by the sixth year of follow-up, with only
5% of patients remaining stable or reverting to normal after the sixth year of follow up [37-
39]. Early AD dementia can be diagnosed according to the criteria described on table 2.
Further progression of the disease leads to late-stage AD (LAD) characterized by severe
dementia with profound global cognitive deficits and motor compromise [19].
262 Rodrigo Pizarro
Table 10-1. National Institute of Neurological and Communicative Diseases and

Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA)
Diagnostic Criteria for Alzheimer’s Disease [35].
Symptoms Exclusion criteria

Dementia diagnose*1 including clinical dementia Disturbance in consciousness
rating scale score of 0.5-1
Decline in one or more areas of cognition*2 Onset age outside 40-90
Progressive worsening of cognitive function*3 Systemic or neurological condition that could
otherwise explain dementia
Impaired ADLs
*1 Established by clinical evaluation and mental status tests.

*2 In addition to memory.
*3 From a previous higher level.
Table 10-2. Diagnostic Criteria Required for the diagnose of Mild Cognitive
Impairment [34].
Symptoms Preserved functions

Memory complaints Intact general cognitive function
Objective memory impairment* Intact activities of daily living (ADLs)
Clinical exclusion of dementia
*According to age and education based on a score of 1.5 standard deviations from the mean of controls
on the CERAD Word List Learning Task [182].
2. Pathophysiology
2.1. Histopathology and Molecular Biology
Hyperphosphorylated forms of Tau proteins and Aβ peptides are responsible for the
classic histological hallmarks of the disease: the presence of neurofibrillary tangles (NFT)
and the accumulation of cerebral senile plaques (SP) of Aβ deposit. These findings are also
accompanied by loss of neurons (cholinergic in particular) and synapses, in the forebrain, and
depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Other frequent
histological findings are neuropil thread formation, proliferation of reactive astrocytes in the
entorhinal cortex, hippocampus, amygdala and association areas of frontal temporal, parietal
and occipital cortex [12, 19, 33, 40]. The etiology of the disease is still unknown, but current
hypotheses focus on synaptic and neuronal loss and the role of amyloidogenic and/or Tau
proteins tightly collaborating with oxidative stress, mitochondrial and vascular dysfunction as
primary events of the disease [41, 42].
Tau is a neuronal protein present in axons and dendrites where it promotes tubulin
polymerization and stabilizes microtubules and thus contributes to cell structure, axon
transport and cellular growth. Neurofibrillary tangles consist of intracellular filaments
deposits of the hyperphosphorylated form of Tau protein. The hyperphosphorilation of Tau
prevents the protein from binding to microtubules, causing destabilization of cell structure
and thereby, likely contributing to the loss of axons, dendrites and synapses [43-45].
Alzheimer β-amyloid is a 39–42 amino acid peptide generated by sequential proteolytic
cleavage of amyloid β-protein precursor protein (APP), a large transmembrane glycoprotein
that is initially cleaved by the β-site APP cleaving enzyme 1 (BACE1) and subsequently by
membrane bound proteolytic enzymes, called secretases in the transmembrane domain [46-
48]. β- and γ-secretase, are the two main proteases that cleave APP at the amino and
carboxyl-terminus of the Aβ peptide, respectively and are hence, directly responsible for Aβ
peptide generation. On the other hand, a less abundant α-secretase promotes a non-
amyloidogenic pathway of APP proteolysis. Alpha-Secretase activation may even have the
additional advantage of generating the putatively neuroprotective sAPP-α, as well as
preventing neurotoxic Aβ peptide formation [49]. Its potential therapeutic properties will be
discussed further on.
The resulting length of the Aβ protein is dependent on initial cleavage of the extracellular
domain generating the amyloidogenic end products Aβ 1-42 and Aβ 1-40 [50]. Low
molecular weight oligomers, particularly Aβ 1-42 have been found to be the most toxic for
neurons and form aggregates that appear to be the predominant species in SP. The ratio
between Aβ 1-42 and Aβ 1-40 in CSF correlates directly with the onset age of AD [51]. It has
been suggested that Aβ peptides might have a toxic effect, and impair synaptic plasticity long
before its SP formation and deposition [52]. Aβ appears to promote neuronal death, at least in
part by the generation of oxidative stress. Indeed, this process seems to be dependent of the
predominant β-sheet conformation. It has been proposed that soluble Aβ might be the initial
event that triggers the neurodegenerative cascade. However, whether oxidative stress is cause
or consequence of the amyloid deposition is still in dispute [53]. It is also highly unlikely to
be the sole element contributing to the progressive nature of the disease. Nevertheless, the
exact mechanism through which Aβ can interfere with normal synaptic physiology and
contribute to cognitive deficit through a preferentially basal forebrain cholinergic cell loss is
yet to be determined [44].
Senile plaques can be differentiated into two histological forms; a) diffuse plaques of
amorphous extracellular neurite lacking, deposits of Aβ or b) neuritic plaques, composed of
extracellular deposits of insoluble Aβ surrounded by dystrophic neurites, activated astrocytes
and microglia. Recent studies suggest that Aβ can impair synaptic plasticity through
mechanisms that might contribute to cognitive decline in AD. Evidence is mounting that Aβ
oligomers can mediate these effects, possibly accounting for why plaque number is such a
poor predictor of cognitive impairment. The deposit of SP is a frequent finding in the elder,
not always linked to cognitive decline. Early studies suggested that there was a direct relation
between the deposit of SP and cognitive decline. Further studies have shown that the
presences of NFT and synapse loss are better histological markers of the disease. Indeed, the
density of terminals containing the acetylcholine-synthesizing enzyme, choline acetyl-
transferase in the neocortex shows a negative correlation with the severity of dementia and is
already altered in the early disease [17].
Interestingly, there is a direct link between the function of cholinergic neurons, the
cerebrovascular system and amyloid pathology. Acetylcholine is a potent vasodilator
affecting cerebral blood flow [54]. Amyloid deposits are also found around cortical vessels
264 Rodrigo Pizarro
where they promote Aβ deposition that consequently contributes to brain hypoperfusion or

vice versa [40]. Also, endothelial dysfunction of cells participating in the BBB may
contribute to inadequate supply of nutrients to the brain. Indeed, increased hypertension,
leading to augmented BBB permeability in cortex and hippocampus has been directly
associated with Aβ deposit in murine models [55]. Current evidence also supports a role for
cholinergic innervation in the non-amyloidogenic maturation of APP, whereas amyloidogenic
related peptides depress the activity of cholinergic neurons. These cholinergic neurons
innervate the hippocampus and neocortex, being central to the loss of cognitive function in
AD. It has been shown that Aβ peptides may have acute detrimental effects on acetylcholine
synthesis and release and are neurotoxic on the long term [40].
For a long time researchers have been trying to elucidate If Aβ and Tau modifications are
related, or represent parallel mechanisms in the pathogenesis of AD. There’s evidence to
support that Aβ toxicity can induce Tau phosphorylation and production of 17 kD Tau
fragments. Moreover, there’s evidence that Tau-deficient neurons might be more resilient to
Aβ toxicity independently of Aβ deposit, or dystrophic neurites [56]. Thus, Tau appears to be
essential for the Aβ-mediated neurotoxicity, linking the two pathological features
mechanistically to the loss of cholinergic neurons in AD [57]. Also very interesting are recent
findings revealing that NFT and Aβ may be linked by their interaction through the Wnt
pathway [58].
2.2. Oxidative Stress in Alzheimer’s Disease
Multiple studies have underlined the importance of oxidative stress in the pathogenesis
of several ND diseases such as AD. However, the origin of the initial insult has remained
elusive to these days. Numerous studies have shown evidence of increased levels of oxidative
damage in brain tissue and cerebrospinal fluid (CSF) of patients with ND [59]. We will
explore the source of ROS in the CNS and the antioxidant endogenous defense, to finally
review the most relevant biological markers of oxidative stress.
2.2.1. Sources of Reactive Oxygen Species
Besides ROS produced in the CNS from normal and altered mitochondrial function, and
the presence of transition metals, also recent studies now recognize the enzyme NADPH and
Aβ peptides per se as very relevant ROS producing agents [60, 61]. It is possible that all
these elements interact, especially at early stages of the disease, where Aβ could enter the
mitochondria increasing ROS generation. Recent post-mortem AD studies and transgenic-
mice studies have revealed that APP can be found in mitochondrial membranes where they
can disrupt the electron transport chain leading to oxidative stress-mediated irreversible cell
injury [60]. There is evidence to support the role of APP and Aβ in this organelle malfunction
as they are both present in the mitochondrial membrane, interacting with mitochondrial
proteins and disrupting its import channels, intracellular transport and altering the electron
transfer chain, leading to a ROS overproduction and further mitochondrial damage [62].
Recent evidence supports the establishment of oxidative stress before the appearance of
Aβ plaques. Researchers have found that levels of 8-hydroxyguanosine (8OHG), an oxidized
derivative of guanosine, and protein nitrotyrosilation, are quantitatively increased at early

stages of the disease, and decline with disease progression. Also there was an inverse
relationship between histological findings (Aβ deposition and NFT formation) and duration
of dementia. Furthermore, oxidative stress was found to be much lower in familiar forms of
AD (ApoE e4 allele) and Down syndrome, in which abundant Aβ deposits were found. All
these findings support the role of oxidative stress as a putative primary event in the
pathogenesis of the neurodegenerative process [13].
Alzheimer β-amyloid deposits have been related to the activation of microglia through
binding to scavenger receptors, which leads to a pro-inflammatory response, characterized by
the release of cytokines and ROS. Microglial activation also leads to an increase in the
clearance of Aβ deposits, compatible with the role of oxidative stress as a primary event in
the pathogenesis of AD [63, 64]. In addition to the Aβ induced oxidative stress and
inflammatory damage by microglia activation there’s also free radical production though
other sources such as Fenton reaction. All this oxidative stress on the neuron leads to DNA
and RNA oxidation, lipid peroxidation of biological membranes to produce 4-
hydroxynonenal (HNE), malondialdehyde (MDA) and protein oxidation [17, 65, 66]. Pro-
oxidative state also promotes NF-κB activation leading to the transcription of pro-
inflammatory genes. These are responsible for the increased pro-inflammatory cytokine
release and activation of endothelial cell adhesion molecules such as 1,25 monocyte
chemoattractant protein-1,26 and others related to atherogenesis [67].
2.2.2. Antioxidant Defense
Immunohistochemical studies have revealed local increases in CU/Zn superoxide

dismutase (SOD) and catalase over NFT and SP against age-matched controls [68]. The
antioxidant enzyme hemeoxygenase-1 has been shown a local increase at NFT [69]. Also as a
response to oxidative damage, DNA repair in AD has shown increased levels of excision
repair cross-complementing gene products, apparently in an attempt to increase DNA
repairing capacities [70]. However, during the progression of the disease, DNA repairing
capacity might become overwhelmed [71].
Glutathione (GSH) is the most abundant peptide in the CNS. This tripeptide is present
predominantly in its reduced (GSH) form, playing a fundamental role in defense of brain cells
against oxidative stress as a potent intra and extracellular antioxidant, located in epithelial
glial cells and neurons. Apart from its numerous essential cellular functions it also plays vital
roles in the nervous system, such as regulation of glutamatergic transmission and protection
against glutamate-induced exitotoxicity, and participates in the intracellular Ca+2 homeostasis
through the activation of calcium-sensing receptors. It also protects the neuron against Ca+2
mediated cell death, antagonizes neuronal apoptosis, redox modulation of several ionotropic
receptor currents. It also acts possibly as an excitatory neurotransmitter at sites of its own
[72]. The existence and location of these putative GSH receptors has been described in brain
cortex and spinal cord, however not yet in the hippocampus [73]. With such critical roles in
the CNS, its role in the pathogenesis of various ND such as AD, schizophrenia and Parkinson
disease seems feasible [74].
266 Rodrigo Pizarro
Glutathione participates in the modulation of taurine release through complex regulation

of several ionotropic glutamate agonists. Taurine acts as an inhibitory neuromodulator and
shares common features with GSH. Taurine is also critical during SNC development,
participating in functional maturation and cell migration in the cerebellum. There’s evidence
that shows that GSH’s neuroprotective and plastic functions are only possible through its
interaction with taurine. Both GSH and taurine share neuroprotective roles and interact
critically in the developing rodent hippocampus. At low extracellular concentrations (μM),
GSH is capable of inhibiting the NMDA receptor-mediated taurine release, and therefore
affect the development of excitatory neural circuits in the hippocampus. However, at higher
concentrations (mM), GSH enhances kainate-evoked taurine release, providing an
exitotoxicity-attenuating effect. Therefore extracellular GSH, in collaboration with taurine,
appear as essential neuroprotectors during neuronal development. Glutathione is an important
antioxidant, and taurine an inhibitory neuromodulator playing a collaborative role at such
critical stages [75].
It has also been reported that in the striatum of adult mice, reduced (GSH) and oxidized
glutathione (GSSG) may also have an exitotoxicity-attenuating effect by preventing
excessive dopamine depolarization-evoked release and the toxic effects of high glutamate,
and by modulating the redox state of Ca+2 channels [72]. Glutathione additionally seems to
upregulate the glutamatergic-evoked release by regulating the redox state of both non-NMDA
and NMDA receptors. It is thought that the regulation of striatal dopamine release by GSH
may have a neuroprotective role, while preserving normal neurotransmitter release, relevant
to extrapyramidal motor functions [73].
As a consequence of high magnitude, sustained oxidative stress, the antioxidant defense
may become surpassed: GSH gets depleted and/or GSSG augments and therefore, the
GSH/GSSG ratio tends to decrease leaving the cell exposed to oxidative stress induced
irreversible damage. The GSH/GSSG ratio is a sensitive marker of intracellular oxidative
stress [16, 76].
2.3. Animal Models of Alzheimer’s Disease
Since the early nineties, several animal models have been successful in reproducing some
of the pathologic hallmarks of AD. In fact, most of the data from clinical studies has been
validated by experimental data on transgenic mice models [23]. The transgenic (Tg) 2576
mouse model of AD-like amyloidosis manifests evidence of increased brain lipid
peroxidation, before the surge in Aβ levels and deposition [77]. In the same model, there is a
concomitant increase in nitrogen reactive species and antioxidant enzymes with the onset of
the Aβ deposit [78]. Similarly, APP and presenilin 1 (PS1) double knock-in mice (APP/PS1)
manifest features of lipid and protein oxidation at early stages of their phenotype [79]. Also,
deletion of the prostaglandin E2 receptor in the double mutant APP/PS1 mice, results in less
brain lipid peroxidation and a significant decrease in Aβ levels and deposit [80]. Further
recent evidence shows that in a Tg2576 AD-like Aβ mouse model, the use of a thromboxane
(Tx) receptor antagonist, blocks the directly delivered into the brain isoprostane iPF (2α)-III
induced Aβ levels and deposit. This suggests that Tx receptor activation mediates the effects
of iPF (2α)-III on Aβ. This hypothesis was supported by cell culture studies that showed that
Tx receptor activation increased Aβ and secreted APP ectodomains [81].
In a recent triple transgenic murine model (3xTg-AD), which develops SP and NFT,
levels of antioxidants are decreased and by contrast, lipid peroxidation is increased before the
appearance of AD-like pathology [82]. In APP transgenic mice, crossed with Mg-SOD
heterozygous deficient mice, increased brain lipidperoxidation, has been associated to a
significant raise in Aβ levels and plaque deposition [83]. Also, the cross of alpha-tocopherol
transfer protein knockout mice with a AD transgenic model mice (Tg2576) manifests an
earlier, more severe cognitive dysfunction, associated to increased Aβ deposits, succesfully
ameliorated by vitamin E supplementation [84].
An extensive variety of biomarkers of oxidative stress have been related to the
pathogenesis of AD. All these signature markers have been used to support the “oxidative
stress hypothesis” of AD. A simplistic approach to this hypothesis would sustain that
antioxidant supplementation should suffice to stop the ND progression. Conflicting data
regarding this aspect have generated a wave of criticism towards the validity of the
hypothesis, in need to be clarified [23].
2.4. Biological Markers of Alzheimer’s Disease
As most promising effects of treatment against AD have been found when the
intervention takes place at early stages of the disease, there is a pressing challenge to
diagnose it as soon as possible. However, as the disease has a multifactorial pathogenesis it
has not been a simple task [16]. With the increasing prevalence of the disease, clinicians
require the development of reliable diagnostic testing in AD. The general consensus was that
sensitivity and specificity for such instruments shouldn’t be underneath 85% and 75%
respectively and of course, that it could be used in vivo [85]. In order to achieve this, a
peripheral marker (i.e. blood or CSF) to assess the biochemical alterations of the disease
would be also highly desirable.
2.4.1. Protein Oxidation
Proteins are abundant in peripheral blood and targets for ROS oxidation. The advantages
of using protein oxidation in comparison to other markers can be explained due to their early
formation and greater stability. Protein oxidation is an early event in oxidative stress and its
degradation process takes hours to days in comparison to the few minutes taken for the
removal of damaged lipids [16]. The half-life of carbonyl groups in peripheral blood is larger
than markers of lipid peroxidation such as MDA [86]. Increased levels of protein
carbonylation have already been reported in other diseases related to increased oxidative
stress such as diabetes, inflammatory bowel disease, chronic renal failure, sepsis and arthritis
[17, 86]. Increased plasma levels of protein carbonylation can also be observed in MCI, and
to a greater extent, in AD in comparison to healthy age-matched controls, product of the
oxidative stress-inflammation of the disease. In AD and MCI, protein oxidation mostly
occurs in the inferior parietal lobe, the cortex and hippocampus. This is not the case in the
cerebellum where Aβ deposit is scare. In other words, protein oxidation occurs in brain
268 Rodrigo Pizarro
regions where major Aβ deposit can be found during the advanced disease [87, 88]. Levels of
3-nitrotyrosine, another marker of protein oxidation, are elevated in brains of MCI subjects
compared with controls, who also exhibit oxidative modifications of several specific protein
enzymes, including a-enolase and glutamine synthase [89, 90] The accumulation of oxidized
proteins through the loss of their catalytic properties or by interruption of regulatory
pathways reflects a surpassing of the cellular repairing capabilities by oxidative stress [91].
2.4.2. Lipid Peroxidation
Poly-unsaturated fatty acids, compounds particularly rich in double bonds, are very
susceptible to lipid peroxidation [66]. Post-mortem human studies have shown increased
lipidperoxidation in the brain and CSF of AD victims compared to controls, as revealed by
several markers such as MDA and 4-hydroxynonenal [65]. F2-isoprostanes (F2) are
prostaglandin compounds, also product of the ROS-induced PUFA oxidation. These
compounds have been found elevated in typical lesion sites and CSF of AD and MCI
patients, compared to age-matched controls [92]. Furthermore, increased levels of 4-
hydroxynonenal and F2 have been found in plasma of AD patients and thus, the possibility of
using peripheral blood determinations seems feasible [93].
2.4.3. Glutathione
Another already discussed viable marker of systemic redox state that can be determined
in peripheral blood is the GSH/GSSG relation. It has been previously validated as a useful
risk indicator in several human diseases including cancer, atherosclerosis, diabetes, and
preeclampsia [16, 17].
2.4.4. DNA Damage
Even though all macromolecules are susceptible to the attack of ROS, in particular to
OH, DNA is thought to be especially sensitive. Of the two types of DNA, nuclear (nDNA)
and mitochondrial (mtDNA), the latter is more unprotected, as it is closer to ROS sources,
lacks the protection of histones and possesses much more limited reparative capabilities.
These macromolecules are exposed to strand breakage, DNA-DNA and DNA-protein cross-
linking and DNA base modifications such as those derived from oxidation. The most
commonly used marker of DNA damage is 8-OHG, product of C8 hydroxylation of guanosine
[19]. There’s also evidence of augmented levels of 8-OHG in both mtDNA and nDNA in the
brain of AD victims at initial stages (in MCI) [94]. These findings are also coherent to the
hypothesis that oxidative stress might play a primary neurodegenerative role.
2.4.5. Hyperhomocysteinemia
Hyperhomocysteinemia (hHcy) has been associated with increased thrombogenicity,

oxidative stress, and activation of pro-inflammatory pathways, impaired endothelial function
and atherogenesis [95]. Hyperhomocysteinemia increases oxidative stress by the induction of
NADPH oxidase and iNOS activity, but also by the impairment in the function of SOD and
glutathione peroxidase (GSHpx). Additionally, oxidation of homocystein leads to further ROS
generation [96, 97]. However, the role of hHcy as an independent cardiovascular risk factor
and the efficacy of homocystein lowering against atherosclerosis have been recently
questioned by major clinical trials [98-100]. Even though both studies successfully and
significantly reduced homocystein levels by similar schemes of supplementation with folic
acid, vitamin B6, and vitamin B12, the HOPE trial was unsuccessful in decreasing the risk of
dying from cardiovascular disease in a primary prevention setting [99]. Neither the NORVIT
trial was capable of lowering the risk of recurrent cardiovascular disease [100]. There have
been no reports yet on randomized trials of folate and/or vitamin B supplementation in
relation to dementia or AD as outcomes [101].
Even though some epidemiological studies have suggested that hHcy is an independent
risk factor for MCI, dementia and AD, results have not been consistent [102]. Some studies
have shown higher homocystein plasma levels in AD patients [101, 103]. A prospective study
also indicated that hHcy is a strong, independent risk factor for the development of dementia
and Alzheimer's disease over an 8-year period in the Framingham Heart Study population
[104]. Other studies, however have found no significant association between plasma
homocystein levels and AD [105, 106].
Elevated homocystein levels have been associated to cognitive decline through both
vascular and non-vascular mechanisms [107], and with hippocampal atrophy and cognitive
decline in absence of cerebrovascular disease [108]. In another neuroimaging study, higher
plasma Hcy levels were associated with smaller frontal and temporal lobe volumes and the
presence of silent brain infarcts at MRI, even in healthy, middle-aged adults [107]. Also, rats
exposed to hHcy develop deficits in spatial learning and hippocampal signaling [109].
With the current evidence, the relation between hHcy and AD seems controversial, and
thus, it does not appear as reliable as other biomarkers.
2.4.6. Asymmetric Dimethylarginine
Hyperhomocysteinemia is frequently associated to increased asymmetric

dimethylarginine (ADMA) levels [67]. Asymmetric dimethylarginine is an endogenous
competitive inhibitor of NOS, derived from its substrate L-arginine, inhibiting the NO
production. Asymmetric dimethylarginine has been determined as an independent risk factor
of cardiovascular disease [110]. Significant increase in the ADMA levels has been reported
in patients with AD, independent of other elements such as gender, age, BMI, hypertension,
hyperlipidemia, renal function, tabaquism and diabetes [111]. Cerebrospinal fluid (CSF)
ADMA levels have been reported decreased in AD, related inversely to cognitive impairment
[111, 112]. This suggests that the blood-brain barrier remains intact in AD and therefore CSF
and ADMA levels are regulated independently [111]. Decreased ADMA levels might lead to
increased NOS activity and NO production, leading to increased peroxynitrite formation that
might explain the extensive protein oxidative damage reported in AD. Another hypothesis
proposes that elevated systemic ADMA levels may collaborate in the pathogenesis of AD
through chronic cerebral hypoperfusion. Therefore, ADMA should be considered a reliable
predictor of AD [111].
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2.4.7. Genetic Markers
In sporadic as in late-onset familiar AD, ApoE e4 allele has been characterized as a

major genetic risk factor, that lowers the average onset age, decreases neuronal metabolism,
and increases Aβ deposit [13]. This apolipoprotein binds directly to Aβ promoting
fibrillogenesis [113]. Prodromes such as decreased glucose metabolism have been found in
AD victims more than two decades before the onset of the disease. Also metal binding
properties have been related to isoforms of ApoE [13].
Mutations in the gene encoding amyloid APP and in genes of presenilin-1 and -2 (PS2)
have been found to play crucial roles in the pathogenesis of early-onset familial AD [101].
Mutations in the APP gene result in early onset autosomal dominant AD. Mutations in
presinilisn also cause early onset autosomal dominant AD [113]. Presinilin-1 is an integral
membrane protein that forms the catalytic core of the γ-secretase complex, and is thus related
to the accumulation of the Aβ 1-42 protein. The association of an intronic polymorphism
(rs165932) of the PS1 gene with late-onset AD has been documented. However,
contradicting results have been shown in different populations [114]. It seems that the PS1,
PS2 genotype might confer a small level of risk of causing AD in the European population,
but results have remained conflicting [115].
2.4.8. Cardiovascular Risk Factors
The roles of cardiovascular risk factors such as atherosclerosis, hypercholesterolemia,

hypertension, insulin resistance syndrome, and diabetes mellitus on the pathogenesis of AD
are yet to be clarified. There are some elements such as Apo E4 allele, associated with
hypercholesterolemia and coronary artery disease, with a well-established strong risk factor
for AD. However, there is growing evidence that their mechanisms in AD might differ from
those of atherogenesis. On the other hand, there has been mixed results regarding the relation
between other cardiovascular risk factors implicated in AD such as hHcy, cholesterol levels
and the impact of statins on the development of AD [101].
3. Antioxidants in Alzheimer’s Disease

3.1. Statins
Well-established antioxidant properties have been attributed to statins. One of the

proposed mechanisms to explain such benefits is the prevention of Rac-translocation-
mediated NADPH oxidase activation [116]. The use of statins has been associated in some
epidemiologic studies with reduced risk of Alzheimer disease (AD). Recently, the use of
statins has also been associated with decreased typical AD pathological findings [117]. The
Adult Changes in Thought (ACT) revealed a diminished risk for dementia, for subjects who
begun statin intake before the age of 80 [118]. However, a large randomized placebo-
controlled trial, with onset of the intervention at 65 years old, did not reveal a relationship
between statin use and subsequent onset of dementia or AD [119].
3.2. Antioxidant Supplementation
Of the therapeutic approaches to AD, one of the most though of, yet underestimated, is
the role of general nutrition. The brain is acutely influenced and maintained by our diet.
Approximately 60% of this organ’s dry weight is composed of lipids [1]. The aging cellular
membrane is characterized by higher levels of cholesterol, decreased cholesterol turnover and
decreased levels of PUFA. This may be related to poor uptake of PUFA over the BBB,
decreased incorporation into the membrane and/or reduced enzymatic activity [2]. Fats
obtained from our diet directly affect the composition and structure of cell membranes [120].
Obtaining data from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a
total of 2,258 community-based non demented individuals were evaluated regarding the
following of a mediterranean diet consisting of a higher intake of vitamin C, vitamin E,
flavonoids, unsaturated fatty acids, fish, higher levels of vitamin B12 and folate, modest to
moderate ethanol intake, and lower total fat consumption. Adherence to mediterranean diet
was associated with a significant reduction in the risk for AD [121].
Even though epidemiological studies have reported conflicting data, the evidence to date
supports a contribution of nutrition in the risk, and particularly prevention of AD. Increasing
evidence reveals that nutrients are far from being a mere energy substrate, rather, stimulating
neuronal plasticity, and ameliorating ongoing neurodegenerative processes [40].
3.2.1. Animal Models
Several studies using antioxidants have been also been published in AD transgenic mice
models, all of which show a consistent beneficial effect towards their behavioral and
amyloidotic phenotype. Experimental thiamin deficiency, an established model of altered
oxidative metabolism, exacerbates amyloid pathology in Tg19959 mice, by inducing
oxidative stress [122]. Dietary copper, by stabilizing brain SOD activity, reduces Aβ
production in APP23 transgenic mice [123]. On the contrary, dietary aluminum modulates
brain amyloidosis by increasing oxidative stress in Tg2576 mice [124].
Curcumin, is a well-know antioxidant and anti-inflammatory compound, (a polyphenol)
found in Turmeric, the powder obtained from the root of Curcuma longa, frequently used in
Indian cuisine. Curcumin treatment, in low-dose (160 ppm) and high dose (5000 ppm),
significantly lowered oxidized proteins and interleukin-1beta, in Tg2576 mice brains. With
low-dose, but not high-dose curcumin treatment, the astrocytes marker glial fibrillary acidic
protein (GFAP) was reduced, and insoluble Aβ, soluble Aβ, and plaque burden were
significantly decreased by 43-50%. However, levels of APP in the membrane fraction
remained unchanged [125].
Also in Tg2576 mice, vitamin E supplementation, early during the evolution of their
disease phenotype (before SP are deposited), showed quite revealing results. One group of
Tg2576 mice received vitamin E starting at 5 months of age until they were 13 months old,
the second group started at 14 months of age until they were 20 months old. Brain levels of
8,12-iso-iPF2alpha-VI, a specific marker of lipid peroxidation, were significantly reduced in
both groups of mice receiving vitamin E compared to placebo. Trangenic mice Tg2576
administered with vitamin E at a younger age showed a significant reduction in Aβ levels and
272 Rodrigo Pizarro
amyloid deposition. By contrast, mice receiving the diet supplemented with vitamin E at a
later age, when amyloid plaques are already deposited, did not show any significant
difference in either marker when compared with placebo. These results support the
hypothesis that oxidative stress is an important early event in AD pathogenesis, and
antioxidant therapy may be beneficial only if given at this stage of the disease process [126].
Numerous studies have shown that Melatonin is decreased during the aging process and
that AD patients suffer a profound reduction of this pineal hormone. It has also been
demonstrated that melatonin protects neuronal cells from Aβ-mediated oxidative damage and
inhibits the formation of β-sheets and amyloid fibrils. Again in Tg2576 mice, administration
of melatonin partially inhibited the expected time-dependent elevation of Aβ, reduced
abnormal nitration of proteins, and increased survival [53]. Also in wild type (WT) mice,
intraperitoneally injected with Aβ 25–35 peptides, melatonin therapy significantly enhanced
the activities of oxidative scavenging enzymes such as SOD, catalase and GSH levels in the
astrocytes, lymphocytes and hepatocytes. Immunohistochemistry studies reveal that
melatonin prevents the activation of GFAP in neocortex and transcription factor NF-κB in
liver and neocortex of Aβ injected mice. It also prevents the elevation of dopamine depletion
and its degradation products. These findings suggest that melatonin could act as a protective
agent against oxidative damage in AD by scavenging ROS and thus by maintaining the
activities of the antioxidant enzymes, regulating the GSH levels, and preventing Aβ-induced
dopamine turnover [127].
(−)-Epigallocatechin-3-gallate (EGCG), the main flavonoid in green tea has been found
to decrease Aβ-mediated toxicity through an increase in secreted levels of the soluble form of
APP (sAPP-α) in Tg APP overexpressing mice. In further experiments, EGCG or placebo
was administered to mice of the same type, at 8 months of age for 6 months. Several
histopathological analyses revealed that plaque burdens were significantly reduced in the
cingulate cortex, hippocampus, and entorhinal cortex. ELISA of brain homogenates revealed
consistent reductions in both Aβ 1–40 and 1–42 soluble and insoluble forms. In the cognitive
experiments, animals treated with EGCG also showed better performance in comparison to
placebo [128].
Retinoic acid (RA) has been shown to control the expression of genes related to APP
processing by regulating gene expression through its nuclear receptors. In an APP and PS1
double Tg mice, RA supplementation was associated with a significant decrease in Aβ
deposition and Tau phosphorylation. Mice treated with RA also revealed decreased activation
of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning
and memory in comparison to placebo [129].
3.2.2. Experience on Humans
To analyze the experience gathered with humans, we’ll categorize the studies in cohort,
cross-sectional and clinical trials.
Cohort Studies
Five cross-sectional studies have been reported so far. The MoVIES project
supplemented 1,059 rural, non-institutionalized elder residents of the southwestern
Pennsylvania area from year 1989 to 1991. The mean age of the participants was 74.5 years.
The use of nutritional supplements containing vitamin A, C, or E, β-carotene, zinc, or
selenium was measured through self-report. Women and persons with higher levels of
education were more often antioxidant users. Antioxidant users scored better than nonusers
on several cognitive tests, but after data were controlled for age, sex, and education, there
were no statistically significant relations between antioxidant use and cognitive test
performance [130].
More data came from a study that investigated the association between serum antioxidant
(vitamins E, C, A, carotenoids, selenium) levels and poor memory performance in an elderly,
multiethnic sample of 4,809 North Americans. The data for this analysis came from the Third
National Health and Nutrition Examination Survey (NHANES III) a national cross-sectional
survey conducted from 1988 to 1994. The study finally concluded that decreased serum
levels of vitamin E were consistently associated with increasing levels of poor memory after
adjustment for age, education, income, and vascular risk factors. However, serum levels of
vitamins A and C, β-carotene and selenium were not associated with decreasing memory
performance [131].
The Honolulu–Asia Aging Study, is a longitudinal study of Japanese-American men
living in Hawaii. Data for this study were obtained from a cohort of 3,385 men, aged 71 to 93
years followed from 1982 to 1993. Results for this trial were mixed. In the 1988 report, no
protective effect was found for Alzheimer’s dementia. Among those without dementia, use of
either vitamin E or C supplements alone in 1988 was associated significantly with better
cognitive test performance at the 1991 to 1993 examination, and use of both vitamin E and C
together had borderline significance [132].
The Chicago Health and Aging Project (CHAP) is an ongoing study that begun on 1999,
following a cohort of 815, AD free volunteers over 65 years old. After a mean of 3.9 years of
follow up, vitamin E intake from foods was associated with a decreased risk of developing
AD after adjustment for age, education, sex, race, ApoE e4 gene, and length of follow-up.
The protective association of vitamin E was only observed among the ApoE e4 negative.
Surprisingly, the intake of vitamin C, β-carotene, and vitamin E from supplements was not
significantly associated with risk of AD [133]. In a second report, searching for a possible
explanation for the inconsistency of previous studies, the investigators examined the roles of
the different tocopherol forms in the protective vitamin E association with AD and cognitive
decline. They hypothesized that the protective effect was not due to α-tocopherol alone but to
another tocopherol form or to a combination of tocopherol forms. Higher intakes of vitamin E
and α–tocopherol were associated with a reduced incidence of AD. In separate mixed
models, a slower rate of cognitive decline was associated with intakes of vitamin E, α-
tocopherol equivalents, and α - and γ-tocopherols, which suggests that various tocopherol
forms rather than α- tocopherol alone may be important in the vitamin protective association
with AD [134].
274 Rodrigo Pizarro
Finally, the Nurses’ Health Study included 14,968 women, from 70–79 years of age, who
participated in the study between 1995 and 2000. The study concluded that the use of specific
vitamin E supplements, but not vitamin C supplements was related to modest cognitive
benefits in older women. Interestingly, there was a trend for increasingly higher mean scores
with increasing durations of use. Also, benefits were less consistent for women taking
vitamin E alone [135].
The results of the cohort studies are not surprising. It has been well established that
oxidative stress is an early event in AD. If we begin antioxidant supplementation at an
advanced age we would arrive late, as the pathologic changes should had taken place and the
neurodegenerative process already taking course. This is coherent with experimental
evidence of the benefits of early antioxidant supplementation [126], and the benefit of
prolonged antioxidant intake [135].
Prospective Studies
Analyzing the prospective studies we find an even more complex perspective. The
Rotterdam Study, a prospective cohort study conducted in the Netherlands followed 5,395
participants from 1990 to 1999, aged 55 years or older, free of dementia at the moment of
enrolment. After adjustment for potential confounding factors, the use of high intake of
vitamin C and vitamin E was associated with lower risk of AD. This relationship was most
pronounced among current smokers, and did not vary by education or ApoE e4 genotype
[136].
The Canadian Study of Health and Aging (CSHA) conducted a population-based,
prospective 5-year investigation on 894 Canadians over 65 years of age with no evidence of
dementia at baseline. Subjects reporting a combined use of vitamin E and C supplements
and/or multivitamin consumption were significantly less likely to experience cognitive
decline during the follow-up period. However, a reduced risk for incident dementia or AD
was not observed [137].
The Washington Heights-Inwood Columbia Aging Project (WHICAP) studied 980
subjects over 65 years old, free of dementia at baseline for a mean period of 4 years. Neither
intake of carotenes and vitamin C, nor vitamin E in supplemental or dietary form or in both
forms, was related to a decreased risk of AD [138].
The Cache County, cross-sectional and prospective study analyzed 4,540 volunteers, 65
years or older from 1995 to 2000. The use of vitamin E and C supplements in combination
was associated with reduced AD and incidence. A trend toward lower AD risk was also
evident in users of vitamin E and multivitamins containing vitamin C, but no evidence of a
protective effect with use of vitamin E or vitamin C supplements alone, with multivitamins
alone, or with vitamin B–complex supplements [139].
Another report from the Honolulu-Asia Aging Study analyzed 2,459 men, free of
dementia at the first assessment in 1991–1993 and reassessed them between 1991 and 1999.
The intake of β-carotene, flavonoids, and vitamins E and C again were not associated with
the risk of dementia or its subtypes [140].
A subgroup of Duke’s EPESE (Established Populations for Epidemiologic Studies of the
Elderly) followed 616 volunteers from 1986 to 2000 sharing the same profile as previously
described studies. Neither the use of vitamins C and/or E reduced the time to the onset of
dementia or AD [141].
Finally, the most recent evidence came from the prospective-cohort Adult Changes in
Thought (ACT) study. The study involved 2,969 participants of similar characteristics. Over
a mean follow-up of 5.5 years, the use of vitamin E was not associated with the risk of
developing dementia or AD. No association was found between vitamin C alone or
concurrent use of vitamin C and E on either outcome [142].
Clinical Trials
The Alzheimer’s disease Cooperative Study (ADCS) was the first clinical trial to address
the AD burden. The researchers conducted a 2-year, double-blind, placebo-controlled,
randomized, multicenter trial in patients with Alzheimer’s disease of moderate severity. A
total of 341 patients received the selective monoamine oxidase inhibitor selegiline (10 mg a
day), α-tocopherol (2000 IU a day), both selegiline and α-tocopherol, or placebo for two
years. The primary outcome was the time to the occurrence of any of the following: death,
institutionalization, loss of the ability to perform basic activities of daily living, or severe
dementia. However, despite random assignment, the baseline score on the Mini–Mental State
Examination was higher in the placebo group than in the other three groups, and this variable
was highly predictive of the primary outcome. After Mini–Mental State base-line score
adjustments, there was a significant delay in the time to the primary outcome for the patients
treated with selegiline, α-tocopherol, or combination therapy days, as compared with the
placebo group [143].
Later Petersen et al enrolled 769 subjects with MCI from 69 ADCS sites in the United
States and Canada. Following the same controlled trial standards, subjects were randomly
assigned to receive 2000 IU of vitamin E daily, 10 mg of donepezil daily (the most widely
used cholinesterase inhibitor available at the time the study), or placebo for three years. The
primary outcome was clinically possible or probable AD. Secondary outcomes were
cognition and function. As compared with the placebo group, there were no significant
differences in the probability of progression to AD in the vitamin E group or the donepezil
group during the three years of treatment [144].
A large randomized trial was conducted as a part of the Age-Related Eye Disease Study
(AREDS). Between 1992 and 1998, 11 clinical centers randomized 3,640 participants to
receive daily antioxidants (vitamin C, 500 mg; vitamin E, 400 IU; β-carotene, 15 mg), zinc
and copper (zinc, 80 mg; cupric oxide, 2 mg), antioxidants plus zinc and copper, or placebo.
There was no significant effect of intake of these antioxidants on the likelihood of having
cognitive impairment [145].
Finally, the group of Kang et al tested the effect of vitamin E supplementation on
cognitive function using data from the Women’s Health Study (WHS), placebo-controlled
trial. From 1998 to 2008, 6,377 women 65 years or older participated in the cognitive sub-
study. There were no differences in cognitive tests between the vitamin E and placebo groups
at the first assessment (5.6 years after randomization) or at the last assessment (9.6 years of
treatment). Mean cognitive change over time was also similar in the vitamin E group
compared with the placebo group [146].
276 Rodrigo Pizarro
Parkinson’s Disease
Parkinson’s disease (PD) is the most common cause of motor disorder and the second
most frequent age-related neurodegenerative disorder after AD. It affects over 1% of the
population of 65 years or older, with over one million cases in the United States alone.
Parkinson’s disease is a progressive disease with a median age-of-onset of 55 years. Its
incidence increases markedly with age, from 20/100,000 overall to 120/100,000 at age 70
[147,148,149]. As in virtually every ND, the etiology of the disease is still unknown but
probably involves a combination of genetic and environmental factors [150].
Parkinson’s disease is another appealing entity to the “Oxidative stress hypothesis”
paradigm. As we will discuss in this section, it is not the biomarkers of oxidative damage in
CNS tissue samples or experimental models of PD in support of the occurrence of oxidative
stress that are missing. However, similar to other ND disorders, there is still lack of
understanding about the triggering events of the condition and the sites of origin of ROS and
RNS [151].
Clinicians have learned that it is more accurate to refer to “Parkinsonism” or
“Parkinsonian syndromes,” as PD seems more of a collection of distinct neurological
disorders sharing a similar clinical phenotype than a single neurological entity. There more
than 40 distinct entities than can share the clinical features of PD. Clinic-pathological studies
have revealed that 20% of patients with an initial PD diagnose had pathological changes
suggesting other diagnose, and 5-10% of patients with Parkinsonism had the PD histological
hallmarks at the moment of autopsy [1, 151].
There is still much to come to light regarding the pathogenesis of the disease. So much
indeed that the question now is whether Parkinson’s disease is a single clinical entity, or the
common denominator for several Parkinsonian disorders is the formation of Lewy bodies and
the loss of dopaminergic neurons. Are we facing several disorders sharing a common
pathogenic process that may converge at a certain point to become the same disease? Most
researchers agree that the pathogenic cascade may, in fact, consist of common mechanisms. If
this is the case, one may propose that similar molecular machinery is recruited, regardless of
the nature of the initiating pathological factor, somehow facilitating the task of relieving the
burden of these patients. Following this premise, it is possible that oxidative stress is a
common event to such dopaminergic disorders [151, 152]. We will try to address some of
these questions in order to identify the role of oxidative stress in PD, a main event in the
neurodegenerative process, or merely an epiphenomenon in the pathophysiology of the
disease.
2. Pathophysiology
2.1. Histopathology and Molecular Biology
The classical pathological feature of PD is the loss of the nigrostriatal dopaminergic

pathway neurons; the substantia nigra pars compacta (SNpc). The loss of SNpc neurons leads
to striatal dopamine (DA) deficiency, responsible for the motor symptoms of PD.
Characteristic clinical motor features of the disease include: resting-tremor, spasticity,

postural reflex compromise and bradykinesia. Clinical symptoms of PD appear only when
dopamine levels are reduced to greater than 60% that of normal [154]. Replenishment of
striatal DA through the oral administration of the DA precursor levodopa (L-3,4-
dihydroxyphenylalanine) alleviates most of the motor symptoms [147, 153, 154]. The
dopaminergic deficit though, is not sufficient to explain other signs and symptoms associated
with PD. Pathologic findings in other brainstem in subcortical and cortical structures are also
prominent, where widespread neuronal loss has been detected in other catecholaminergic and
non-catecholaminergic nuclei. These less specific neurodegenerative processes are thought to
be responsible for the complex, and non-motor symptoms including disturbances of
autonomic functions and deterioration of cognition [149, 153].
Other distinguishing pathological features of PD include the accumulation of neuronal
intra-cytoplasmic inclusions known as Lewy bodies (LB), found within nearly the entire
characteristic affected brain areas, and gliosis. The role of LB in PD remains unclear.
Hypotheses are very distinct, ranging from a possible contribution in cell death, to non-
significant relevance. Others postulate that their occurrence might indicate that the cell is
evading death by successfully rendering misfolded proteins harmless. Lewy bodies are
particularly rich in aggregated α-synuclein, but also contain several other proteins, including
components of the ubiqiuitin–proteasome system and molecular chaperones, and lipids. The
proportion of neurons containing LB in PD remains relatively constant at 4% regardless of
disease stage. It has therefore been suggested that LB are continuously formed during the
course of the disease and disappear when the affected neuron dies [149, 151].
Alpha-Synuclein is normally abundant in nerve terminals where it’s involved in synaptic
function. Alpha-Synuclein is lipid-associated under physiological conditions but tends to
aggregate in the lipid-free state into higher molecular weight oligomers. Formation of these
aggregates has been directly linked to neurodegeneration in PD. Two mutations in the α-
synuclein gene (A30P and A53T) have been associated with familial PD, but the biochemical
consequences of these mutations are not completely understood. What is known is that during
normal aging and in PD, levels of natively folded α-synuclein increase in the neuronal
cytoplasm [155].
Parkinson’s disease can be classified as primary when it’s due to hereditary or idiopathic
causes or secondary when it has an identifiable underlying pathology. Parkinson’s disease is
a sporadic disease in 95% of the cases; however several genes that lead to a hereditary
parkinsonian disorder have been identified in the last few years. Rarely occurring genetic
defects include mutations in genes such as α-synuclein, parkin, DJ1, PINK1, and LRRK2.
These mutations are transmitted as either dominant or recessive Mendelian traits. Clinically
both, sporadic and familial forms of PD are almost indistinguishable sharing the same basic
biochemical hallmark of a profound dopaminergic deficit [151, 152].
Considering the biochemical function of these genes and mutations, three main,
interrelated, underlying pathogenic pathways can be identified. These are: altered protein
quality control; oxidative stress and mitochondrial dysfunction; and disturbed kinase activity.
The common pathway to these pathologic processes is cellular death, executed by
exitotoxicity, apoptosis and autophagy, being apparently facilitated by the pro-oxidant,
neuroinflammatory progression [152].
278 Rodrigo Pizarro
Prior to the last 5 years, most of the generated evidence regarding the etiology and
pathogenesis of PD was derived from postmortem tissue of animals models exposed to the
neurotoxic compound 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Exposure to
MPTP induces a dopaminergic neurodegeneration that causes a syndrome that mimics PD,
also common to humans. The more recent discovery that mutations in the gene for α-
synuclein can cause an inherited form of PD changed the field completely. Several genes
involved in the pathogenesis of PD have been described to these days. As in AD, these rare
PD genes appear to operate through common molecular pathways. This has led to the
development of novel animal models for the study the disease [147]. We will now revise the
role of oxidative stress in the disease, gathered thanks to this previous knowledge.
2.2. Oxidative Stress in Parkinson’s Disease
As in Alzheimer’s disease, there is growing evidence regarding the importance of

oxidative stress in the pathogenesis of PD. However, there is still much discussion whether
oxidative stress is the primary insult or a secondary event. We will once again explore the
source of ROS in these neurodegenerative processes, and their relation to the antioxidant
endogenous defense. Finally, we’ll review the most relevant oxidative stress-derived
biological markers of the disease.
2.2.1. Sources of Reactive Oxygen Species and Antioxidant Defense
Evidence now exists to support a role for alterations in mitochondrial form and function,
as well as increased oxidative stress in the pathogenesis of PD [153]. Factors contributing to
the increased oxidative stress in PD include: high basal levels of aerobic activity in the brain,
auto-oxidation of DA, its precursors, and metabolites, to form quinones and semiquinones
capable of adducting protein sulfhydryl groups, including GSH, and increased iron levels in
the SNpc contributing to Fenton chemistry through the reduction of hydrogen peroxide
(H2O2) to produce the highly reactive hydroxyl radical (OH•). Histological findings in
postmortem brains from PD patients, confirm high levels of oxidative stress in the SNpc, as
revealed by increased iron concentrations, decreased levels of GSH, increased lipid
peroxidation, and DNA and protein oxidation [148]. However, the earliest reported event of
oxidative stress in PD is depletion of GSH. The GSH depletion precedes mitochondrial
damage and dopamine (DA) loss and the degree of its loss correlates with the disease’s
severity [156].
Proteasome inhibition is another phenomenon of main relevancy affecting neuronal
viability in PD. Proteasomal inhibition in animal models results in most of the biochemical
hallmarks and selective brain damage of PD [157]. Proteasome inhibition has also been
associated to altered mitochondrial lysosomal-mediated degradation and homeostasis, and in
combination with alterations in GSH metabolism [158, 159].
A growing number of clinical and experimental evidence implicate mitochondrial
dysfunction as a fundamental aspect of the increased oxidative stress in the pathogenesis of
PD [150]. Mitochondria isolated from PD patients displays reduced complex I activity [160].
Impairment of the respiratory chain may lead to increased ROS generation, leading to more
oxidative stress and macromolecular damage and selective death of SNpc of PD patients. A
complicated interplay occurs between mitochondria and other cellular machinery that
dramatically affects cellular survival [148, 153].
Mitochondrial dysfunction also leads to increased DA oxidation in its reservoirs,
increasing the damage by liberation of the oxidized neurotransmitter. The oxidative stress-
inflammation cycle can also lead to microglial activation. Microglia activation has been
associated with upregulation of iNOS and NADPH oxidase, leading to increased nitrous
oxide and superoxide anion production respectively. In this sense, augmented protein
nitration has been observed in Lewy bodies’ together with more lipid peroxidation and DNA
strand breakage in areas affected by PD [148]. Accordingly, mutant mice defective in
NADPH-oxidase exhibit less SNpc dopaminergic neuronal loss and protein oxidation than
their wild type littermates after MPTP injections [161]. Also, pharmacologic inhibition of
NOS significantly protects MPTP-injected mice against injury to the SNpc [162].
However, there is still much discussion whether oxidative stress is a primary or
secondary event to the genetic injury. Several PD-associated genes have been found to
influence the balance of mitochondrial fission and fusion, affecting the maintenance of
dynamic networks of these organelle’s tubular structures [153]. Genetic studies additionally
have linked mitochondrial dysfunction with several genes expressing their phenotype in PD
such as α-synuclein, parkin, DJ-1, PINK1, and LRRK2 [163].
2.2.2. Oxidative Stress and Genopathies
Clinically, both sporadic and inherited forms of PD are virtually indistinguishable.

Sharing the biochemical stamp of a profound DA deficit in the SNpc, they have provided
with deeper understanding of PD’s neurodegenerative mechanisms [151].
Mutations to the autosomal dominant LRRK2 gene represent the most frequent cause of
hereditary PD, affecting up to 7% of all PD patients in Europe and up to 40% in Ashkenazi
Jews and North African Arabs [152, 163]. The LRRK2 gene possesses two catalytic domains:
a Ras/GTPase super family, and as serine-threonine kinase. Disinhibited kinase activity
associated to such mutations appears to induce a progressive reduction in neurite length and
branching, both in vitro and in vivo. The PD-associated LRRK2 mutations additionally
harbor prominent phospho-tau-positive inclusions with lysosomal characteristics and
ultimately undergo apoptosis [164].
The PINK1 gene codifies for a serine-threonine kinase with a mitochondrial-targeting
motif at its N-terminal end [165]. This protein has been localized in mitochondrial fractions,
where it is thought to play a role in normal cellular physiology, protecting mitochondria from
oxidative stress and apoptosis [148]. The loss of PINK1 function in mice does not cause
major ultrastructural changes in their mitochondria, but instead leads to functional deficits in
a dopaminergic circuit and age-specific manner with increased sensitivity to oxidative stress
[150].
Mutations of DJ-1 have also been related to the pathogenesis of PD. This putative
antioxidant expresses ubiquitously and localizes to the mitochondrial matrix where it is
capable of reducing ROS formation directly and stabilizing the major antioxidant gene
280 Rodrigo Pizarro
regulating the transcription factor Nrf2 [148]. Over expression of DJ-1 confers enhanced
protection against oxidative stress, while the genetic DJ-1 knockout has an increased
sensibility to ROS [166, 167].
2.3. Biological Markers of Parkinson’s Disease
The same general guidelines as in Alzheimer’s disease apply for PD. The brain is a high-
metabolism organ, rich in fatty acids. Damage to virtually all macromolecules is ubiquitously
seen in autopsy materials obtained from PD patients and evidenced by increased lipid
peroxidation, protein carbonyl modifications, and DNA oxidation. Nitration and nitrosylation
of proteins, particularly α-synuclein and parkin has been documented in PD [151]. Common
deletions of mitochondrial DNA have been reported in DA neurons of the SNpc [168]. These
findings have proved useful in the understanding of the mechanisms of the disease. However,
the challenge now is to develop biomarkers useful in everyday practice. In this regard,
several researchers have validated pathologic features of PD in peripheral samples such as
blood and CSF.
The diagnosis of sporadic PD has remained basically clinical. Accuracy of clinical
diagnoses of PD can reach 90% within experienced specialists [169]. The development of
biomarkers for PD for identifying individuals in risk has been highly desired as in any other
ND, particularly for the important implications of an early, accurate diagnosis and
consequential treatment, as to prevent, and monitorize the progression of the disease.
Furthermore, biomarkers could give further insight of the mechanisms of the disease for a
better understanding of PD, but also of atypical Parkinsonian disorders, generally
unresponsive to current treatments [170].
Markers of oxidative stress in peripheral blood have been recently determined in PD
patients and age-matched controls. Patients with PD had significantly higher SOD activity
and increased lipid peroxidation products (TBARS content) [171]. In a prospective
population-based cohort study among 4,695 participants aged 55 years and older, with an
average of 9.4 years of follow-up, serum levels of uric acid were also associated with a
significantly decreased risk of Parkinson disease [172]. Oxidative damage to proteins is also
significantly increased. On the other hand, levels of 8-OHG in serum and urine have not
revealed to be trust-worthy markers of the disease. Neither has the GSH/GSSG relation, who
has not significantly shown differences between PD patients and age-matched controls [170,
171].
3. Antioxidants in Parkinson’s Disease

3.1. Antioxidant Properties of Current Treatments
Several therapeutic options are available or being tested for the treatment of PD.
However, none except the MAO-B inhibitor rasagiline and coenzyme Q10 have been capable
of modifying the course of the disease [152]. Most approaches have been targeted to cell
death mechanisms, not the initial events of the disease. Monoamine oxidase (MAO) appears
as a relevant source of ROS in PD. Pharmacologic MAO-B inhibition (iMAO-B) with

deprenyl, is a widely used clinical approach. The iMAO-B rasgiline has proved antiparkinson
activity in classical MPTP neurotoxin mice models, where it prevents the degeneration of
SNpc dopaminergic neurons. However, there is evidence that the neuroprotective activity of
rasagiline is independent of MAO-B, and yet its actual mechanism is yet to be elucidated
[173]. Recent reports from clinical trials have confirmed the protective role of new iMAO-B,
suggesting that MAO-B could be a relevant source of ROS in PD [154].
3.2. Promising New Antioxidants Compounds
Another important pharmacologic therapy being studied to diminish oxidative stress in

PD is iron chelation. The potent iron chelator VK-28 is capable of crossing the BBB. It has
proven neurotoxin protective activity in rats. The use of iron chelators in combination with
iMAO has yielded promising results [154, 173]. Further findings came from the iron-
neuromelanin relationship. Neuromelanin binds iron and accumulates during the aging
process. In PD, dopaminergic neurons containing neuromelanin selectively degenerate.
Neuromelanin blocks hydroxyl radical production by Fenton’s reaction, in a dose-dependent
manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus
sparing this major antioxidant molecule in the brain. Blockade of iron into a stable iron-
neuromelanin complex prevented DA oxidation, inhibiting the formation of neurotoxic DA
quinones. This process occurs intracellularly during aging and PD, thus providing evidence
of the neuroprotective properties of neuromelanin [174].
There is also increasing interest in the micronutrient coenzyme Q10 (CoQ10) to treat
neurodegenerative processes. CoQ10 is a fundamental component of the mitochondrial
electron transport chain, accepting electrons from complexes I and II. It serves an important
antioxidant role in both, mitochondria and lipid membranes through its interactions with α-
tocopherol. Coenzyme Q10 is also an obligatory co-factor for mitochondrial uncoupling
proteins. Activation of these proteins reduces mitochondrial uncoupling in the SNpc of
primates, providing neuroprotection against MPTP toxicity [110]. A multicenter, randomized,
placebo-controlled clinical trial analyzed the neuroprotective role of CoQ10. Eighty patients,
diagnosed with PD in the previous five years were randomized to CoQ10 at doses of 300-1200
mg/d or placebo. They were then followed up for up to 16 months. The main endpoint to
measure was tremor score of the United Parkinson’s Disease Rating Scale (UPDRS).
Coenzyme Q10 was well tolerated under the studied dosage and associated with significantly
better UPDRS scores [175]. Even though the results are promising, further larger studies,
with a longer follow-up time will be needed to evaluate the actual benefit of CoQ10 in
delaying the progression of PD.
Other antioxidant substances under current evaluation and possible antiparkinsonian
properties are ginko biloba, L-carnitine, cannabis, estrogen and nicotinamide. The potent
antioxidant properties of the polyphenols found in green tea are also being tested [154]. In
large Asian population cohort studies, neither green tea drinking, nor diet was related to PD
risk [176]. The putative antioxidant role of cannabinoids has been previously questioned.
However, it has been recently demonstrated that the cannabinoid CP55,940 significantly
282 Rodrigo Pizarro
protects and rescues D. melanogaster against a neurotoxic injury through a receptor-

independent mechanism [177].
3.3. Antioxidant Supplementation
3.3.1. Glutathione
Glutathione levels can be raised by slowing its degradation, or by supplementation with

GSH itself or analogs. As GSH does not easily cross the BBB due to its charged cysteine-SH
group, GSH esters have been explored as an alternative. In vivo and in vitro studies have
demonstrated that the GSH precursor glutamyl cisteine ester (GCEE) and glutathione ethyl
ester (GEE) are capable of elevating intracellular GSH levels, and provide protection to
dopaminergic neurons against MPTP induced neurotoxicity [154, 178]. In the only published
clinical trial, the effects of GSH supplementation were studied in nine patients with early,
untreated PD. Glutathione was administered intravenous, 600 mg twice daily, for 30 days, in
an open label fashion. The drug was then discontinued and a follow-up examination carried-
out at 1-month interval for 2-4 months. Thereafter, the patients were treated with carbidopa-
levodopa. Clinical disability was assessed by different rating scales at baseline, and at 1-
month intervals for 4-6 months. All patients improved significantly after GSH therapy, with a
42% decline in disability. Once GSH was stopped the therapeutic effect lasted for 2-4 months
[179].
3.3.2. Antioxidant Vitamin Supplementation
Only a few clinical trials with antioxidant vitamin supplementation have been directed
with limited success. A combination of high doses of α-tocopherol and ascorbate were
administered to patients with early PD in an open-labeled pilot study. Patients were allowed
the concomitant administration of amantadine and anticholinergics, but not levodopa. The
primary endpoint was time until patients needed treatment with levodopa or a dopaminergic
agonist. The time when levodopa became necessary was extended by 2.5 years in the group
under antioxidant supplementation [180]. In 1987 the Deprenyl and Tocopherol
Antioxidative Therapy of Parkinsonism (DATATOP) was initiated to examine the eventual
benefits of the iMAO-B deperenyl and α-tocopherol in slowing the progression of PD. After
14 months of observation, deperenyl significantly delayed the necessity of levodopa. The
effect was largely sustained after 8.2 years of follow-up. However, supplementation with α-
tocopherol did not show any benefits in delaying the progression of PD [181].

Neurodegenerative diseases and Alzheimer’s disease in particular have changed the
epidemiological situation, as we knew it. As the principal risk factor for developing AD is
aging, the disease presents a heavy social and economic burden to our society.
With a poorly understood pathogenesis, the task to develop novel therapeutic approaches
to prevent or delay the progression of the disease, or even alleviate our patient’s symptoms,
has proved a frustrating endeavor. According to the current evidence about the underlying
mechanisms of the disease we can say, at least, that oxidative stress is a mayor initial event
(if not the most relevant), in the triggering of the neurodegenerative process. In this regard,
several sources of ROS have been revealed in the recent years. Especially important, has been
the identification of Aβ peptides as ROS producing agents per se and also participating as
pro-oxidative mediators in pathologic events such as mitochondrial dysfunction. However,
the presence and relevancy of other pro-oxidative cofactors, and endogenous antioxidants is
yet to be elucidated.
Consequent to a major role for oxidative stress in the pathogenesis of neurodegenerative
processes, human observational epidemiology studies have been in general consistent with
the hypothesis of an inverse relationship between antioxidant defense, cognitive function and
ultimately the risk of developing AD. In spite of disappointing results of clinical trials, there
are several caveats to these findings. Virtually all of them omit important information such as
drug-level monitoring and and/or biomarkers to support the therapeutic effect of the tested
treatment. But also, in all of them the intervention or the follow-up began at 65 years of age
or older. We know from genetic, dietary and pharmacologic approaches, mainly derived from
animal models, but also human, that the most important benefits have been obtained when the
diagnosis of AD is early and the antioxidant supplementation is established at a young age,
before deposition of Aβ plaques. This suggests that longer and earlier antioxidant
supplementation should be stated to address the problem. But then again, we must face the
increased cardiovascular risk that has been recently associated to antioxidant supplementation
with free-radical scavengers such as tocopherol and retinoic acid. In other words, what is it
that represents such a benefit to our cognition but can be so adverse to our cardiovascular
status? In this regard, the increased life expectancy associated to the use of dietary
supplements such as green tea, curcumin, or even better, full mediterranean diet sounds very
promising to come in aid of neurodegenerative diseases.
For Parkinson’s disease, on the other hand, we glimpse a less optimistic panorama. Only
a few current therapeutic interventions are aimed to alter the progression of the disease. On
top of that, our understanding of its pathogenesis is very limited. However great is evidence
that was first obtained from neurotoxic models, and then from the description and
reproduction of genetic defects associated to familiar PD. Many of these anomalies are
neurochemically indistinguishable from the sporadic disorder.
From all gathered evidence, Parkinson’s disease displays as several disorders converging
into a unifying neurodegenerative process with its own recognizable clinical features. Also,
oxidative stress is also an early event in the pathogenesis of PD. This raises the hope that is
possible to intervene with antioxidant supplements early in the progression of the disease, to
provide neuroprotection significant in delaying the progression of PD. However the results of
(very few) clinical trials testing this sort of interventions have not been quite encouraging.
Are we facing the same limitations AD clinical research has had to cope with? Whatever the
answer to these questions it’s not difficult to uphold the thesis that oxidative stress is a main
event of the neurodegenerative process, and as such should be further explored.
284 Rodrigo Pizarro
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Chapter XI
Glaucoma
Leonidas Traipe1, Rodrigo Castillo2 and Ramón Rodrigo2

1
Ophtalmologic Foundation “Los Andes”; Santiago; Chile
2
Abstract
Glaucoma constitutes an increasingly serious public health problem, moreover in
developed countries and is an important cause of blindness after cataracts. It is an optic
neuropathy that implies loss of retinal ganglion cells, including their axons, and a major
tissue remodeling, especially in the optic nerve head. Although increased intraocular
pressure is a major risk factor for glaucomatous optic neuropathy, there is little doubt that
other factors such as ocular blood flow play a role as well. Mechanisms leading to
glaucomatous optic neuropathy are not yet clearly understood. There is, however,
increasing evidence that both activation of glial cells and oxidative stress in the axons
play an important role. The involvement of reactive oxygen species (ROS) in the
pathogenesis of glaucoma is supported by various experimental findings, including: (i)
resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing
trabecular meshwork (TM) degeneration; (ii) TM possesses remarkable antioxidant
potential, mainly explained by superoxide dismutase and catalase activities and
glutathione pathways, all that is found decreased in glaucoma patients; and (iii)
intraocular-pressure increase and severity of visual-field defects in glaucoma patients
paralleled by the amount of oxidative damage of DNA affecting TM. Vascular
alterations, which are often associated with glaucoma, could contribute to the generation
of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells,
appears to be involved in the neuronal cell death affecting the optic nerve in glaucoma.
Despite the major pathogenic role of ROS in the pathophysiology of glaucoma, clinical
trials testing the efficacy of antioxidant drugs for its management are still lacking.
298 Leonidas Traipe, Rodrigo Castillo and Ramón Rodrigo
1. Introduction
Glaucoma is an insidiously progressive optic neuropathy which affects nearly 90 million
people worldwide, and is the leading cause of irreversible blindness after cataracts. It is a
neurodegenerative disorder of aging and occurs with increasing prevalence after the age of 40
[1] . Although data are incomplete and not definitive, primary open angle glaucoma (POAG)
has been estimated to affect about 2% of the population of Italy [2]. It is estimated that only
40% of glaucoma-affected subjects undergo adequate therapy, the absence of early severe
symptoms often contribute to delay the diagnosis [3]. Indeed, POAG is a chronic-
degenerative disease in which the clinical manifestations are preceded by a long latency. This
is of major relevance due to once the damage affecting visual capacity arises; it is no longer
reversible [4]. There are important risk factors for POAG such as presence of elevated
intraocular pressure as yet in the absence of visual-field damage (prevalence 6%) [5].
Advanced age represents a fundamental risk factor for this disease [6].
Increasing experimental evidence indicates that oxidative stress plays a major role in the
pathogenesis of the main cause of irreversible blindness, the degenerative primary open angle
glaucoma (POAG) [7, 8]. Indeed, tissues from the trabecular meshwork (TM) in patients with
primary open angle glaucoma have been reported to contain a high amount of metabolic
products of lipid peroxidation suggesting a role for oxidative stress in the pathophysiology of
this disease [9]. Recently, some experimental models have associated the elevation of
intraocular pressure with the occurrence of oxidative retinal damage thus giving a clue for the
use of antioxidant administration in order to attenuate the progression of the structural and
functional irreversible damage. Despite the major pathogenic role of ROS in the
pathophysiology of glaucoma, clinical trials testing the efficacy of antioxidant drugs for its
management are still lacking.
2. Pathophysiology of Glaucoma
2.1. General Concepts
Glaucoma is an optic neuropathy characterized by a specific structural alteration of the

head of the optic nerve accompanied by a progressive damage to the visual field. Although
increased intraocular pressure (IOP) is a major risk factor for POAG, other concomitant
factors affecting the eye play important roles including increased glutamate levels [10],
alterations in nitric oxide (NO) metabolism [11], vascular alterations [12] and oxidative
damage caused by ROS [13].
2.1.1. Outflow Pathway

The conventional outflow pathway consists of trabecular lamellae covered with human
trabecular meshwork (TM) cells, in front of a resistor consisting of juxtacanalicular human
TM cells and the inner wall of Schlemm’s canal. The outermost juxtacanalicular or cribriform
region has no collagenous beams, but rather several cell layers which some authors claim to
be immersed in loose extracellular material/matrix [14].
Glaucoma 299
The main resistance to the aqueous humour outflow is located in the TM directly
underneath the inner wall of Schlemm’s canal [15, 16]. Ultrastructural changes in
glaucomatous TM are similar to, but much more intense than, those observed in the normal
TM in the elderly [17]. These changes include the thickening of basal membranes and
trabecular beams, their enlargement or collapse, partial loss of endothelial cells and the
accumulation of materials such as pigment granules and calcium precipitates [18], central
nucleus changes such as an increase in electrodense plaques and collagen, and loss of
endothelial cells [18]. Furthermore, outflow resistance increases with age [20].
Thus, the increase in oxidative DNA damage in the cellular component of the TM could
directly affect regulation of the extracellular matrix structure and the associated regulation of
intraocular pressure, leading to the clinical onset of glaucoma [21, 22] When the intraocular
pressure (IOP) exceeds the pressure in the episcleral venous plexus, the endothelial cells that
line the lumen of Schlemm’s canal (SCEs) form ‘‘giant vacuoles’’ that facilitate the aqueous
outflow in this condition [23]. Similarly, SCEs discourage the reflux of blood by preventing
the formation of giant vacuoles when the episcleral venous pressure exceeds the IOP [24].
Indeed, these endothelial cells release vasoactive cytokines and other factors able to increase
the permeability of the endothelial barrier of the Schlemm’s canal. Alterations in these
cytokines may not allow sufficient flow through Schlemm’s canal and, consequently, the IOP
may rise to abnormal levels [25].
This interpretation of glaucoma pathophysiology is in agreement with the view that
increased intraocular pressure (IOP) is secondary to a decline in trabecular meshwork
cellularity [26]. Lutjen-Drecoll [19] has recently claimed that ‘‘common factors are involved
in the pathogenesis of both the TM and the optic nerve changes’’. The common denominator
involved in the cellular alterations of the TM structure and optic nerve damage is on one hand
the oxidative stress, and on the other the vascular damage described both in glaucoma [27]
and aging.
2.1.2. Retinal Glutamate/Glutamine Cycle Activity

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when
present in elevated concentrations. Retinal tissue is in fact an established paradigm for
glutamate neurotoxicity for several reasons: (i) insult leads to accumulation of relatively high
levels of glutamate in the extracellular fluid [28]; (ii) administration of glutamate leads to
neuronal cell death [29]; and (iii) glutamate receptor antagonists can protect against neuronal
degeneration [30]. Thus, an appropriate clearance of synaptic glutamate is required for the
normal function of retinal excitatory synapses and for the prevention of neurotoxicity. Glial
cells, mainly astrocytes and Müller glia, surround glutamatergic synapses and express
glutamate transporters and the glutamate-metabolizing enzyme glutamine synthetase [31, 32].
Glutamate is transported into glial cells and amidated by glutamine synthetase to the non-
toxic aminoacid glutamine. Glutamine is then released by the glial cells and taken up by
neurons, where it is hydrolyzed by glutaminase to form glutamate again, completing the
retinal glutamate/glutamine cycle [33, 34]. In this way, the neurotransmitter pool is
replenished and glutamate neurotoxicity is prevented.
Glutamatergic injury has been proposed to contribute to the death of retinal ganglion
cells in glaucoma. This hypothesis is supported by the demonstration that vitreal glutamate is
elevated in glaucomatous dogs [35, 36] and quail with congenital glaucoma [37]. In addition,
high glutamine levels have been found in retinal Müller cells of glaucomatous rat eyes [38].
In contrast, other authors showed no significant elevation of glutamate in the vitreous humor
of patients with glaucoma [39], or in rats [40] and monkeys with anatomic and functional
damage from experimental glaucoma [41]. In any case, it seems limited to assume that high
levels of glutamate in the vitreous are a necessary condition for excitotoxicity to be involved
in glaucomatous neuropathy. The local concentration of glutamate at the membrane receptors
of ganglion cells is the important issue for toxicity. This could be very different from the
level in samples of vitreous humor. Vitreous humor must be removed for experimental
measurement by a process that inevitably disturbs its state before removal. These
manipulations could themselves alter the measured glutamate concentration.
With the only exception of glutamine synthetase, the changes of glutamate/glutamine
cycle parameters were transitory. Although this issue has no ready explanation, it is possible
that these changes were provoked by a reversible injury to Müller or other retinal cells more
than with cell death. The changes in glutamate recycling preceded functional and histological
alterations induced by ocular hypertension [42]. Therefore, it is tempting to speculate that the
changes in glutamate/glutamine cycle activity could be a causal factor in ocular hypertension-
induced neuropathy. Furthermore, it seems possible that the increase in synaptic levels of
glutamate could represent an initial (and probably reversible) insult responsible for the
initiation of damage that is followed by a slower secondary degeneration that ultimately
results in cell death. It has been previously described that decreased in the retinal antioxidant
defense system potential at 6 weeks of treatment with hyaluronic acid [13, 44], and other
authors have postulated that excessive levels of NO may contribute to this optic neuropathy
[45]. With respect to these data, the hypothesis that the retinal damage induced by ocular
hypertension may result, at least in part, from oxidative stress induced by a
glutamate/mediated pathway, as shown in other neuronal injury models [46, 47].
2.1.3. Nitric Oxide and Neurotoxicity

Nitric oxide is an important messenger intra and extra molecular implicated in
vasodilatation, contractility, neurotransmission, neurotoxicity and inflammation. Nitric oxide
has a demonstrate role in many neurodegenerative diseases like: glaucoma, Alzheimer
disease, multiple sclerosis and cerebral-cardio-vascular diseases [48]. In this point, Ferreira et
al. [49] analyzed the implication of NO and nitrosative stress in the pathophysiological
mechanism of glaucoma, emphasizing the importance of biochemical markers in the aqueous
humor to evaluate the progression of the glaucomatous optic neuropathy. A significant
increase in the NO concentration, malondialdehyde levels, and a significant decrease of the
total antioxidant status were detected in patients having trabeculectomy. This study is in
agreement with other findings in some experimental models such as retinal ganglionar cell
line [50] and rats [51]. Recently, it has been proposed that the increase in NO formation in
the aqueous humor in those patients with glaucoma, in the presence of oxidants, should
induce the formation of peroxynitrite and it is very possible that nitrosative stress is induced
as a result of this. This point is an essential goal to be handled when it comes to future
experimentation within this line of research [52]. In this view, besides of evaluating the
sources of NO in anterior chamber, the trabecular distribution of NOS suggests an important
Glaucoma 301
role of nitric oxide in the future therapies for the glaucoma. The increase of nitric oxide
produces vasodilation and improves contractility in the TM; the final effect being the
decrease of intraocular pressure and on the other hand the contra-apoptotic effect giving
neuroprotection [53].
2.2. Role of Oxidative Stress in the Development of Glaucoma
2.2.1. Defenses Against Free Radicals and the Eye

Both vitamin C and glutathione operate in fluid both outside the cell and within the cell
[54], whereas vitamin E prevents endogenous mitochondrial production of ROS [55]. This
may be important in maintaining cellular homeostasis, which is relevant to counteract the
mechanism leading to the development of POAG [56]. Indeed, vitamin E prevents apoptosis
during hypoxia and oxygen reperfusion [57], and it may reduce apoptosis by means other
than antioxidation [58].
Ascorbic acid is thought to be a primary substrate in ocular protection because of its high
concentration in the eye. Within the cell, vitamin C helps to protect membrane lipids from
peroxidation by recycling vitamin E [59]. It is present at high concentrations in vitreous
humor [60], cornea [61] and tear film [62]. One of ascorbate’s presumed functions is to
protect the lens and retina from the damaging effects of ultraviolet radiation [63]. In addition,
it exerts a filter-like function against UV radiation in both the central corneal epithelium and
aqueous humor [64] and reacts with O2 to form H2O2. The direct correlation between the
concentrations of ascorbic acid and H2O2 in aqueous humour suggests that ascorbic acid is
the primary source of H2O2 in this fluid [65], although it has been claimed that H2O2 levels
can be overestimated [66]. A high level of ascorbic acid is necessary to maintain oxidative
balance in the aqueous humor, while vitamin E deficiency increases H2O2 levels [55].
Unfortunately, these processes are not able to eliminate free radicals completely, and, if
oxidative stress is very severe, it may ultimately cause cell death [67].
On the other hand, the GSH redox system is believed to protect ocular tissues from the
damage induced by low H2O2 concentrations, whereas catalase is thought to protect ocular
tissues from the damage induced by higher H2O2 concentrations [68, 69]. A decline of
catalase activity with age has been observed in the iris and in the corneal endothelium of
rabbits [70, 71]. Both glutathione and ascorbate have been detected in aqueous humour [72,
73]. These antioxidants seem to play a particularly important role in glaucomatous disease.
Indeed, patients with glaucoma exhibit low levels of circulating glutathione, suggesting a
general compromise of antioxidant defenses [74]. Moreover, genetic polymorphisms have
been detected for GSH transferase isoenzyme, and the GSTM1-null genotype has been found
to be significantly more common in patients with POAG than in controls [75].
On the basis of the currently available literature, free radicals seem to play an important
role in the pathogenesis of ocular diseases. Indeed, ROS are a cause of cataract [10, 76], are
implicated in age-related macular degeneration [77, 78], and also may play a significant role
in the pathophysiology of glaucoma [49, 79]. Vascular damage and neuronal cell death
associated with glaucoma and role of oxidative stress is show in figure 11-1.
Figure 11-1. Intraocular pressure increase in glaucoma progression and relationship with oxidative-
related degenerative processes affecting TM. ONOO-: peroxynitrite; TM: Trabecular meshwork;
IOP: intraocular pressure.
2.2.2. Oxidative Stress and Intraocular Pressure Elevation

The pathogenic role of oxidative stress in increasing IOP by reducing aqueous outflow
facility is supported by various experimental studies performed in vitro and in vivo. In vitro
treatment of human TM cells with H2O2 alters cellular adhesion and integrity [80]. In an
animal model of study, perfusion of TM with peroxide has shown to reduce aqueous humor
drainage from the anterior chamber of the calf’s eye [81]. In humans, oxidative DNA damage
has been reported to be significantly higher in the TM cells of glaucoma patients than in
those of age-matched controls [75]. Further studies demonstrated abundant oxidative
nucleotide modification (8-OH-dG) levels in human TM to be significantly correlated to the
increase in IOP and to visual field damage [79]. Further evidence suggests that patients with
POAG exert mitochondrial abnormalities implicating that mitochondrial dysfunction is most
probably a consequence of oxidative stress [82]. Free radicals, contained in the aqueous
humor, contribute to pathogenic alterations in the TM [20]. It was shown increased resistance
to the outflow of aqueous humor of calf is a result of TM cytoskeletal rearrangements and
cellular loss, in the presence of increased levels of H2O2. The damage caused by pro-
oxidants to the aqueous humor outflow system accounts for reports signaling that radiologists
suffer from ocular hypertension more frequently [83]. At the molecular level, human TM
endothelium has been reported to be an enriched site of NO synthesis. Nitric oxide can
interact with oxygen or metals, such as copper or iron, to modulate outflow resistance of the
TM [84].
Glaucoma 303
2.2.3. Oxidative Stress and Glaucomatous Optic Neuropathy

Glaucomatous optic neuropathy (GON) is the optic nerve damage secondary to
progressive and chronic enhancement of IOP in glaucoma patients. However, not all
glaucoma patients suffer from elevated IOP, along with the observation that the majority of
these patients show signs of reduced ocular blood flow as well as ischemic signs in the eye
(e.g., upregulation of the ischemia inducible factor 1-α) [85] suggesting that hemodynamic
factors are involved in this process.
Glaucoma patients have reduced ocular blood flow, what has a predictive power for the
progression of GON [86]. On the other hand, a blood flow reduction by atherosclerosis,
multiple sclerosis, or other similar ischemic diseases, increases the risk for GON
insignificantly. The solution of this seemingly paradox observation is simply the fact that it is
not a stable reduction in ocular blood flow but rather the instability in blood flow that may
lead to GON [87]. Such an instability in ocular blood flow leads to a repeated mild
reperfusion injury [88]. This hypothesis is supported by the observation that IOP fluctuation
is more damaging than a stable increase in IOP [89, 90] and by observations that patients that
progress despite a normalized IOP suffer from a disturbed autoregulation [91]. The main
cause for this insufficient autoregulation is a primary vascular dysregulation syndrome [92].
The term dysregulation simply means that blood flow is not properly adapted to this need.
Dysregulative mechanisms can lead to an over- or underperfusion. A steady overperfusion
may be less critical for long-term damage. A constant underperfusion, however, can lead to
some tissue atrophy or in extreme situations to infarction. Unstable perfusion (underperfusion
followed by reperfusion) leads to oxidative stress [93].
As described previously, oxidative stress occurs under a condition of high energy
consumption, light exposure, or age-dependent decline of coping capacity to deal with free
radicals. In glaucoma, an additional major factor is most likely a repeated mild reperfusion
injury. This hypothesis is supported by observations on circulating lymphocytes of human
glaucoma patients. Activated astrocytes express MHC-II capable of communicating with
lymphocytes and lymphocytes also communicate with the capillary endothelial cells during
reperfusion [94]. Indeed, the lymphocytes of glaucoma eyes express neural thread protein
[95], indicating axonal damage, but they also reveal upregulation of p53 and of proteosome
20 S subunits, along with an over-expression of metalloproteinase-9. Moreover, glaucoma
patients present higher plasma lipid peroxidation products concentration in comparison to
controls [96]. These reports together with observations of increased DNA breaks in
circulating lymphocytes [97] support the assumption of the occurrence of oxidative stress in
glaucoma patients [98].
On the other hand, in the optic nerve head, blood flow is particularly unstable as a result
of mechanical stress and an insufficient blood--brain barrier giving access of vasoactive
substances to the pericytes and smooth muscle cells [89] . In addition, there is very high
energy consumption in the axons of the optic nerve head due to the lack of myelin sheaths
and therefore a high concentration of mitochondria in this area. Therefore, it is very
important to describe the role of the pathological process derived from ischemia-reperfusion
cycle in eyes, mainly the retina cells. During reperfusion the main source of free radicals in
cells lacking xanthine oxidase (all neural cells) are the mitochondria [ 99]. Whereas oxidative
stress damages all types of molecules and thereby reduces the probability of cellular survival,
the question arises as to why in glaucoma specifically the retinal ganglion cells and their
axons die by apoptosis.
Reperfusion injury in glaucoma patients, particularly in the optic nerve head, is very mild
but occurs repeatedly. The assumption of reperfusion injury being involved in the
pathogenesis also explains why sleep apnea [100] or reversible shock--like states [101] can
lead to GON. Reactive oxygen species damage biomolecules such as proteins or lipids of
plasma membranes. The damage to the cells, in turn, causes the release of more free radicals.
In prolonged ischemia hypoxanthine is formed as a result of the breakdown of ATP and the
enzyme xanthine dehydrogenase is converted to xanthine oxidase. This also results in
molecular oxygen being converted to the highly reactive superoxide and hydroxyl radicals,
further resulting in tissue damage [102]. The nerve cells of CNS, however, lack xanthine
oxidase (with the exception of blood vessels). In the central nervous system, ROS formation
therefore does not occur via xanthine oxidase. The major source of oxidative stress in
reperfusion stems from the mitochondria which are very crowded in the optic nerve head due
to high energy consumption in these nerve fibers lacking myelin sheaths [103] . The role of
oxidative stress is further supported by findings of weaker antioxidant defense systems in
POAG patients.
2.2.4. Experimental Models of Glaucoma and Oxidative Stress

Rats are becoming an increasingly used model system for understanding the mechanisms
of optic nerve injury in POAG. Although the anatomy of the rat optic nerve head is different
from that of humans, the ultrastructural relationships between astrocytes and axons are quite
similar, making likely that cellular processes of axonal damage in these models will be
relevant to human glaucoma [104]. All of these models rely on elevating IOP, a major risk
factor for glaucoma. Methods that produce increased resistance to aqueous humor outflow at
the anterior chamber angle, specifically hypertonic saline injection of aqueous outflow
pathways and laser treatment of the limbal tissues, appear to produce a specific regional
pattern of injury that may have a particular relevance to understand the regional injury in
human glaucoma. Increased pressure and its fluctuations are a characteristic of such models
and the rodent optic nerve head appears to have high susceptibility to elevated IOP. Special
instrumentation and measurement techniques are required to document pressure exposure in
these eyes. With these techniques, it is possible to obtain an excellent correlation between
pressure and the extent of nerve damage [105].
2.2.4.1. Steroid-Induced Ocular Hypertension

Ocular hypertension is induced by glucocorticoids, such as dexamethasone, in
experimental animal models and a subset of patients treated systemically or topically (steroid-
induced glaucoma) [106, 107]. Ocular hypertension in both POAG and steroid-induced
glaucoma results from an increased aqueous outflow resistance across the TM, and
histopathological findings of the TM in POAG and steroid-induced glaucoma share some
similarities [108]. It has been demonstrated that this model shows down-regulation of
antioxidant enzymes such as SOD and CAT, causing increased vulnerability to oxidative
stress in ocular hypertensive retinas [7]. In addition, Ko et al., [109] demonstrated amplified
generation of superoxide anion and increased lipid peroxidation in the retina of a rat ocular
Glaucoma 305
hypertensive model generated by cauterization of episcleral veins. In another rat model,

generated by intraocular hyaluronic acid injection, Moreno et al. [13] also observed increased
lipid peroxidation, what was associated with variable retinal SOD activity among studies.
Furthermore, retinal SOD response may vary depending on the study protocol [110]. In this
model, the results suggest potential vulnerability to oxidative stress in the retina due to a
diminution of SOD activity, which is directly involved in cellular protection against oxidative
stress of the retinas. However, it remains to be clarified whether these protein alterations are
due to the ocular hypertension or to the direct effects of dexamethasone, and future studies
should address this important issue. For example, it should be examined whether these
alterations take place in other ocular hypertension models.
2.2.4.2. Others Models that Induce Ocular Hypertension

The injection of hypertonic saline into episcleral vein [111, 112], cauterization of
episcleral veins [113, 114], and laser photocoagulation at TM with or without injection of
India ink to enhance laser energy uptake [40] are the currently adopted approaches to produce
ocular hypertension models. Except using laser photocoagulation at TM, Wolde- Mussie et
al. [115] applied laser treatment at episcleral veins and limbal veins to decrease aqueous
outflow and elevate the IOP. However, the understanding of the characteristics of
pathological retinal changes in specific models is fundamental for the investigation of
glaucoma using those models.
Loss of retinal ganglion cells (RGCs) is an important consequence of glaucomatous
damage [116]. The ocular hypertension model using cauterization of episcleral veins showed
a RGCs loss of approximately 4% per week after IOP elevation [117]. In another model using
the injection of hypertonic saline into episcleral veins demonstrated a higher loss of RGC of
about 9% per week [118 ]. In a recent study, ocular hypertension induced by unilaterally laser
photocoagulation at episcleral veins and limbal veins in adult rats [119 ] show that the loss of
RGCs was about 3% per week (25% RGC loss at 8 weeks), which was consistent with
previous findings [120] that used the same model. Although the level of IOP elevation in this
model was similar to those of injecting saline into episcleral veins, resulted in lower extent of
RGC loss. This is probably due to the different experimental approaches adopted.
3. Effects of Antioxidants in Glaucoma

There is still much debate about the value of natural antioxidant or vitamin
supplementation on ocular diseases such as glaucoma [121, 122].Because the pathogenesis of
glaucoma involves various factors, one of which may be oxidative stress, the possibility that
natural dietary antioxidants or vitamin supplementations may be beneficial becomes
plausible. If diet proves to be beneficial it will be one of the most cost-effective treatments
for glaucoma, the incidence of which is expected to increase by 2030, mostly because of the
aging population [123]. This disease may in fact be an even more relevant problem in
developing countries with social or economical problems. Unfortunately, however, the effect
of diet cannot be easily measured due to confounding factors such as timing of diet exposure,
level of diet, or intake of other nutrients [124] .
A number of studies deal with the antioxidant role of polyphenolic compounds in our
health. Results are difficult to evaluate and interpret because of the large number of phenolic
compounds and the fact that the phenolic content of most foods is not well established.
Although there have been major research advancements in the identification and
characterization of specific polyphenols [125, 126], many of them remain unidentified.
Whereas certain studies reported a significant improvement between green tea consumption
and cancer risk [127], others along with a large meta-analysis of epidemiological studies
found no such association [128]. This may be a problem of methodology. A close scrutiny of
the polyphenolic studies shows that most were in vitro assessments, [129] and only a few are
done in vivo. Moreover, the bioavailability or delivery of polyphenols to a specific tissue site
is also often not accounted for. Although results of in vitro assessments may be very
promising, they do not necessarily reflect in vivo changes. The functional significance of the
reports is therefore often not clearly established. Various potential antioxidant compounds are
presented in table 11-1.
Table 11-1. Antioxidant compounds in glaucoma
Compound Mechanism Effects in Human

Scavenging properties ↓ IOP
Tea Chelating properties Wei., et al 1999
Vasodilating factors ↓IOP,↓neovascularization
Wine
Inhibition VEFG Garvin et al., 2006
Ubiquinone Scavenging properties ↓lOP, ↓ lipid peroxidation
(Coenzyme Q 10) Nieminen et al., 2005
↓ NADPH activity ↓LDL oxidation, ↓lOP
Vitamin C
↑ NOS activity Elmore, 2005
↓ Mitochondrial ROS ↓IOP
Vitamin E ↓ TM remodeling Desmettre, 2005
↓ TM cell apoptosis ↓ IOP
PUFA n-3
Antiinflammatory Nguyen et al., 2007
IOP: intraocular pressure; NADPH: nicotinamide adenine dinucleotide phosphate-oxidase; LDL: low
density lipoprotein; ROS: Reactive oxygen species; PUFA: polyunsaturated fatty acid; TM
trabecular meshwork .
3.1. Polyphenolic Flavonoids
3.1.1. Tea
Tea flavonoids have been reported to have powerful antioxidant properties, as a result of
their free radical scavenging properties. In fact, the polyphenolic compounds in tea have been
shown to act as efficient scavengers for the superoxide anion [130], H2O2 and thereby
partially inhibit ultraviolet-induced oxidative DNA damage [131]. Flavonoids present in
green tea are able to inhibit the formation of lipid peroxyl radical species and to act as
inhibitors of low-density--lipoprotein peroxidation [132]. Myricetin, for example, also
functions as a potent and effective neuroprotective agent for photoreceptor cells against
oxidative and light damage associated with a diminution of inflammatory and apoptotic
biomarkers [133]. Due to their neuroprotective effects [134], the use of green and black tea
may prove to be of therapeutic value in the treatment of glaucoma [135].
Glaucoma 307
Recent in vitro studies on brain membranes showed that epigallocatechin gallate was
approximately 10 times more potent than trolox (vitamin E analogue) in attenuating lipid
peroxidation caused by the NO donor, sodium nitroprusside. Subsequent
immunohistochemical studies revealed that following an intraocular injection of sodium
nitroprusside retinal photoreceptors are affected [136]. When epigallocatechin gallate was co-
injected, the detrimental effects to the retina caused by sodium nitroprusside were
significantly blunted [137]. In agreement with these data, the daily intake of epigallocatechin
gallate may help individuals suffering from retinal diseases where oxidative stress is
implicated.
3.1.2. Wine
Red wines exhibit a stronger antioxidant capacity than white wines due to the higher
phenolic content of the first [138]. Polyphenolic flavonoids in wine have been reported to
improve endothelial dysfunction and lower the susceptibility of low-density--lipoprotein
lipids to oxidation [139]. Impairment in endothelial function may lead to damage to vascular
cells and the surrounding tissue. Endothelial dysfunction plays a role in the pathogenesis of a
variety of disorders, including glaucoma [140] and cardiovascular diseases [141]. Indeed, red
wines strongly inhibit the synthesis of endothelin-1 [142], a vasoactive peptide that plays a
crucial role in the pathogenesis of glaucoma. Animal studies have also shown that
polyphenolic flavonoids in wine potentially prevent the initiation of atherosclerotic plaque
development [143, 144]. Moreover, resveratrol, a polyphenol found in grapes and wine, has
been shown to reduce extracellular levels of vascular endothelial growth factor [145].The
mechanisms by which polyphenols affect endothelial function is due to their ability to
stimulate the production of endothelial NO synthase (eNOS) and promote the production of
NO, which induces vasodilation [146].
Red wine polyphenolic flavonoids are available in plant and vegetable sources and have
several biological actions, which make these compounds a potentially important agent in the
glaucoma therapy. Some problems with respect to bioavailability in clinical trial need to be
clarified.
3.2. Antioxidant Vitamins
3.2.1. Thiamin (Vitamin B1)

Levels of thiamin have been found to be lower in some glaucoma patients in comparison
to controls, and a deficient absorption of this vitamin in these patients has been postulated
[147]. Thiamine deficiency has been reported to cause neurodegeneration by inducing
various changes in microglia [148], astrocytes [149], endothelial cells, and mast cells [150],
leading to neuronal cell death. The brains of thiamine deficient mice show vascular changes,
inflammatory responses, and oxidative stress, similar to the brains from patients who die from
common neurodegenerative diseases [151]. Recently, the hypothesis that in thiamine-
deficient brain [152], vascular factors constitute a critical part of a cascade of events leading
to increases in blood-brain barrier permeability to non-neuronal proteins and iron, leading to
inflammation and oxidative stress. Inflammatory cells may release deleterious compounds or
cytokines that exacerbate the oxidative damage to metabolically compromised neurons.

Similar mechanisms may operate in the pathophysiology of glaucoma and neurodegenerative
diseases in which vascular factors, inflammation and oxidative stress are implicated [153].
3.2.2. Vitamin C
The potential protective effect of vitamin supplementation has not been thoroughly
evaluated. Because physiological systems of antioxidant defense are complex it makes sense
that optimal functioning requires the availability of numerous antioxidants. Foods provide an
even greater array of antioxidants than supplements and a correctly dosed combination of
antioxidants, as found naturally, in foods, appears to be more effective [154].
Ascorbate protects low-density lipoprotein cholesterol from oxidative damage and
reduces platelet aggregation [155]. By enhancing NO synthase activity, vitamin C is
potentially important in lowering blood pressure [156] . The administration of ascorbic acid
appears to have a transient hypotonic effect on intraocular pressure in normal eyes. Its effect
on glaucomatous eyes is not well defined particularly in the case of the rabbit model [157].
Osmolarity is suggested as a possible hypotensive mechanism and a differential fluid
transport rate between the blood and the aqueous humor for the different genotypes is
suggested as a possible mechanism for the difference in duration [158]. With respect to
doses, the weight of available evidence supports the role of vitamin C in prevention of lens
opacities, and the adverse reactions reported to have occurred above the dosage of 1000 mg
per day [159]. Vitamin C has been included at 40 mg, equal to the amount of protein, vitamin
or mineral that is sufficient for almost every individual.
3.2.3. Vitamin E
Mitochondria also produce ROS as a by-product of oxidative phosphorylation. These
factors make the mitochondria more susceptible to damage in vitamin E deficiency [160].
Besides serving as an antioxidant, vitamin E has been suggested to be involved in the direct
modulation of regulatory proteins, and in the activity of key regulatory enzymes [55].
Apoptosis during hypoxia and oxygen reperfusion can be prevented by vitamin E [57]. There
is also some evidence that suggests that vitamin E may reduce apoptosis by means other than
antioxidation [161, 162].
In a recent clinical trial, glaucomatous patients were divided into three groups. One
group was not supplemented in their therapy. Patients included in the other two groups
received 300 and 600 mg/day of oral alpha-tocopherol acetate, respectively. The average
differences between the pulsatility indexes and resistivity indexes of both ophthalmic arteries
and posterior ciliary arteries of supplemented groups were significantly lower than those not
treated at months 6th and 12th. In trial groups, resistivity indexes decreased in posterior
ciliary arteries at months 6th and 12th and pulsatility indexes decreased in ophthalmic arteries
at the 6th month. In conclusion, alpha-tocopherol deserves attention beyond its antioxidant
properties for protecting retina from glaucomatous damage [163, 164]. Recently, some
authors described that a mitochondrial complex I defect is associated with the degeneration of
TM cells in patients with POAG, and vitamin E and N-acetylcysteine inhibitors can reduce
the progression of this condition [165].
Glaucoma 309
On the other hand, collagen remodeling and apoptosis (associated with an increase in
intraocular pressure) are mainly influenced by water-soluble antioxidants such as glutathione
[166]. In the case of one matrix collagen type such as elastin, apoptosis and remodeling
(correlated with the occurrence of optic atrophy) are particularly influenced by lipid-soluble
liposoluble antioxidants such as vitamin E.
In addition, the dietary ratio of omega3/omega6 PUFA intake could influence the balance
of intraocular pressure. Omega-3 PUFA could influence cyclooxygenase competition through
an increase in intraocular pressure reducing synthesis of PG-F2, leading to a decrease in
uveo-scleral outflow [167]. The true importance of these factors has not yet been solidly
determined and studies are in progress to clarify the real implication of these nutritional
factors.
3.2.4. Ubiquinone (Coenzyme Q10)

Coenzyme Q10 is a coenzyme for the inner mitochondrial enzyme complexes involved in
energy production within the cell. Coenzyme Q10 has been demonstrated to prevent lipid
peroxidation and DNA damage induced by oxidative stress [168]. In vivo supplementation
with coenzyme Q10 enhances the recovery of human lymphocytes from oxidative DNA
damage [169]. These properties are due to ubiquinones free radical scavenging activity [170].
In one study of glaucoma patients, administration of oral ubiquinone was shown to be useful
in mitigating cardiovascular side-effects without affecting IOP [171] . Unfortunately, studies
examining the effect of ubiquinone on glaucoma are lacking and limit the use of this agent.

The final common pathway of vision loss in glaucoma is the apoptotic loss of retinal
ganglion cells with subsequent degeneration of the optic nerve head. Despite numerous
cellular events and pathways have been argued to play major roles in the development of the
disease, the exact cause responsible for this alteration is still essentially unknown. The
elevated intraocular pressure is not an obligate factor and has been removed from the
definition. However, this neuropathy is sensitive to the intraocular pressure; therefore, the
medical and surgical treatment is aimed to reduce this parameter, as it halts the progression of
the disease in the majority of patients.
In primary open angle glaucoma, it is still unknown the exact mechanism that lead to
damage of the trabecular meshwork and thereby to an increase in intraocular pressure, nor do
we know the exact mechanisms leading to glaucomatous optic neuropathy. Obviously there
are a number of factors and mechanisms involved. One of these factors is oxidative stress. At
present, studies on the mechanism linking oxidative stress with the development of glaucoma
are just arising. Preliminary evidence in glaucoma experimental models indicates that various
antioxidant compounds reduce intraocular pressure together with ameliorating the retinal
damage. Unfortunately, at this time a pharmacological treatment in humans is not still
availability to be recommended for clinical use.
Future intensive studies on the effect of these compounds may open up a new therapeutic
era in glaucoma. Moreover, the development of randomized controlled double-blind clinical
trials could help to ascertain the potential therapeutic efficacy of antioxidants in the
prevention and treatment of this deleterious disease.
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Index
acute, vii, 17, 54, 64, 72, 97, 103, 114, 116, 117,
4
118, 119, 122, 124, 127, 128, 129, 130, 131, 132,
4-hydroxynonenal, 14, 21, 234, 265, 268, 285, 286 133, 189, 216, 264, 291
4-hydroxynonenal (HNE), 265 acute interstitial nephritis, 119
acute kidney injury, 128
A acute renal failure, vii, 17, 122, 124, 127, 128, 129,
130, 131, 132, 133
acute tubular necrosis, 114, 128, 129
Aβ, 259, 267, 270, 271, 272, 283
acyl transferase, 227
AA, 7, 45, 53, 113, 116, 118, 133, 134, 209, 214, Adams, 89, 90, 219
250, 254, 285, 313 adaptation, 28
abnormalities, 14, 97, 160, 161, 167, 169, 173, 181, adducts, 82, 142, 178
200, 209, 245, 249, 252, 285, 317 adenine, 8, 76, 86, 101, 106, 207, 308
absorption, 165, 216, 309 adenocarcinoma, 21
AC, 48, 62, 85, 86, 90, 151, 156, 178, 179, 213, 216, adenosine, 36, 76, 244
284, 315 adenosine triphosphate, 76, 244
acceptor, 39, 230 adenylyl cyclase, 35
accounting, viii, ix, 1, 29, 48, 91, 94, 96, 103, 146, adhesion, ix, 13, 17, 27, 33, 42, 63, 67, 69, 70, 71,
260, 263 72, 73, 77, 79, 80, 85, 86, 89, 90, 116, 122, 137,
accuracy, 226, 296 146, 150, 151, 166, 219, 235, 236, 265, 304
ACE, 30, 34, 43, 50, 58, 60, 117, 118, 169, 205, 207, adhesive interaction, 146
219 adipocyte, 162, 163, 169, 178, 179
ACE inhibitors, 43, 50, 60, 117, 118, 205, 207 adipocytokines, 180
acetate, 310 adiponectin, 72, 163, 169, 181, 219
acetylation, 68 adipose, 41, 162, 163, 165, 169, 170, 181, 183, 185,
acetylcholine, 13, 19, 26, 29, 34, 51, 53, 70, 85, 120, 186, 226, 227, 233, 240, 247
190, 204, 263, 264 adipose tissue, 162, 163, 165, 169, 170, 181, 183,
acquired immunodeficiency syndrome, 117, 129 185, 186, 226, 227, 233, 240, 247
actin, 73, 116, 128, 313 adiposity, 162, 164, 179, 180
action potential, 93, 95 adjustment, 273, 274
activated receptors, 182, 237 administration, 30, 31, 44, 47, 48, 61, 100, 102, 103,
activators, 181, 188 104, 109, 117, 118, 125, 126, 133, 145, 148, 170,
active site, 40 172, 173, 174, 187, 203, 204, 217, 244, 272, 277,
282, 300, 301, 310, 311
adrenal gland, 170
322 Index
adrenal glands, 170 alanine aminotransferase, 174

adrenoceptors, 33 albumin, 178, 197, 202, 215, 285
adriamycin, 254 albuminuria, 204, 206, 218, 220
adult, 62, 162, 182, 242, 266, 307, 313 alcohol, 22, 49, 166, 173, 224, 231, 232, 245
adults, 22, 47, 50, 160, 177, 180, 189, 190, 203, 216, alcohol abuse, 224
248, 261, 269, 294 alcohol consumption, 224
advanced glycation end products, xi, 123, 193, 194, alcoholic liver disease, 184, 255
207, 211, 212, 219, 220, 221, 252 alcoholics, 255
advanced glycation end products (AGEs), xi, 193 alcohols, 8, 11
adventitia, 27, 29, 38, 42, 52, 66, 74, 86 aldehydes, 21, 234, 238
AE, 23, 86, 186, 189, 250, 284 aldosterone, 35, 94
aerobic, 3, 120, 233, 242, 278 allele, 265, 270
aetiology, 286 alleles, 195
AF, 57, 89, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, allergic reaction, 119
104, 106, 107, 219, 317 allograft, 104
age, 10, 17, 21, 50, 65, 74, 92, 112, 136, 139, 144, allopurinol, 7, 39, 119, 124, 125, 133
150, 160, 188, 198, 201, 218, 258, 261, 262, 263, allosteric, 34, 54
265, 267, 268, 269, 270, 271, 272, 273, 274, 276, alpha, 89, 90, 97, 108, 133, 153, 155, 156, 165, 173,
280, 283, 284, 287, 292, 300, 301, 303, 304, 305, 182, 184, 187, 188, 189, 202, 215, 217, 236, 250,
316, 322 252, 253, 255, 267, 294, 295, 297, 310, 317, 322
age related macular degeneration, 316 alpha-tocopherol, 89, 90, 133, 153, 155, 173, 189,
ageing, 112, 210, 258, 261, 284, 296 202, 215, 253, 255, 267, 294, 297, 310, 322
agent, viii, 2, 4, 6, 7, 11, 18, 26, 32, 33, 40, 44, 79, alteplase, 103
100, 118, 125, 183, 205, 206, 207, 232, 239, 241, alternative, 19, 103, 176, 240, 245, 282, 288, 319
244, 256, 272, 308, 309, 311 alternative medicine, 319
agents, viii, ix, x, xii, 8, 12, 18, 25, 27, 32, 37, 38, alters, 40, 184, 189, 295, 304
40, 43, 50, 91, 92, 99, 101, 108, 117, 118, 119, aluminum, 271
120, 129, 132, 135, 144, 166, 200, 201, 202, 219, Aluminum, 293
221, 224, 225, 231, 240, 241, 242, 246, 251, 259, Alzheimer disease, 271, 284, 285, 289, 290, 291,
261, 264, 283, 294, 320 292, 293, 294, 302
age-related macular degeneration, 303 Amadori, 178, 197
AGEs, xi, 193, 197, 198, 206 amelioration, 18, 104, 175, 240, 253
aggregates, 263, 277, 288 American Heart Association, 82, 177
aggregation, 71, 73, 100, 200 American Psychiatric Association, 286
aggression, 233, 235 amine, 9, 20, 197, 220, 231, 293
aging, vii, xii, 7, 10, 12, 17, 21, 22, 24, 160, 194, amines, xi, 8, 193
198, 257, 258, 260, 271, 272, 277, 281, 283, 284, amino, 5, 6, 10, 31, 32, 40, 225, 232, 243, 244, 263,
285, 288, 290, 291, 292, 296, 300, 301, 307, 316 314
aging population, xii, 160, 257, 258, 307 amino acid, 5, 6, 10, 31, 32, 40, 225, 243, 244, 263,
aging process, 17, 22, 24, 258, 260, 272, 281 314
agonist, 33, 34, 38, 49, 70, 167, 282 aminoglycosides, 123
aid, 260, 283 amniotic, 153
AIDS, 117, 118 amniotic fluid, 153
AJ, 54, 57, 59, 84, 85, 88, 89, 128, 149, 181, 182, amorphous, 263
190, 208, 211, 215, 219, 247, 248, 250, 251, 252, amygdala, 262
253, 255, 285, 291, 293, 295, 296, 322 amyloid, 15, 170, 263, 264, 270, 271, 272, 286, 287,
AKT, 252 288, 289, 293
AL, 23, 52, 106, 107, 150, 151, 152, 154, 156, 217, Amyloid, 264, 287, 288
218, 219, 287, 295, 317 amyloid beta, 15, 286, 287, 288, 289
alanine, 132, 137, 174 amyloid deposits, 265
Index 323
amyloid fibrils, 272 anti-inflammatory drugs, 117, 118

amyloid plaques, 272 Antioxidative, 254, 282, 297
amyloid precursor protein, 287, 288, 289 antioxidative activity, 202
amyloid β, 263 antioxidative potential, 87
amyloidosis, 266, 271, 289, 293 antiphospholipid antibodies, 136, 143
AN, 155, 317 Antiretroviral, 178
anaerobic, 120 anti-sense, 31
analytical techniques, 171 antitumor, 126
anatomy, 105, 306 anuria, 117
Andes, 299 aorta, 30, 32, 33, 47, 54, 62, 83, 93, 145, 200, 204
androgen, 189 AP, 85, 96, 107, 130, 133, 179, 186, 211, 235, 250,
anemia, 125 289
angina, 183 APC, 195
angiogenesis, x, 37, 135, 144, 218, 321 Apo E, 270
angiogenic, 138 APOE, 292
angioplasty, 37, 43 apoptosis, ix, 10, 20, 28, 29, 33, 42, 73, 77, 91, 92,
Angiotensin, 7, 30, 51, 75, 83, 94, 106, 118, 166, 96, 102, 107, 109, 114, 116, 126, 145, 146, 148,
207, 214, 233, 238, 251 156, 157, 165, 182, 201, 210, 219, 229, 232, 234,
angiotensin converting enzyme, 30, 34, 117, 221 235, 236, 241, 245, 249, 250, 251, 255, 265, 278,
angiotensin II, viii, 13, 23, 25, 26, 45, 51, 52, 53, 55, 279, 280, 285, 286, 287, 303, 306, 308, 310, 311,
57, 58, 69, 75, 93, 94, 98, 113, 152, 206 315, 321, 322
Angiotensin II, 7, 51, 75, 83, 94, 106, 118, 166, 207, apoptotic, ix, 91, 109, 140, 171, 207, 225, 237, 238,
214, 233, 238, 251 303, 308, 311
angiotensin receptor blockers, 118 Apoptotic, 235
angiotensin-converting enzyme, 48, 54, 221 apoptotic effect, 303
animal models, viii, xiii, 25, 42, 102, 125, 126, 145, APP, 259, 261, 263, 264, 266, 267, 270, 271, 272,
168, 196, 200, 205, 207, 226, 228, 231, 241, 243, 287, 289, 293
255, 257, 266, 278, 279, 283, 306 aqueous humor, xiii, 16, 299, 302, 303, 304, 306,
animal studies, 13, 126, 175, 198, 205, 206, 244, 245 310, 312, 314, 315, 316
animals, 3, 30, 32, 124, 132, 171, 197, 200, 232, aqueous solution, 19
243, 272, 278 Arabs, 279
Anion, 131 arachidonic acid, 7, 10, 35, 36, 77, 101, 116, 142,
anoxia, 132 198, 201
ANP, 120 ARF, ix, x, 111, 112, 113, 114, 115, 116, 117, 118,
antagonism, 53 119, 120, 121, 122, 123, 125, 126, 127, 128
antagonist, 31, 40, 185, 206, 267 arginine, 5, 6, 13, 30, 34, 39, 48, 52, 54, 57, 61, 70,
antagonists, 43, 50, 207, 301 76, 90, 121, 138, 146, 269, 314
anterograde amnesia, 261 argon, 313, 319
anthracene, 202 arrest, 28
anti-angiogenic, 143, 144 arrhythmia, ix, 91, 92, 102, 103, 104, 105, 107
antiapoptotic, 217, 319 arrhythmias, 100, 102, 107, 110
antibiotics, 117, 123 arsenic, 255
anticancer, 133 arterial hypertension, 32, 57, 61, 92, 166, 173, 288
anticancer drug, 133 arteries, x, 16, 30, 32, 36, 43, 47, 53, 58, 64, 66, 72,
antidiabetic, 320 82, 135, 137, 139, 142, 146, 148, 154, 204, 214,
antifibrotic, 245 218, 310, 320
antigen, 195 artery, 13, 19, 32, 37, 41, 53, 54, 55, 58, 69, 70, 85,
antigen-presenting cell, 195 87, 92, 96, 99, 103, 108, 110, 115, 131, 139, 143,
antihypertensive agents, 43, 50 144, 145, 146, 148, 154, 157, 173, 183, 189, 190,
antihypertensive drugs, 48, 54, 58, 137 194, 203, 214, 216, 270, 292, 321
324 Index
arthritis, 267 autonomic nervous system, 106

ascorbic, 14, 43, 44, 58, 79, 90, 103, 108, 110, 143, autooxidation, xi, 193, 194
155, 170, 187, 204, 218, 255, 281, 303, 310, 315, autophagy, 278
321 autopsy, 276, 280
ascorbic acid, 14, 43, 44, 58, 79, 90, 103, 108, 110, autoreactive T cells, 195
143, 155, 170, 187, 204, 218, 255, 281, 303, 310, autosomal dominant, 270, 279
315, 321 autosomal recessive, 15
Ashkenazi Jews, 279 availability, 14, 39, 100, 143, 145, 167, 204, 214,
Asia, 273, 275, 294 218, 231, 310, 311
Asian, 282 awareness, 22
aspartate, 137, 167 axon, 262, 314
assessment, 65, 72, 81, 148, 152, 154, 169, 213, 275, axonal, 258, 305, 306, 319, 322
276, 286, 297, 312, 322 axons, xiii, 262, 299, 305, 306
astrocytes, 205, 219, 262, 263, 271, 272, 301, 305, azotemia, 112, 113, 115
306, 309 Aβ, 261, 262, 263, 264, 265, 266, 268, 270, 272
Asymmetric dimethylarginine (ADMA), 76, 88
asymptomatic, 12
B
asynchronous, 94
atherogenesis, ix, 22, 23, 63, 64, 67, 68, 69, 70, 71,
B vitamins, 291
74, 75, 76, 77, 80, 82, 84, 86, 88, 89, 90, 161,
bacteria, 6, 165
176, 204, 219, 265, 269, 270
bacterial, 72, 122, 165, 182
atherosclerosis, vii, viii, 1, 10, 12, 13, 21, 28, 29, 37,
bariatric surgery, 179, 180
41, 43, 49, 55, 57, 60, 63, 64, 65, 66, 67, 68, 69,
barrier, 27, 66, 70, 165, 219, 235, 258, 269, 301,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
305, 310, 318
83, 84, 85, 86, 87, 88, 89, 90, 122, 160, 166, 168,
basal forebrain, 263
169, 170, 171, 176, 178, 180, 183, 184, 185, 187,
basal lamina, 236
188, 194, 200, 203, 212, 213, 218, 253, 268, 269,
basement membrane, 113, 117, 138
270, 288, 305, 321
basic research, 187
atherosclerotic plaque, 69, 74, 75, 83, 87, 99, 198,
battery, 149
309
BBB, 258, 264, 271, 281, 282
atoms, 7
BD, 287
ATP, 76, 95, 116, 120, 228, 230, 306
beams, 300, 301
ATPase, 109, 116, 128
beef, 57
atria, 93, 94, 95, 97
behavior, 2, 296
atrial fibrillation, ix, 91, 92, 95, 98, 104, 105, 106,
beneficial effect, 33, 43, 46, 49, 80, 92, 125, 133,
107, 108, 109, 110
145, 170, 173, 174, 175, 202, 205, 208, 271
Atrial fibrillation, ix, 91, 92, 105
benefits, x, 58, 104, 136, 171, 174, 197, 240, 244,
atrial fibrillation (AF), 104
270, 274, 282, 283
Atrial fibrillation (AF), 92
benign, 225, 246
atrial natriuretic peptide, 120
benzo(a)pyrene, 202
atrioventricular node, 93
beta cell, 161, 209, 213, 214, 217, 218
atrium, 93
beta-blockers, 110
atrophy, 269, 305, 311
beta-carotene, 90, 155, 202, 215
autoantibodies, 31, 45
bile, 231, 242, 245, 255
autoantigens, 195
bile acids, 245
autocrine, 51
bilirubin, 11
autoimmune, 119, 161, 195, 196, 209, 217
binding, 2, 34, 35, 42, 74, 76, 83, 116, 122, 123, 165,
autoimmune disease, 119
182, 185, 199, 206, 227, 263, 265, 270, 287, 289
autoimmune diseases, 119
bioactive compounds, 240
autoimmunity, 196, 315
Index 325
bioavailability, viii, 13, 25, 26, 29, 32, 35, 36, 37, bradykinesia, 277
41, 42, 43, 44, 48, 50, 57, 70, 74, 76, 77, 121, bradykinin, 34, 36, 71, 118
138, 184, 200, 308, 309 brain, 12, 14, 33, 41, 64, 108, 137, 184, 258, 259,
biochemistry, viii, 1, 21, 191, 210, 285 260, 264, 265, 266, 267, 268, 269, 271, 272, 277,
biogenic amines, 8 278, 279, 280, 281, 285, 288, 289, 290, 291, 292,
biological activity, 4, 19, 79, 252 293, 296, 305, 309, 315, 321
biological markers, 264, 278 brain damage, 279
biological processes, 26 brain injury, 291
biological systems, 1, 9, 18 brainstem, 277
biomarker, 37, 86, 165, 198, 226, 259 branching, 279
biomarkers, x, xiii, 37, 42, 72, 94, 102, 119, 135, Brazilian, 146
140, 141, 142, 153, 187, 189, 199, 200, 207, 257, breakdown, 11, 37, 39, 120, 306
267, 269, 276, 280, 283, 286, 290, 296, 309 breast cancer, 320, 321
Biometals, 251 bubbles, 34
biomolecules, viii, 25, 77, 225, 234, 306 buffer, 48
biopsies, 97, 150, 199, 234, 242, 243, 244, 245 bypass, 92, 96, 99, 103, 107, 108, 110, 131
biopsy, 165, 224, 226, 242, 243, 244, 245 by-products, 228, 234
biosynthesis, 218
births, 140
C
black tea, 309, 320
blastocyst, 150
Ca2+, 29, 32, 34, 43, 49, 50, 70, 95, 98, 106, 109,
blepharitis, 316
129, 130, 230, 236
blindness, xiii, 16, 299, 300
calcium, 14, 26, 34, 49, 55, 62, 76, 95, 102, 103,
blocks, 7, 13, 58, 132, 206, 210, 220, 229, 267, 281
113, 116, 120, 129, 131, 230, 265, 301
blood flow, xiii, 28, 66, 75, 115, 120, 121, 139, 140,
calcium channels, 55
141, 142, 150, 152, 299, 305, 317
caliber, 28
blood glucose, 58, 199, 207
calmodulin, 34, 54, 76
blood plasma, 83
caloric restriction, 171, 188
blood pressure, viii, 25, 26, 27, 28, 30, 33, 34, 37,
calorie, 188, 242
39, 40, 42, 44, 45, 46, 47, 48, 49, 52, 56, 58, 59,
cAMP, 35
60, 61, 62, 65, 109, 136, 169, 171, 172, 176, 190,
Canada, 275
237, 310, 317
cancer, 12, 17, 18, 59, 60, 61, 117, 189, 190, 231,
Blood pressure, 168, 175
268, 308
blood pressure reduction, 35
cancer cells, 320
blood supply, x, 64, 135
candidates, 92, 99, 140
blood urea, 113
cannabinoids, 282
blood urea nitrogen, 113
cannabis, 282
blood vessels, 13, 28, 29, 33, 36, 37, 40, 55, 108,
capillary, 113, 136, 236, 305
109, 156, 166, 211, 219, 306, 313
capsule, 113, 137
blood-brain barrier, 258, 269, 310, 318
carbohydrate, 227
BMI, 269
carbohydrates, 36, 119, 173, 174, 227, 233
body fat, 162, 233
carbon, 4, 7, 44, 79, 118, 231, 232, 253
body mass index, 136, 143, 144, 162, 164, 224, 242
carbon atoms, 7
body weight, 48, 169, 175, 260
carbon tetrachloride, 118
Bohr, 289
carbonyl groups, 198, 267, 290
bonds, 97, 230
carboxyl, 263
borderline, 273
carcinogen, 21
Boston, 128
carcinogenesis, 7
bovine, 2, 54, 55, 108, 315, 320
carcinogenic, 8, 9, 19
bowel, 165
carcinogens, 231
326 Index
carcinoma, xii, 9, 223, 224, 252 CDK4, 184

cardiac function, 109 CE, 87, 90, 181, 209, 214, 216, 247
cardiac myocytes, 33 cell adhesion, 33, 84, 85, 89, 150, 212, 236
cardiac output, 118, 136 cell culture, 267, 295, 320
cardiac surgery, 94, 95, 99, 104, 105, 106, 107, 108, cell death, xii, xiii, 16, 20, 96, 103, 122, 129, 145,
125, 128, 133 148, 181, 203, 224, 225, 228, 235, 246, 250, 265,
cardiomyocytes, ix, 38, 91, 93, 95, 102, 103, 109, 277, 281, 285, 299, 301, 302, 303, 309, 319, 320,
181 322
cardiomyopathy, 109, 213, 254 cell differentiation, 86
cardiopulmonary, 107 cell growth, 26, 28, 30, 37, 55
cardiopulmonary bypass, 107 cell line, 205, 213, 241, 250, 302
cardiovascular disease, viii, 12, 14, 25, 26, 30, 34, cell membranes, 271
37, 41, 43, 46, 47, 48, 50, 55, 59, 60, 63, 78, 81, cell metabolism, 260
82, 87, 94, 130, 163, 164, 171, 173, 175, 176, cell signaling, 184
177, 180, 186, 188, 189, 190, 199, 209, 213, 269, cell surface, 35, 42, 150, 168, 177, 195, 235
291, 292, 309, 320 cellular adhesion, 71, 236, 304
Cardiovascular disease, 12, 64 cellular homeostasis, 10, 229, 303
cardiovascular function, 185 Cellular response, 316
cardiovascular physiology, 36 cellulosic, 122
cardiovascular protection, 99, 171 central nervous system, 32, 33, 53, 137, 260, 306
cardiovascular risk, 13, 30, 43, 46, 55, 57, 65, 72, central obesity, 225
81, 167, 172, 173, 174, 176, 177, 186, 269, 270, cereals, 49
283 cerebellum, 266, 268, 290
cardiovascular system, 31, 34, 49, 50, 62, 100 cerebral blood flow, 258, 264
carotene, 59, 78, 79, 80, 153, 273, 275 cerebral cortex, 262
carotenoids, 48, 79, 90, 143, 153, 154, 273 cerebral function, 258, 260
carotid arteries, 65 cerebral hypoperfusion, 270
carrier, 122, 174, 228 cerebral ischemia, 315
caspase, 229, 230 cerebrospinal fluid, 264, 292
caspases, 10 cerebrovascular, viii, 63, 64, 264, 269
CAT, 11, 16, 124, 184, 306 cerebrovascular disease, viii, 63, 64, 269
catabolism, 5, 113, 185, 293 ceruloplasmin, 153
catalase, xiii, 11, 41, 42, 92, 124, 132, 139, 142, 150, channel blocker, 43, 50
172, 188, 196, 203, 217, 232, 234, 265, 272, 299, channels, 34, 36, 40, 49, 55, 57, 70, 96, 230, 265,
303, 316 266, 288
Catalase, 11, 40 chaperones, 230, 277
catalysis, 5 cheese, 49
catalyst, 4, 84, 123, 217 chelators, 281
catalytic activity, 35, 61 chemical properties, 19
catalytic properties, 268 chemical stability, 21
cataract, 303, 321 chemiluminescence, 122
cataracts, xi, xiii, 193, 198, 299, 300 chemoattractant, ix, 17, 63, 69, 71, 77, 236, 265
catechins, 320 chemokine, 137, 259
catecholamines, 260 chemokines, 71, 73, 97
cathepsin B, 228, 241 chemotherapeutic agent, 118
cation, 230 chemotherapy, 118
cats, 170, 186 CHF, 109
causal relationship, vii, 1 childhood, 64, 162
cauterization, 307 children, 81, 119, 173, 189, 215, 242, 253
CD8+, 72
Index 327
Chile, 1, 25, 63, 91, 111, 135, 159, 193, 223, 257, 151, 173, 174, 175, 190, 200, 205, 206, 208, 224,
299 225, 240, 241, 243, 244, 245, 247, 257, 261, 269,
Chinese medicine, 240 273, 281, 282, 283, 284, 288, 299, 300, 312
Chitosan, 177 clinics, 318
chloride, 6, 77 clustering, 160
chloride anion, 6 CNS, 260, 264, 265, 276, 306
CHO cells, 227 Co, 53, 128
cholestasis, 244, 255 coagulation, 27, 55, 70, 137
cholesterol, 10, 65, 68, 77, 81, 83, 86, 88, 99, 168, cocaine, 119
169, 172, 200, 224, 270, 271, 285, 310 Cochrane, 155, 156, 248
cholinergic, 29, 262, 263, 264, 288 coenzyme, 40, 87, 143, 153, 188, 190, 234, 281, 292,
cholinergic neurons, 264 296, 311, 322
cholinesterase, 275 co-existence, 93
chromatography, 199 cofactors, 5, 19, 34, 39, 76, 100, 283
chromium, 174, 191, 202 coffee, 319
chromosome, 169, 186, 195, 209 cognition, 262, 275, 277, 283, 291, 294
chromosomes, 169 cognitive deficit, 262, 263
chronic disease, viii, 12, 25, 81, 167, 202, 207, 215 cognitive deficits, 262
chronic diseases, viii, 12, 25, 81, 202 cognitive disorders, 288
chronic illness, 194 cognitive dysfunction, 267
chronic kidney disease, 173 cognitive function, 262, 264, 276, 283, 293, 294
chronic renal failure, 12, 17, 41, 122, 123, 213, 267 cognitive impairment, 261, 263, 269, 275, 285, 286,
cigarette smoke, 211 290, 291, 293, 294
cigarette smokers, 211 cognitive test, 273, 276
cimetidine, 119 cohort, 64, 173, 187, 189, 190, 273, 274, 275, 280,
ciprofloxacin, 119 282, 292, 293
circulation, ix, 20, 91, 92, 94, 104, 118, 122, 138, collaboration, 266
140, 143, 151, 156, 165, 226, 317 collagen, xi, 29, 66, 73, 74, 86, 161, 172, 193, 198,
cirrhosis, xii, 223, 224, 232, 238, 247, 253, 255 206, 237, 244, 252, 301, 311
cis, 77 colony-stimulating factor, 69, 84, 86, 236
cisplatin, 117, 125, 133 Columbia, 271, 274
Cisplatin, 129 combination therapy, 275
CK, 59, 84, 85, 95, 98, 154, 251, 254, 315, 318, 319 combined effect, 195
CL, 57, 122, 130, 150, 155, 156, 249, 314 communication, 6, 36, 163
classical, 32, 43, 50, 103, 127, 202, 226, 235, 236, communities, 176
238, 277, 281 community, 202, 215, 258, 271, 286, 292, 293, 294
classification, 67, 82, 114, 149, 226 competition, 311
cleavage, 6, 263, 287 complement, 107, 122, 202, 241
clinical approach, 281 complement system, 107
clinical diagnosis, 259, 296 complex systems, 27
clinical presentation, 114, 195 complexity, 43, 160
clinical syndrome, 136 compliance, 189, 198
clinical trial, vii, viii, ix, x, xii, xiii, 16, 25, 43, 44, complications, xi, 14, 23, 30, 64, 89, 137, 143, 154,
46, 50, 62, 63, 79, 80, 102, 104, 111, 136, 145, 156, 160, 166, 171, 172, 176, 179, 183, 193, 194,
146, 149, 151, 173, 174, 175, 189, 190, 200, 202, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206,
205, 206, 208, 224, 225, 231, 240, 241, 243, 244, 208, 209, 210, 211, 213, 214, 215, 217
245, 247, 255, 257, 261, 269, 273, 275, 281, 282, components, 2, 3, 7, 9, 23, 27, 28, 46, 50, 52, 64, 68,
283, 284, 288, 299, 300, 309, 310, 312 72, 73, 77, 100, 104, 141, 160, 164, 168, 169,
clinical trials, vii, viii, ix, x, xii, xiii, 16, 25, 43, 44, 170, 172, 174, 177, 202, 237, 238, 277, 286
46, 50, 63, 79, 80, 102, 104, 111, 136, 146, 149, composition, 28, 164, 271
328 Index
compounds, viii, x, xi, 4, 8, 17, 18, 25, 27, 48, 112, cPLA2, 287
113, 122, 135, 136, 173, 174, 193, 197, 203, 228, CPR, 6
231, 240, 245, 268, 308, 309, 310, 311, 312 CR, 108, 129, 131, 150, 181, 185, 218, 251, 289,
computed tomography, 117 315, 318
concentration, xi, 2, 3, 4, 10, 16, 26, 31, 32, 44, 48, cranial nerve, 259
94, 95, 96, 102, 103, 112, 115, 116, 121, 124, CRC, 55
125, 137, 142, 143, 150, 159, 161, 165, 196, 203, C-reactive protein, 72, 86, 97, 98, 107, 164, 166,
206, 209, 214, 230, 261, 302, 303, 305, 314, 315 172, 173, 175, 195, 209, 221
conception, 139 creatine, 95, 98, 107
conduction, ix, 91, 92, 93, 94, 95, 97, 105, 206 creatine kinase, 95, 98, 107
conductive, 36 creatinine, 112, 113, 114, 115, 125, 126, 137, 206
conductivity, 93 credentials, 160
confirmatory factor analysis, 177 critically ill, 112, 117, 119, 128
confounders, 47 criticism, 267
congestive heart failure, 13 crossing over, 8
Congress, iv cross-linking, xi, 20, 193, 198, 268
connective tissue, 28, 29, 66, 67, 237, 238 cross-sectional, 181, 196, 209, 216, 273, 274
consciousness, 262 cross-sectional study, 196
consensus, 239, 267, 284 crosstalk, 206
consumption, ix, xii, 48, 50, 62, 63, 80, 94, 115, 121, cross-talk, 107
128, 137, 172, 173, 177, 178, 190, 203, 216, 223, CRP, 72, 195
224, 233, 245, 260, 271, 274, 305, 306, 308, 319, crystalline, 197
320 crystalluria, 118
contaminants, 122 CSF, 263, 264, 267, 268, 269, 280, 285
contractions, 31, 214 CT, 24, 248, 322
control, xi, 12, 14, 19, 22, 23, 27, 28, 30, 34, 49, 51, C-terminal, 287
53, 55, 57, 70, 79, 81, 103, 106, 118, 142, 144, culture, 313
161, 169, 170, 171, 176, 183, 193, 195, 198, 199, culture conditions, 313
200, 202, 204, 206, 240, 272 curcumin, 48, 215, 271, 283, 293
control group, 103, 142, 144, 204 Curcumin, 202, 271
controlled studies, 149 CVD, 64, 65, 68, 70, 78, 80
controlled trials, 81 cycles, 137, 140
conversion, 8, 11, 34, 37, 75, 77, 120, 139, 141, 244 cycling, 231
convulsion, 136 cyclooxygenase, 29, 36, 59, 101, 109, 120, 171, 214,
copper, 2, 18, 21, 151, 203, 251, 271, 275, 294, 304 311
cornea, 303, 316 cyclooxygenase-2, 59, 101, 109
corneal epithelium, 303, 315 cyclophosphamide, 117
coronary arteries, 33, 65, 166, 250, 290 cyclosporine, 119
coronary artery bypass graft, 92, 96, 103, 108, 110 Cyclosporine A, 118
coronary artery disease, 41, 85, 87, 183, 190, 194, cystathionine, 183
203, 216, 270, 292 cysteine, 9, 10, 47, 167, 214, 239, 243, 244, 255, 282
coronary heart disease, 65, 79, 81, 90, 92, 172, 189 cystine, 255
correlation, 20, 21, 48, 102, 142, 149, 162, 202, 263, cytochrome, xii, 6, 10, 15, 18, 20, 36, 37, 39, 40, 76,
303, 306 108, 164, 223, 227, 228, 230, 231, 232, 233, 241,
cortex, 259, 264, 266, 268, 272 243, 315, 322
corticosteroids, 318 cytokine, 6, 72, 84, 137, 146, 164, 210, 217, 225,
cortisol, 187 236, 265
cosmetics, 322 cytokines, xi, xii, 17, 29, 32, 67, 72, 73, 75, 96, 97,
cost-effective, 307 120, 164, 193, 203, 223, 234, 236, 237, 265, 301,
CP, 22, 107, 248, 254, 255, 284 310
Index 329
cytoplasm, 40, 71, 225, 277 dementia, 258, 259, 261, 262, 263, 265, 269, 271,
cytoprotective, 218, 242, 251 273, 274, 275, 284, 285, 291, 292, 293, 294
cytosine, 9, 20 demographic factors, 143
Cytoskeletal, 312 demographics, 284
cytoskeleton, 38, 116, 120, 128, 152 dendrites, 262
cytosol, 57, 95, 230 dendritic cell, 72
cytosolic, 5, 38, 74, 95, 121, 129, 131, 204, 236, density, 10, 60, 68, 69, 77, 81, 82, 83, 84, 85, 89,
244, 315, 322 138, 167, 175, 184, 190, 237, 242, 253, 263, 308,
cytotoxic, 6, 7, 33, 114, 149, 237 309
cytotoxicity, 151, 205, 232 deoxyribonucleic acid, 316
deoxyribose, 7
Department of Health and Human Services, 286
D
depolarization, 10, 93, 228, 266
deposition, 29, 37, 83, 206, 225, 237, 259, 263, 264,
D. melanogaster, 282
265, 266, 267, 272, 283, 288, 293
daily living, 262, 275
deposits, 66, 263, 264, 265, 267
dairy, 49
depressed, 171, 186
dairy products, 49
depression, 201, 203
data set, 162
deprivation, 75, 116, 128, 185
de novo, 7, 32, 226, 227, 232
derivatives, xi, 68, 94, 159, 232, 289, 296, 313
death, 12, 14, 15, 26, 64, 112, 114, 224, 225, 228,
desensitization, 109
235, 258, 260, 261, 263, 275, 277, 278, 279, 286,
destruction, 48, 119, 137, 195, 205, 217, 237
291, 301, 302, 315
detachment, 13, 129
death rate, 258
detection, xiii, 17, 33, 72, 143, 144, 216, 245, 251,
deaths, 64
257
decomposition, 6, 217
detoxification, 180, 233
defects, xiii, 16, 194, 201, 295, 299
detoxifying, 40, 176
defense, vii, ix, 1, 2, 7, 10, 11, 12, 18, 36, 48, 83, 91,
developed countries, xiii, 12, 15, 136, 299
101, 104, 137, 145, 170, 172, 176, 181, 182, 184,
developing countries, 307
199, 200, 210, 233, 235, 246, 260, 264, 265, 266,
dexamethasone, 52, 306
278, 283, 302, 306, 310
diabetes, vii, xi, 1, 7, 12, 13, 14, 22, 23, 30, 37, 41,
defense mechanisms, 18, 137, 145, 199
46, 48, 51, 54, 55, 57, 60, 61, 65, 70, 92, 117,
defenses, 83, 132, 141, 167, 229, 230, 233, 234, 244,
129, 132, 136, 140, 143, 160, 161, 162, 166, 168,
246, 303
170, 171, 173, 174, 175, 176, 178, 179, 180, 183,
deficiency, 29, 34, 39, 56, 79, 115, 119, 122, 133,
184, 189, 193, 194, 195, 196, 197, 198, 199, 200,
160, 171, 202, 231, 254, 255, 277, 292, 303, 309,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
310, 321, 322
211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
deficit, 13, 14, 96, 260, 277, 278, 279
221, 232, 233, 244, 250, 267, 268, 269, 270
deficits, 262, 269, 280
diabetes mellitus, 14, 22, 23, 30, 54, 57, 65, 92, 118,
definition, 82, 128, 149, 168, 178, 311
129, 161, 162, 194, 198, 200, 201, 203, 208, 210,
degenerate, 281
212, 213, 215, 216, 217, 219, 270
degenerative disease, 300
diabetic nephropathy, 123, 132, 202, 204, 211, 214,
degradation, xi, 35, 41, 75, 83, 122, 124, 193, 206,
220, 221
241, 250, 253, 267, 272, 279, 282
diabetic patients, xi, 14, 23, 65, 89, 163, 171, 193,
degradation process, 267
199, 200, 201, 202, 206, 207, 208, 209, 212, 213,
degrading, 42, 316
214, 215, 216, 219, 232
dehydration, 259
diabetic retinopathy, 219, 220
dehydrogenase, 5, 37, 39, 41, 75, 141, 152, 306
diacylglycerol, 206
delivery, xi, 115, 116, 120, 125, 136, 137, 159, 177,
Diagnostic and Statistical Manual of Mental
226, 227, 308
Disorders, 286
Delphi, 284
330 Index
dialysis, 122, 124, 125, 133 DNA repair, 9, 21, 265

diaphragm, 236 DNA strand breaks, 194
diastolic blood pressure, 37, 50 dogs, 99, 103, 108, 170, 186, 302, 314
dichotomy, 315 domain structure, 53
diet, 11, 17, 48, 49, 77, 79, 84, 88, 163, 168, 169, donor, 38, 40, 44, 47, 74, 79, 120, 174, 205, 245,
171, 173, 174, 176, 180, 181, 183, 185, 187, 188, 309
190, 198, 211, 226, 231, 240, 241, 243, 251, 271, donors, 168
272, 282, 283, 292, 307 dopamine, 266, 272, 277, 278, 296
diet composition, 168 dopaminergic, 15, 276, 277, 278, 279, 280, 281, 282,
dietary, 14, 45, 49, 61, 62, 89, 112, 124, 160, 164, 295
170, 173, 174, 181, 185, 189, 201, 202, 203, 208, dopaminergic neurons, 15, 276, 281, 282
212, 215, 216, 226, 227, 253, 254, 255, 256, 271, Doppler, 143, 144, 146, 148, 154
274, 283, 291, 292, 294, 307, 311 dosage, 60, 90, 281, 310
dietary fat, 173, 226, 227 dosing, 145, 148
dietary intake, 14, 112, 174, 189, 202, 216, 294 double bonds, 7, 268
dietary supplementation, 89, 202, 253 Down syndrome, 265
diets, viii, 26, 50, 79, 107, 169, 174, 198, 227 down-regulation, x, 43, 44, 45, 46, 100, 136, 141,
differential diagnosis, 114, 260 145, 163, 207, 306
differentiation, 29, 33, 37, 69, 71, 86, 139, 162, 178, drainage, 304
181, 232, 235 dream, 106
diffusion, 4, 7, 27 drinking, 282, 312
dihydroxyphenylalanine, 277 Drosophila, 297
dilated cardiomyopathy, 23 drug reactions, 108
dilation, 26, 29, 42, 55, 93, 94, 219 drug targets, 183
dimer, 200 drug treatment, 103
dimeric, 76, 243 drug use, 99
Dimethylarginine, 269 drugs, xiii, 16, 65, 92, 99, 100, 103, 117, 118, 119,
disability, 282 122, 129, 155, 166, 176, 199, 201, 205, 219, 231,
disease model, 123, 132, 290 240, 243, 299, 300
disease progression, 195, 265 DSM, 286
diseases, vii, viii, xii, xiii, 10, 12, 18, 26, 34, 48, 63, DSM-IV, 286
65, 94, 119, 163, 171, 175, 176, 188, 198, 206, duration, 35, 80, 124, 213, 265, 310
223, 232, 235, 240, 242, 244, 245, 257, 258, 260, dyslipidemia, xii, 16, 160, 161, 168, 169, 180, 220,
261, 264, 267, 268, 283, 292, 307, 309, 310, 317 223, 224, 225, 226
disorder, 14, 15, 24, 92, 93, 104, 106, 160, 226, 261, dysmetabolic, 180
276, 277, 283, 300, 314 dysphagia, 284
displacement, 34, 76 dysregulated, 60, 120
distribution, 31, 41, 95, 106, 115, 179, 187, 302 dysregulation, 17, 163, 225, 250, 305, 317, 322
disulfide, 230
disulfide bonds, 230
E
diuretics, 119
diversity, 185
EB, 59, 215, 292, 314
DNA, xi, xiii, 7, 8, 9, 14, 16, 17, 20, 21, 41, 44, 48,
eclampsia, x, 15, 24, 135, 136, 137, 139, 149, 150,
76, 96, 97, 107, 138, 141, 142, 152, 177, 193,
153, 154, 156
194, 198, 199, 212, 217, 234, 244, 251, 255, 260,
ECM, 73, 77, 207
265, 268, 278, 279, 280, 285, 289, 299, 301, 304,
edema, 114, 136, 138, 195
305, 308, 311, 318, 320, 322
Education, 105, 160
DNA damage, 7, 8, 9, 16, 20, 21, 77, 96, 152, 212,
eicosanoids, 116
217, 251, 255, 268, 285, 301, 304, 308, 311, 320,
elaboration, xi, 193
322
elastin, 66, 311
Index 331
elderly, ix, 59, 111, 112, 126, 258, 261, 273, 291, endothelium, 3, 7, 13, 16, 19, 23, 26, 27, 28, 29, 31,
293, 294, 301 33, 35, 36, 37, 38, 39, 41, 44, 45, 48, 66, 67, 70,
elderly population, ix, 111 71, 73, 79, 85, 90, 113, 116, 120, 122, 131, 139,
elders, 294 140, 145, 155, 166, 173, 183, 189, 194, 204, 205,
electric potential, 95 208, 213, 214, 236, 303, 304, 316, 320, 321
electrical cardioversion, 103, 108, 109 endotoxemia, 165
electrolyte, 114, 117 end-stage renal disease, 61, 114
electrolytes, 112 energy, 10, 15, 116, 160, 161, 162, 164, 179, 185,
electron, 4, 5, 6, 7, 10, 35, 37, 38, 39, 40, 44, 46, 74, 225, 226, 227, 228, 230, 271, 284, 305, 306, 307,
76, 79, 96, 100, 129, 130, 152, 174, 196, 198, 311
200, 207, 228, 230, 232, 236, 264, 281 energy consumption, 305, 306
electron spin resonance, 200 England, 154
electrons, 2, 5, 39, 41, 48, 74, 75, 76, 228, 229, 230, enlargement, 178, 301
231, 281 enolase, 268
electrophysiological properties, 93, 97, 103 entorhinal cortex, 262, 272
electrophysiology, 93, 102, 105 environment, 9, 10, 11, 35, 112, 139, 160, 171, 208,
elephant, 130, 297 230
ELISA, 272 environmental factors, 225, 276
EM, 83, 128, 181, 185, 249, 251, 252, 286, 288, 294, enzymatic, 2, 8, 9, 12, 35, 37, 45, 50, 104, 131, 197,
296 236, 271
emigration, 71 enzymatic activity, 2, 35, 45, 131, 271
emulsions, 183 enzyme inhibitors, 48, 207
encephalopathy, 321 enzymes, viii, 4, 5, 9, 11, 17, 24, 26, 32, 34, 35, 37,
encoding, 227, 270 38, 41, 42, 43, 75, 76, 77, 80, 92, 94, 100, 101,
endocrine, 27, 32, 169, 227 113, 116, 124, 131, 132, 139, 141, 142, 145, 150,
endometriosis, 155 155, 166, 171, 176, 181, 182, 185, 196, 201, 203,
endometrium, 150 208, 227, 230, 231, 232, 233, 234, 243, 258, 261,
endoplasmic reticulum, xii, 49, 168, 184, 223, 228 263, 266, 268, 272, 306, 310, 317
endothelial cell, xii, 7, 10, 12, 13, 16, 17, 19, 24, 27, eosinophils, 71
29, 31, 33, 35, 37, 38, 41, 46, 49, 53, 56, 59, 66, epidemic, 160
67, 68, 69, 70, 71, 73, 74, 75, 76, 79, 83, 84, 85, epidemiologic studies, 81, 271
87, 88, 89, 90, 109, 131, 138, 141, 145, 149, 152, epidemiology, 81, 105, 128, 283
156, 166, 173, 218, 224, 228, 232, 235, 236, 238, epidermal growth factor, 178
251, 265, 301, 305, 309, 315, 317, 321 epigallocatechin gallate, 309, 319, 320
endothelial cells, xii, 7, 10, 12, 13, 17, 19, 27, 29, epithelial cell, 113, 114, 116, 130
31, 33, 35, 37, 38, 46, 49, 53, 56, 59, 66, 67, 68, epithelial cells, 113, 114, 128, 130
69, 70, 71, 73, 74, 75, 76, 79, 83, 84, 85, 87, 89, epithelium, 16, 116, 303, 315, 321
90, 109, 131, 138, 141, 145, 149, 156, 166, 218, epitopes, 84, 209
224, 228, 232, 235, 236, 238, 301, 305, 309, 315, equilibrium, 27, 168
321 ER, 55, 60, 87, 90, 108, 221, 228, 230, 231, 233,
endothelial dysfunction, ix, x, 13, 20, 22, 23, 27, 30, 250, 254, 290, 292
36, 37, 38, 39, 41, 46, 47, 49, 51, 52, 53, 54, 57, ERK1, 198, 205
58, 62, 63, 67, 70, 71, 75, 77, 79, 80, 85, 88, 89, erosion, 73
100, 109, 135, 137, 138, 143, 145, 148, 152, 154, erythrocyte, 2, 121, 125, 200, 216
155, 160, 167, 172, 176, 187, 188, 200, 201, 206, erythrocytes, 11, 124, 142, 198, 200, 203
209, 213, 218, 221, 232, 250, 264, 290, 309 erythroid, 125
endothelial progenitor cells, 13 erythropoietin, 125
Endothelin, 31, 52, 62, 156, 320 ester, 30, 61, 282
endothelin-1, viii, 25, 26, 45, 51, 53, 58, 75, 152, esterification, 226
309, 320 esters, 10, 68, 71, 282
332 Index
estrogen, 171, 282 fat, viii, xi, xii, 26, 49, 50, 62, 159, 162, 163, 164,
estrogens, 119 166, 168, 169, 173, 174, 178, 179, 180, 187, 190,
ET, 26, 28, 30, 31, 38, 44, 45, 49, 50, 53, 85, 87, 146 216, 223, 225, 226, 227, 243, 249, 271
ETA, 31, 45, 53 fats, 173, 227
ethanol, 61, 119, 255, 271 fatty acids, xii, 11, 36, 55, 68, 77, 116, 129, 152,
Ethanol, 184 162, 164, 167, 170, 173, 180, 194, 208, 223, 226,
ethnicity, 55 228, 229, 233, 241, 249, 268, 271, 280, 322
ethylene, 118 Fatty liver, 251
ethylene glycol, 118 FD, 53, 130, 163
etiology, 14, 15, 16, 137, 260, 262, 276, 278 FDA, 125
EU, 213 fear, 259
Europe, 279 feedback, 7, 96, 116, 229
evolution, xii, 68, 184, 213, 223, 226, 238, 272 feeding, 47, 172
excision, 265, 289 females, 65, 169, 258
excitability, 93 ferritin, 126
excitatory synapses, 301 ferrous ion, 41
excitotoxicity, 302 fetal, x, 135, 137, 139, 145, 148, 154, 156
exclusion, 47, 262 fetal growth, 137, 139, 145, 148, 154
excretion, 30, 32, 107, 112, 114, 137, 144 fetus, 15, 136
executive function, 261 fiber, 237
exercise, 28, 175, 176, 203, 216, 240 fibers, 49, 66, 237, 306
exposure, ix, 21, 67, 69, 73, 91, 104, 121, 133, 139, fibrillar, 288
145, 198, 199, 200, 207, 291, 295, 305, 306, 307 fibrillation, ix, 91, 92, 96, 98, 105
Exposure, 75, 278 fibrinogen, 72
extracellular matrix, 26, 28, 37, 66, 67, 73, 94, 97, fibrinolysis, 27, 70, 71
123, 237, 301 fibroblast, 29, 74, 86, 237
extraction, 320 fibroblast proliferation, 74
extrusion, 18 fibroblasts, 5, 29, 31, 38, 66, 69, 74, 77, 87, 237
eyes, 302, 305, 306, 310, 313, 316, 318 fibrogenesis, 181, 237, 238, 252
fibronectin, 73, 150, 152
fibrosis, xii, 17, 32, 37, 92, 93, 94, 97, 102, 106, 165,
F
172, 184, 207, 223, 224, 225, 226, 233, 237, 238,
241, 242, 243, 244, 245, 246, 248, 252, 253, 254
FA, 56, 170, 184, 218, 252, 285, 296, 312
Fibrosis, 237
factor analysis, 162, 177
fibrous cap, 66, 67
FAD, 5, 6, 34, 74, 76
fibrous tissue, 237
failure, x, 37, 94, 128, 135, 140, 146, 148, 150, 155,
film, 303
162, 228, 287
filtration, 113, 115
false positive, 144
Finland, 209
familial, 15, 68, 177, 186, 270, 277, 278, 287, 296
fish, 11, 32, 149, 173, 271
familial combined hyperlipidemia, 186
FISH, 153
familial hypercholesterolemia, 68
fish oil, 11, 149
family, xi, 2, 5, 34, 36, 40, 60, 70, 77, 159, 171, 215,
fission, 279
230, 241, 248, 279, 288
FL, 21, 190, 320
family history, 70
flavonoid, 62, 203, 272, 319
family physician, 248
flavonoids, 48, 61, 62, 203, 216, 271, 275, 308, 309,
Fas, 146, 157
320
FasL, 146, 157
flow, xiii, 28, 66, 70, 71, 74, 75, 85, 115, 118, 120,
fasting, 113, 169, 181, 194, 196, 199, 216
121, 139, 140, 141, 142, 150, 152, 219, 229, 258,
Fasting, 152, 210, 252
264, 299, 301, 305, 313, 317
fasting glucose, 199
Index 333
fluctuations, 306 gas, 4, 34, 199

fluid, 14, 19, 70, 113, 114, 122, 153, 264, 269, 292, gas chromatograph, 199
301, 303, 310, 313 gastric, 240, 319
fluid transport, 310 gastric ulcer, 319
fluorescence, 18, 220 gastrointestinal, 113
fluoride, 108 gastrointestinal bleeding, 113
focusing, 46, 65, 246 GC, 20, 83, 88, 181, 211, 247
folate, 167, 184, 244, 269, 271, 288, 291 GE, 123, 194, 254, 286, 292, 321
folding, 230 gender, 55, 57, 162, 269
folic acid, 202, 269, 291 gene, xi, 9, 20, 21, 22, 48, 55, 61, 75, 77, 85, 86, 88,
food, xi, 159, 162, 174, 185, 198, 211, 231, 292 89, 96, 132, 144, 151, 152, 159, 160, 165, 169,
Ford, 177, 180 173, 179, 182, 184, 185, 186, 193, 194, 200, 210,
forebrain, 262 213, 214, 217, 218, 227, 232, 249, 265, 270, 272,
foreigner, 195 273, 277, 278, 279, 280, 292, 296, 317, 321
formaldehyde, 220 gene expression, xi, 22, 48, 55, 61, 85, 86, 96, 144,
fortification, 288 151, 152, 159, 160, 165, 169, 179, 182, 185, 186,
Fox, 181, 286 193, 194, 200, 213, 214, 217, 232, 249, 272, 317
FP, 185, 321 gene promoter, 210
France, 216 gene transfer, 184, 218
free radical, vii, 1, 2, 4, 10, 14, 15, 17, 18, 20, 21, generation, vii, ix, xi, xii, xiii, 1, 2, 3, 4, 8, 12, 15,
22, 27, 35, 38, 39, 41, 48, 50, 55, 56, 68, 83, 84, 16, 18, 19, 23, 27, 30, 31, 35, 36, 38, 39, 41, 43,
105, 108, 123, 130, 131, 132, 143, 144, 160, 166, 45, 46, 55, 58, 59, 63, 69, 77, 93, 94, 120, 122,
170, 171, 174, 176, 187, 201, 203, 216, 219, 241, 123, 124, 126, 130, 141, 153, 155, 164, 166, 167,
243, 244, 251, 265, 303, 305, 306, 308, 311, 315, 170, 171, 176, 183, 193, 198, 204, 223, 228, 229,
321, 322 230, 231, 232, 233, 235, 237, 240, 241, 243, 249,
free radical oxidation, 83 255, 263, 264, 269, 279, 288, 299, 306, 315
free radical scavenger, 219 genes, xi, 6, 9, 15, 37, 71, 73, 85, 96, 97, 124, 159,
free radicals, vii, 1, 4, 10, 15, 17, 20, 22, 27, 35, 48, 163, 171, 195, 227, 249, 259, 265, 270, 272, 277,
50, 56, 68, 84, 105, 130, 132, 144, 166, 170, 174, 278, 279, 289, 318, 320
176, 187, 201, 216, 219, 243, 303, 305, 306 genetic defect, 277, 283
free-radical, 176, 261, 283 genetic factors, 225, 246
fructose, 171, 185, 187, 197 genetic instability, 7
fruits, 49, 50, 124, 173 Geneva, 81
FS, 152 genistein, 241, 245, 256
functional aspects, 251 Genistein, 239, 245
functional changes, 30, 207 genome, 168, 170, 209
funding, 145 genomic, 8, 96
furan, 11 genomic instability, 8
fusion, 279 genotoxic, 20
genotype, 270, 274, 292, 303, 316
genotypes, 310
G
gentamicin, 123, 125, 126, 132, 133
Germany, 312
G protein, 31, 33
gestation, 136, 139, 142, 143, 144, 145, 146, 150,
gallbladder, 170
154
gallbladder disease, 170
gestational age, 144, 150
gametes, 149
gestational diabetes, 140
Gamma, 189
GFAP, 272
gamma-tocopherol, 153, 173
GH, 293
ganglion, xiii, 299, 301, 306, 307, 311, 315, 318,
GL, 59, 130, 180, 209, 249, 287, 314
319, 322
334 Index
glaucoma, xiii, 16, 24, 299, 300, 301, 302, 303, 304, glycosyl, 232
305, 306, 307, 308, 309, 310, 311, 312, 313, 314, glycosylated, 161, 197
315, 316, 317, 318, 319, 320, 321, 322 glycosylated hemoglobin, 197
glia, 301, 313 glycosylation, 170, 196, 207, 211, 220
glial, xiii, 265, 271, 299, 301, 313, 314 gold, 103
Glial, 301 gold standard, 103
glial cells, xiii, 265, 299, 301, 313 government, iv
glial fibrillary acidic protein, 271 G-protein, 29, 31
glial fibrillary acidic protein (GFAP), 271 gracilis, 55
gliosis, 277 grading, 249
glomerulonephritis, 17, 24, 206 grafting, 96, 103, 108, 110
glomerulus, 17, 113, 118, 119, 120, 122 granules, 301
glucocorticoids, 306 granulocyte, 84, 236
gluconeogenesis, 113, 196, 210 grapes, 126, 309
glucose, xi, 44, 47, 58, 61, 75, 79, 113, 123, 159, green tea, 187, 272, 282, 283, 297, 308, 320
161, 163, 168, 169, 170, 173, 174, 176, 179, 185, groups, x, 9, 10, 11, 14, 35, 48, 66, 75, 80, 99, 103,
187, 193, 194, 196, 197, 199, 200, 202, 207, 208, 114, 119, 121, 125, 135, 145, 198, 200, 202, 203,
209, 210, 211, 213, 214, 216, 227, 233, 238, 240, 231, 242, 267, 272, 275, 276, 278, 290, 310
244, 248, 251, 270 growth, 15, 16, 26, 28, 30, 33, 37, 55, 67, 69, 70, 73,
glucose metabolism, xi, 123, 159, 169, 170, 199, 75, 77, 88, 93, 97, 137, 138, 139, 144, 145, 147,
210, 238, 248, 270 148, 150, 154, 178, 194, 238, 253, 263, 309
glucose tolerance, 163, 168 growth factor, 28, 33, 67, 69, 75, 88, 93, 97, 138,
glucose-induced insulin secretion, 200 139, 144, 147, 178, 194, 238, 253, 309
GLUT, 196 growth factors, 28, 33, 67, 75
glutamate, 265, 266, 285, 289, 300, 301, 302, 313, guanine, 8, 9, 21, 212
314 guidelines, 65, 81, 280
glutamate receptor antagonists, 301 Guillain-Barré syndrome, 117
glutamatergic, 265, 266, 301 gut, 165, 182
glutamic acid, 195
glutamine, 268, 301, 302, 312, 314
H
glutathione, xiii, 9, 11, 16, 41, 42, 43, 48, 92, 119,
124, 125, 132, 139, 142, 152, 166, 167, 172, 173,
H1, 53
174, 177, 180, 188, 190, 196, 199, 200, 202, 204,
H2, 35, 55
212, 213, 215, 230, 233, 234, 239, 241, 243, 244,
HA, 57, 60, 188, 314, 319
253, 254, 255, 266, 269, 282, 285, 289, 295, 297,
Haj, 131
299, 303, 311, 315, 316
half-life, 4, 35, 267
glutathione peroxidase, 11, 41, 92, 124, 139, 142,
halogenated, 231
173, 196, 200, 202, 204, 213, 215, 269, 316
handling, 230, 260
glycation, xi, 15, 187, 193, 194, 197, 198, 208, 210,
hands, 259
211, 212, 220, 221, 233, 238
harm, 179
Glycation, 197, 205, 210
harmony, 179
glycemia, xi, 123, 174, 193
Hawaii, 273
glycerol, 126
HDL, 10, 65, 168, 203
glycine, 132
HE, 176, 209, 216
glycogen, 173, 196
healing, 106
glycol, 119
health, xi, 17, 33, 90, 170, 187, 188, 193, 308
glycolysis, 197
Health and Human Services, 286
glycoprotein, 263
health effects, xi, 170, 193
glycoside, 240
hearing, 125
glycosides, 319
hearing loss, 125
Index 335
heart, ix, 7, 12, 13, 22, 30, 32, 33, 40, 41, 46, 48, 53, heterogeneous, 69, 115, 208, 258
57, 58, 61, 64, 65, 79, 81, 90, 91, 92, 93, 94, 95, high blood pressure, 13, 27, 38, 49, 136, 172
96, 97, 103, 104, 108, 115, 172, 174, 189, 242 high fat, 231, 241
Heart, 53, 55, 56, 61, 64, 65, 81, 82, 83, 105, 106, high pressure, 3
108, 109, 110, 131, 177, 178, 181, 218, 250, 269, high resolution, 54
288, 290, 291 high risk, 14, 126, 145, 149, 156, 161, 173
heart attack, 64 high-density lipoprotein, 10, 184
heart disease, viii, 61, 63, 64, 65, 79, 81, 93, 172 high-fat, 163, 168, 169, 180, 190, 226, 227
heart failure, 12, 13, 22, 41, 58, 108, 115, 174 high-performance liquid chromatography, 199
heart rate, 46, 48 high-risk, 144, 145, 146, 176
heat, 72, 86, 315 hip, 162
heat shock protein, 72, 86, 315 hippocampal, 261, 269, 287, 291
heaths, 305, 306 hippocampus, 262, 264, 266, 268, 272, 288, 289, 290
heating, 9, 198 histamine, 34, 35
heavy metal, 118 histological, 82, 126, 140, 180, 238, 240, 241, 244,
heavy metals, 118 249, 254, 262, 263, 265, 276, 302
Helicobacter pylori, 316 histology, 174, 226, 242, 253
hematocrit, 125 histopathology, 290
hematologic, 119 HIV, 177, 178
hematological, 117, 118, 137 HK, 149, 212, 288
hematoma, 66 HLA, 195
hematopoietic, 125, 137 Holland, 51, 56, 87, 88
heme, 6, 15, 34, 35, 40, 74, 76, 77, 122, 126, 134, homeostasis, viii, 12, 26, 27, 29, 41, 63, 64, 70, 74,
231 95, 113, 114, 117, 161, 163, 179, 187, 265, 279,
heme oxygenase, 15, 126, 134 286, 295, 315
hemodialysis, 17, 48, 61, 122, 131, 132, 133, 215 homocysteine, 51, 152, 166, 167, 171, 174, 184, 245,
hemodynamic, 184, 305 290, 291
hemodynamics, 53, 118, 128, 149 Homocysteine, 167, 183, 184, 288, 291
hemoglobin, 121, 122, 137, 197, 198, 205 Honda, 180, 284
hemorrhage, 66, 115 honey, 49
hemostasis, 27, 70 hormone, 113, 171, 179, 185, 188, 272
hepatic fibrosis, 238, 252, 253 hormones, 26, 27, 29, 33, 113, 225
hepatic injury, 241, 243 hospital, ix, 91, 92, 128, 146
hepatic stellate cells, 182, 237, 238, 252 hospital stays, ix, 91
hepatitis, 184, 235, 244, 252 hospitalization, 112
hepatitis a, 235 hospitalized, ix, 111, 112, 122, 126
hepatitis C, 184, 252 host, 7, 83
hepatocellular, xii, 223, 224, 225, 229, 252 HR, 54, 87, 133, 185, 316
hepatocellular carcinoma, xii, 223, 224, 252 HSP60, 72
hepatocyte, xii, 164, 224, 225, 226, 228, 229, 230, human condition, 167
233, 234, 235, 236, 238, 241, 242 human immunodeficiency virus, 162
hepatocytes, xii, 8, 11, 162, 164, 165, 223, 226, 228, human neutrophils, 202
232, 233, 236, 237, 245, 272 humans, viii, xi, 5, 9, 25, 32, 33, 36, 39, 43, 44, 48,
hepatoma, 250 51, 55, 58, 69, 76, 79, 89, 125, 126, 163, 164,
hepatorenal syndrome, 115 168, 170, 174, 175, 189, 193, 198, 200, 207, 226,
hepatotoxic drugs, 224 231, 244, 246, 273, 278, 304, 306, 311, 321
hepatotoxicity, 251, 252, 253, 255 hyaline, 224, 242, 252
herbal, 320 hybrids, 169
heredity, 55, 177 hydatid, 150
heterogeneity, 50, 80 hydatid mole, 150
336 Index
hydro, 11, 112, 190, 231 hypothesis, 9, 17, 23, 24, 38, 40, 45, 47, 55, 67, 68,
hydrocarbons, 231 70, 78, 82, 84, 94, 98, 99, 140, 143, 144, 148,
hydrogen, viii, xiii, 5, 7, 10, 23, 25, 26, 28, 35, 39, 160, 162, 168, 175, 176, 189, 196, 201, 203, 225,
41, 46, 48, 51, 55, 56, 77, 117, 120, 163, 194, 245, 251, 267, 268, 270, 272, 276, 283, 285, 287,
212, 228, 229, 231, 278, 299, 315, 316, 320 288, 301, 302, 305, 309
hydrogen peroxide, viii, xiii, 5, 23, 25, 26, 28, 35, hypovolemia, 118
39, 41, 46, 48, 51, 55, 56, 77, 120, 163, 194, 212, hypoxemia, 260
228, 229, 278, 299, 315, 316, 320 hypoxia, x, 32, 75, 96, 102, 103, 109, 115, 116, 121,
hydrolases, 231 129, 131, 135, 138, 140, 141, 303, 310
hydrolysis, 33 Hypoxia, 129, 139, 317
hydrolyzed, 301 hypoxic, 106, 117, 139
hydroperoxides, 11, 199
hydrophilic, 11, 190
I
hydrophobic, 4, 11, 166, 242
hydrostatic pressure, 115, 116, 318
iatrogenic, 136
hydroxide, 4
IB, 57, 60, 154, 314
hydroxyl, 4, 9, 37, 41, 48, 108, 120, 126, 194, 205,
ICAM, 71, 73, 85, 145, 156, 179, 236
228, 278, 281, 288, 306, 319
ice, 272
hydroxylation, 39, 75, 234, 244, 268
ICU, 128
hyperactivity, 170
id, 146
hypercholesterolemia, 13, 57, 70, 79, 86, 88, 89, 90,
identification, 33, 140, 160, 283, 290, 308
168, 254, 270
identity, 3
hyperglycaemia, 180
idiopathic, 23, 277
hyperglycemia, 123, 160, 161, 170, 172, 174, 179,
IFN, 73, 236
189, 194, 196, 197, 198, 199, 200, 203, 207, 208,
IGF, 150
210, 211, 212, 214, 225, 227
IGF-I, 150
hyperhomocysteinemia, 138, 168, 183, 290
IgG, 199
hyperinsulinemia, 161, 168, 170, 172, 174, 178, 194,
IL-1, 72, 79
227
IL-6, 44, 72, 98, 145, 196
hyperkalemia, 114, 117, 124
IL-8, 73
hyperlipidemia, 17, 67, 163, 172, 194, 206, 213, 243,
imaging, 70, 291
246, 269
imbalances, 211, 225
Hypertension, v, 12, 22, 23, 25, 26, 27, 42, 43, 49,
immune activation, 72, 240
51, 52, 53, 55, 57, 58, 59, 60, 61, 62, 86, 88, 109,
immune cells, 234, 236, 237, 238
136, 151, 153, 155, 166, 178, 187, 189, 214, 219,
immune response, 33, 166, 195, 232, 235
306, 307
immune system, 2, 86, 225, 233, 234
hypertensive, viii, 22, 23, 25, 26, 30, 37, 39, 40, 42,
immunity, 145, 210
44, 46, 47, 48, 50, 54, 56, 57, 58, 60, 61, 62, 74,
immunoglobulin, 117
102, 108, 109, 145, 149, 153, 154, 155, 156, 169,
immunohistochemical, 150, 309, 321
170, 182, 186, 188, 190, 214, 218, 306, 315
immunological, 67, 233
hypertonic saline, 306, 307
immunomodulation, 236
hypertriglyceridemia, 162, 169, 186, 200
immunomodulatory, 242
hypertrophy, 30, 32, 39, 52, 73, 94, 97, 102, 106,
immunosuppressive, 118, 195
206
immunosuppressive drugs, 195
hypocholesterolemic, 253
impaired glucose tolerance, 168
hyponatremia, 114, 117
in situ, 38, 320
hypoperfusion, x, 115, 135, 137, 139, 146, 264
in utero, 139
hypotension, 31
in vitro, 8, 9, 15, 41, 46, 48, 53, 69, 73, 78, 79, 83,
hypotensive, 30, 48, 172, 310
89, 102, 121, 123, 124, 126, 130, 132, 148, 156,
Index 337
195, 198, 203, 207, 216, 219, 229, 232, 244, 245, inflammatory response, xii, 67, 72, 73, 75, 78, 80,
279, 282, 304, 308, 309, 316, 320 120, 140, 151, 154, 170, 179, 224, 235, 236, 238,
in vivo, vii, 1, 2, 9, 13, 36, 37, 38, 44, 48, 52, 58, 69, 309
77, 78, 79, 83, 88, 89, 116, 126, 163, 171, 184, inflammatory responses, 309
195, 197, 198, 201, 219, 220, 221, 229, 232, 238, ingest, 79, 227
241, 242, 253, 267, 279, 290, 304, 308, 320, 321 ingestion, 24, 203
inactivation, 9, 11, 13, 37, 38, 61, 119, 146, 205, inherited, 185, 258, 278, 279
207, 208, 230, 234 inhibition, xi, 9, 30, 34, 36, 47, 49, 51, 53, 60, 69,
incidence, 12, 15, 45, 59, 64, 99, 103, 104, 105, 112, 70, 71, 76, 77, 79, 87, 89, 99, 102, 118, 121, 124,
118, 133, 136, 145, 149, 173, 202, 215, 274, 276, 132, 139, 159, 199, 205, 206, 219, 220, 221, 228,
307 232, 240, 246, 279, 281, 295, 320, 321
inclusion, 202 inhibitor, 7, 31, 34, 39, 40, 54, 57, 58, 72, 76, 84, 87,
income, 273 88, 118, 124, 126, 139, 150, 169, 202, 216, 218,
India, 153, 307 219, 220, 221, 231, 269, 275, 281, 292, 296, 314
Indian, 271 inhibitors, 17, 29, 31, 48, 75, 108, 117, 118, 162,
Indians, 162, 191 203, 204, 205, 207, 212, 219, 221, 232, 295, 308,
indices, 213 311
indigenous, 202 inhibitory, 97, 232, 266
indirect effect, 29 inhibitory effect, 232
inducer, 172 initial state, 225
induction, 16, 26, 48, 73, 75, 77, 80, 85, 124, 126, initiation, 9, 64, 68, 86, 95, 204, 302, 309
148, 155, 172, 184, 197, 198, 204, 211, 212, 231, injection, 126, 203, 306, 307, 309
232, 233, 236, 238, 260, 269, 319 injections, 279, 314
industrial, 231 injuries, 17, 94, 102, 112, 211
infants, 150 injury, ix, x, 2, 13, 15, 17, 20, 22, 23, 26, 28, 29, 36,
infarction, 141, 305 47, 55, 58, 60, 67, 70, 73, 82, 91, 93, 94, 96, 104,
infection, 48, 119, 235 106, 107, 108, 111, 113, 114, 115, 116, 119, 120,
infectious, 119 121, 122, 123, 124, 125, 126, 127, 129, 130, 131,
Infiltration, 236 132, 134, 151, 164, 165, 182, 184, 188, 194, 197,
inflammation, ix, 2, 3, 9, 18, 19, 33, 37, 42, 55, 61, 204, 206, 207, 208, 211, 225, 229, 234, 236, 237,
66, 70, 71, 72, 86, 91, 92, 94, 96, 97, 99, 104, 241, 243, 246, 249, 251, 252, 253, 260, 261, 264,
105, 109, 137, 145, 156, 160, 164, 165, 168, 171, 279, 282, 301, 302, 305, 306, 313, 315, 317, 318
172, 174, 181, 187, 188, 189, 202, 209, 212, 213, innate immunity, 71
216, 224, 226, 234, 235, 236, 237, 241, 242, 243, innervation, 264
245, 246, 251, 252, 259, 267, 279, 302, 310, 321 inorganic, 4
inflammatory, ix, xii, 3, 17, 26, 27, 29, 33, 55, 63, iNOS, 6, 33, 76, 88, 101, 138, 141, 204, 218, 232,
67, 71, 72, 73, 74, 75, 78, 80, 82, 85, 93, 97, 99, 235, 236, 269, 279
106, 107, 112, 114, 116, 117, 118, 120, 130, 140, inositol, 32
151, 154, 160, 164, 165, 166, 168, 170, 174, 176, INS, 205
179, 183, 185, 194, 198, 203, 205, 207, 209, 219, insertion, 6, 9
221, 223, 225, 227, 233, 234, 235, 236, 237, 238, insight, 26, 280
239, 240, 245, 252, 259, 265, 267, 269, 271, 288, instability, 7, 124, 305
308, 309 institutionalization, 275
inflammatory bowel disease, 267 instruments, 267
inflammatory cells, 74, 116, 198, 237 insulin, xi, xii, 14, 23, 32, 61, 75, 159, 160, 161, 162,
inflammatory disease, 71, 82, 240 163, 164, 166, 168, 169, 170, 172, 174, 175, 176,
inflammatory mediators, ix, xii, 63, 223, 234, 235, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188,
236, 238, 252 189, 190, 193, 194, 195, 196, 197, 200, 202, 203,
204, 208, 210, 211, 213, 214, 215, 216, 217, 219,
338 Index
223, 224, 225, 227, 233, 237, 238, 239, 240, 246, intrinsic, 36, 92, 93, 102, 114, 122, 131, 230, 234
249, 250, 251, 270 invasive, 80, 139, 150
insulin resistance, xi, xii, 61, 159, 160, 161, 162, invertebrates, 174
163, 164, 166, 168, 169, 170, 172, 175, 178, 179, investment, 81
180, 182, 185, 186, 188, 189, 190, 196, 202, 203, ion channels, 96
208, 210, 215, 216, 223, 224, 225, 227, 233, 237, ions, 8, 19, 259
238, 239, 240, 246, 249, 250, 251, 270 IOP, 300, 301, 304, 305, 306, 307, 308, 311
insulin sensitivity, 162, 170, 176, 179, 187, 219 IP, 156, 157
insulin signaling, 186 IR, 186
insulin-producing cells, 194, 210 Iran, 243
insults, 114, 236 iris, 303, 316
integrins, 71, 116, 129, 150 iron, xii, 4, 34, 76, 123, 125, 133, 151, 180, 223,
integrity, viii, 11, 13, 25, 27, 28, 88, 114, 116, 120, 251, 278, 281, 296, 304, 310, 320
128, 165, 190, 208, 230, 304 iron deficiency, 133
interaction, xi, 18, 26, 27, 28, 36, 67, 108, 121, 142, IS, 60, 129, 216
146, 148, 159, 167, 190, 193, 198, 208, 264, 266, ischemia, ix, x, 3, 5, 7, 19, 20, 22, 91, 92, 94, 95, 97,
316 102, 103, 104, 109, 111, 115, 116, 117, 120, 121,
interactions, 22, 23, 27, 32, 54, 55, 70, 85, 146, 157, 124, 126, 128, 130, 131, 132, 134, 137, 141, 150,
160, 181, 182, 281, 316, 317 151, 181, 188, 204, 218, 253, 305, 306, 314, 315
intercellular adhesion molecule, 71 ischemia reperfusion injury, 134
interference, 168, 238 ischemic, viii, 37, 43, 63, 64, 94, 109, 114, 115, 116,
interferon, 72, 195, 203, 236 117, 120, 121, 124, 127, 129, 130, 131, 132, 305
interferon-γ, 72, 195, 203 ischemic heart disease, viii, 63, 64
interleukin, 44, 58, 73, 97, 98, 156, 196, 203, 219, ischemic stroke, 37, 43
235, 236, 271 Islam, 133, 210
interleukin-1, 203, 219, 235, 236, 271 isoenzymes, 6
interleukin-6, 44, 58, 73, 97, 98, 156, 196, 236 isoflavones, 202, 245
intermolecular, 198 isoforms, 5, 34, 38, 40, 56, 76, 123, 214, 231, 270,
interstitial, 27, 116, 119, 130 287
interstitial nephritis, 119, 130 isolation, 176
interval, 282 isomers, 171
intervention, 46, 50, 143, 166, 167, 173, 194, 196, Italy, 224, 248, 300, 312
200, 240, 241, 242, 243, 244, 245, 255, 267, 271, IV collagenase, 156
283
intestine, 3
J
intima, 66, 67, 68, 71, 72, 77, 82, 84
intoxication, 108, 117
JAMA, 59, 60, 81, 90, 108, 128, 177, 286, 293, 294,
intracellular signaling, xi, 17, 126, 193
319
intramuscular, 126
Japan, 224
intramuscular injection, 126
Japanese, 212, 248, 254, 273
intraocular, xiii, 16, 299, 300, 301, 303, 304, 307,
JI, 177
308, 309, 310, 311, 312, 313, 314, 315, 317, 318,
Jordan, 61
319, 322
JT, 18, 56, 87, 150, 182, 217, 220, 254, 285, 287,
intraocular pressure, xiii, 16, 299, 300, 301, 303,
292, 294, 295, 316
304, 308, 310, 311, 312, 313, 314, 315, 317, 318,
Jung, 52
319, 322
intrauterine growth retardation, 150
intravascular, 137 K
intravenous, 44, 59, 125, 133, 282, 297
intravenously, 48 K+, 34, 36, 49, 70, 116
Index 339
kainic acid, 289 lesions, 66, 67, 68, 69, 72, 73, 74, 75, 76, 77, 82, 83,
kappa, 31, 138, 170, 187, 232 84, 87, 88, 126, 166, 206, 249, 319
kappa B, 31, 138, 170, 187, 232 leukocyte, 27, 33, 67, 70, 317
ketones, 164 Leukocyte, 151
KH, 52, 108, 154, 155, 157, 211, 255, 288, 291, 293, leukocytes, 18, 42, 58, 71, 73, 97, 120, 138, 140,
294 141, 237, 318
kidney, 7, 17, 32, 33, 54, 61, 62, 112, 113, 114, 115, leukotrienes, 129
116, 118, 119, 120, 121, 123, 124, 126, 128, 129, levodopa, 277, 282
130, 131, 132, 137, 173, 184, 187, 198, 204, 220 Lewy bodies, 276, 277, 279
kidney stones, 17 LH, 106, 107, 169, 284, 292, 321
kidneys, 115, 130, 134, 148 liberation, 260, 279
kinase, 6, 15, 34, 70, 89, 95, 123, 154, 179, 184, 194, life expectancy, 258, 283
206, 214, 232, 278, 279 life forms, 3
kinase activity, 107, 278, 279 lifespan, 188, 261
kinases, 37, 53, 230 lifestyle, ix, 12, 57, 111, 253
kinetics, 198 lifestyle changes, 12
King, 154, 155, 209, 314, 319, 322 lifetime, 10, 258
kinins, 34 ligand, 35, 71, 157, 188, 315
KL, 22, 83, 130, 156, 179, 185, 189, 249 ligands, 71, 206, 208
knockout, 30, 33, 168, 200, 206, 267, 280, 289 likelihood, 143, 149, 196, 275
Korean, 182 limitation, 235
Krebs cycle, 76 limitations, 168, 259, 284
linear, 164, 173
linkage, 138, 169, 207
L
links, 7, 166
linoleic acid, 10
LA, 53, 59, 61, 107, 153, 169, 177, 186, 190, 217,
lipase, 185
248, 249, 250, 251, 286, 288, 291, 314, 315, 318
lipemia, 169
labor, 155
Lipid, 9, 10, 21, 58, 81, 86, 88, 89, 90, 152, 153,
lamellae, 300
164, 181, 198, 208, 213, 247, 250, 268, 312
lamina, 26, 66, 71, 75, 87, 139, 236, 313
lipid metabolism, 170, 173, 183, 224, 225, 227, 230,
laminar, 26, 71, 75, 87, 313
287
Laminar, 75, 85
lipid oxidation, 41, 77, 171, 198
laminin, 150
lipid peroxidation, x, xi, 4, 7, 9, 11, 14, 21, 22, 24,
language, 261
41, 42, 46, 57, 79, 83, 90, 99, 108, 119, 123, 124,
laser, 306, 307, 313, 319
126, 135, 138, 140, 141, 142, 143, 144, 151, 153,
latency, 300
155, 159, 164, 166, 171, 172, 174, 181, 187, 188,
late-onset, 270, 292
198, 200, 203, 212, 213, 225, 226, 228, 231, 234,
late-onset AD, 270
241, 245, 251, 254, 261, 265, 266, 267, 268, 272,
late-stage, 262, 285
278, 279, 280, 286, 289, 290, 300, 305, 306, 308,
LC, 52, 55, 129, 154, 155, 190, 255, 297, 319
309, 311, 321
L-carnitine, 282
lipid peroxides, 153, 156, 164, 173
LDL, ix, 10, 21, 46, 63, 65, 67, 68, 69, 72, 73, 75,
lipid profile, 213
77, 78, 79, 82, 84, 89, 125, 133, 138, 163, 166,
lipids, xi, xii, 4, 17, 41, 44, 48, 59, 67, 68, 73, 75,
167, 168, 172, 173, 175, 183, 198, 242, 308
76, 83, 116, 119, 142, 163, 171, 174, 189, 193,
leakage, 46, 315
197, 198, 202, 223, 225, 226, 227, 232, 236, 260,
left ventricle, 34
267, 271, 277, 291, 303, 306, 309
lens, xi, 193, 197, 198, 303, 310, 321
lipodystrophy, 162, 178
lenses, 211
lipolysis, 170, 186, 236
leptin, 139, 150, 163, 168, 169, 181, 200, 252
lipooxygenase, 69, 77
340 Index
lipopolysaccharide, 236, 250 lupus, 119

lipoprotein, 10, 67, 81, 82, 83, 84, 85, 86, 87, 89, 90, lutein, 79
161, 167, 175, 184, 189, 212, 230, 253, 308, 310 LV, 86, 218
Lipoprotein, 83 lycopene, 79, 90, 203
lipoproteins, 10, 68, 69, 77, 82, 83, 84, 100, 138, lymph, 195
154, 163, 183, 184, 190, 203, 216, 224, 242, 249 lymph node, 195
liposomes, 90, 181 lymphatic, 169
lipoxygenase, 32, 36, 38, 82, 84, 88, 89, 120 lymphocyte, 195, 254
liquid chromatography, 190, 199 lymphocytes, 67, 71, 272, 286, 305, 311, 317
lithium, 117 lysine, 9, 198, 215
liver, x, xi, xii, 2, 7, 18, 44, 79, 108, 118, 131, 135,
137, 150, 159, 161, 162, 163, 164, 165, 168, 170,
M
172, 174, 177, 178, 179, 180, 181, 182, 184, 185,
188, 190, 207, 210, 223, 224, 225, 226, 227, 228,
M1, 316
229, 232, 233, 235, 236, 237, 238, 239, 240, 241,
machinery, 227, 276, 279
242, 243, 244, 245, 246, 247, 248, 249, 250, 251,
macromolecules, 260, 268, 280
252, 253, 254, 255, 272
macronutrients, 292
liver cells, 225, 228
macrophage, 20, 33, 68, 69, 71, 82, 83, 84, 166, 184,
liver cirrhosis, 255
253
liver damage, 226, 227, 253
macrophages, 8, 29, 33, 37, 64, 66, 67, 68, 69, 70,
liver disease, xii, 118, 180, 181, 182, 184, 185, 190,
71, 72, 74, 76, 77, 84, 85, 87, 141, 166, 183, 198,
223, 224, 225, 229, 232, 237, 238, 239, 241, 243,
203, 216, 234, 236, 237, 251, 252
244, 247, 248, 249, 250, 251, 252, 253, 255
macular degeneration, 303, 316
liver enzymes, 150, 182, 243
magnesium, 137, 202
liver failure, 224
magnetic, 210, 291
liver transplant, xii, 223
magnetic resonance, 18, 291
liver transplantation, xii, 223
magnetic resonance imaging, 291
LM, 22, 51, 57, 82, 86, 90, 109, 130, 149, 150, 180,
Maillard reaction, 197, 198, 211
249, 250, 285, 287, 296, 318, 321
maintenance, 10, 20, 27, 29, 34, 41, 48, 86, 112, 114,
localization, 42, 56, 72, 150, 152, 188
115, 116, 122, 123, 128, 161, 229, 235, 258, 279
location, 38, 93, 100, 106, 108, 265
Maintenance, 230
locomotor activity, 297
maladaptive, 97
locus, 195
males, 169, 258
London, 249, 251
malignant, 234
longitudinal study, 154, 273
malnutrition, 259
losses, 100
malondialdehyde, 9, 15, 21, 152, 200, 201, 204, 234,
lovastatin, 119
265, 302, 316
low fat diet, 226
malondialdehyde (MDA), 9, 265
low risk, 145
Mammalian, 132
low-density, 10, 82, 84, 85, 86, 87, 89, 90, 125, 184,
mammalian cells, 2, 4, 11, 61, 233
188, 189, 212, 226, 308, 309, 310
mammals, 6, 18, 41, 250
low-density lipoprotein, 10, 82, 84, 85, 86, 87, 89,
management, viii, xiii, 16, 25, 43, 49, 81, 128, 129,
90, 125, 184, 188, 189, 212, 226, 310
136, 137, 150, 176, 177, 199, 209, 224, 230, 243,
low-density lipoprotein receptor, 188
247, 286, 299, 300
LPS, 165
manganese, 40, 132, 232, 250
lumen, 64, 66, 67, 113, 116, 182, 230, 236, 301
manganese superoxide, 132, 250
luminal, 28, 31, 66, 70, 113, 165
manganese superoxide dismutase, 132, 250
lung, 9, 20, 21, 108, 132, 211, 320
manipulation, 38, 164, 200
lung cancer, 211, 320
mannitol, 117, 129
lungs, 33
MAO, 281, 296
Index 341
MAPK, 33, 109 mesenteric vessels, 200

mapping, 186 messenger RNA, 82
mass spectrometry, 54, 190, 199 messengers, ix, 26, 37, 91, 92
mast cell, 309, 321 meta-analysis, 46, 90, 99, 105, 146, 173, 177, 189,
mast cells, 309 190, 292, 308, 320
maternal, x, 15, 16, 135, 136, 137, 138, 139, 140, metabolic, xi, xii, 1, 10, 12, 14, 17, 24, 38, 75, 115,
141, 142, 143, 144, 146, 148, 149, 150, 151, 153, 126, 159, 160, 161, 162, 163, 164, 165, 166, 167,
154, 155, 156 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
maternal age, 136 178, 179, 180, 181, 182, 184, 185, 186, 187, 188,
matrix, 26, 28, 37, 40, 66, 67, 72, 73, 94, 97, 123, 189, 190, 194, 202, 208, 223, 224, 225, 226, 227,
147, 237, 280, 300, 301, 311, 313, 317 233, 238, 240, 244, 246, 247, 248, 250, 260, 300,
matrix protein, 123 314
maturation, 171, 264, 266 metabolic disorder, 163, 194, 224
MB, 109, 189, 190, 284, 294, 296, 314, 315, 317, metabolic disturbances, 194
320 metabolic dysfunction, 174
MCI, 261, 267, 268, 269, 275 metabolic pathways, 14, 161, 166, 176, 208
MCP, 69, 71, 73 metabolic rate, 17, 260
MCP-1, 69, 71, 73 metabolic syndrome, xi, xii, 14, 24, 159, 160, 161,
MDA, 9, 14, 142, 144, 245, 267, 268 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
meals, 50, 161, 162 172, 173, 174, 175, 176, 177, 178, 179, 180, 182,
mean arterial blood pressure, 48 184, 185, 186, 187, 188, 189, 190, 223, 224, 225,
measurement, 19, 20, 85, 302, 306 238, 247, 248
measures, 118, 194, 202, 203 metabolism, vii, ix, xi, 2, 4, 7, 10, 15, 24, 36, 39, 75,
meat, 50 83, 95, 111, 112, 116, 119, 120, 123, 128, 159,
mechanical stress, 28, 305 160, 164, 165, 166, 167, 169, 170, 173, 174, 176,
media, 13, 42, 66, 72, 117, 139 183, 184, 186, 187, 193, 194, 198, 199, 205, 210,
median, 276 224, 225, 226, 227, 228, 230, 233, 234, 238, 243,
mediation, 183, 234 248, 260, 261, 270, 271, 279, 280, 284, 285, 287,
mediators, viii, ix, xii, 22, 25, 26, 28, 29, 36, 41, 50, 295, 300, 315, 321
55, 63, 93, 96, 97, 103, 104, 122, 126, 127, 143, metabolite, 36, 241, 245
164, 203, 210, 223, 225, 231, 234, 235, 236, 237, metabolites, 36, 94, 132, 194, 201, 278
238, 249, 252, 283 metabolizing, 301
medications, 61, 113 metalloproteinase, 74, 305
medicinal plants, 240 metalloproteinases, 32, 72, 73
medicine, 99, 202, 240, 319 metals, 4, 18, 194, 260, 264, 304
Mediterranean, 17, 49, 62, 173, 292 metformin, 190, 212, 240, 244, 254
melanin, 316 methionine, 10, 167, 174, 244, 245, 255
melatonin, 62, 272, 319 methyl group, 7, 244
membrane permeability, 117 methylation, 9, 244
membranes, 4, 5, 11, 19, 38, 41, 100, 104, 108, 116, methylprednisolone, 107
125, 129, 166, 171, 230, 231, 232, 241, 259, 264, MHC, 72, 195, 305
265, 281, 289, 301, 309 mice, 22, 30, 32, 33, 43, 51, 52, 54, 55, 77, 85, 87,
memory, 71, 216, 261, 262, 272, 273, 284, 293 88, 89, 125, 132, 133, 163, 168, 169, 171, 179,
memory deficits, 216, 293 180, 182, 183, 184, 185, 187, 188, 200, 201, 204,
memory performance, 273 206, 211, 213, 218, 227, 231, 232, 256, 264, 266,
men, 44, 59, 60, 160, 180, 182, 187, 202, 215, 216, 267, 271, 272, 279, 280, 281, 287, 289, 293, 296,
258, 273, 275, 293 309, 321
menopause, 171 microarray, 107
mercury, 118 microcirculatory, 209
mesangial cells, 123 microdialysis, 321
342 Index
microenvironment, 4, 46, 168, 177 model system, 232, 306

microfilaments, 128 modeling, 44, 296
microglia, 259, 263, 265, 272, 309 models, viii, xiii, 7, 25, 30, 42, 46, 49, 75, 102, 123,
microglial, 279, 321 124, 125, 126, 132, 145, 163, 167, 168, 169, 170,
micronutrients, 170, 202, 215 171, 176, 186, 196, 200, 201, 203, 205, 206, 213,
microparticles, 140 217, 226, 227, 228, 231, 232, 233, 238, 239, 241,
microsomes, 108 243, 244, 245, 255, 257, 261, 264, 266, 271, 274,
microtubule, 116, 128, 287 276, 278, 279, 281, 283, 288, 294, 300, 302, 306,
microtubules, 262 307, 311, 315, 318, 319
microvascular, 40, 109, 200, 201, 204, 205, 206, modulation, viii, 25, 27, 33, 37, 44, 59, 70, 96, 100,
209, 213, 221 104, 107, 124, 132, 166, 168, 178, 203, 265, 266,
middle-aged, 269 310
migration, 26, 28, 29, 33, 37, 67, 69, 70, 71, 73, 77, moieties, 208
86, 116, 146, 150, 166, 238, 252, 266 molecular biology, 254
mild cognitive impairment, 285, 286, 290, 294 molecular changes, 82, 140
milk, 18 molecular mechanisms, 15, 28, 44, 85, 87, 168, 169,
mimicking, 171 235
minerals, 58 molecular oxygen, 5, 11, 34, 37, 38, 39, 40, 75, 76,
MIP, 236 228, 229, 230, 306
miscarriage, 151 molecular weight, 263, 277
misfolded, 277 molecules, vii, ix, 1, 2, 4, 8, 9, 10, 11, 13, 17, 22, 34,
misfolding, 231 35, 36, 42, 44, 55, 63, 71, 72, 73, 77, 79, 85, 89,
mitochondria, xii, 15, 18, 20, 24, 38, 40, 57, 76, 96, 96, 97, 104, 113, 116, 122, 130, 138, 139, 140,
108, 121, 123, 124, 131, 164, 174, 180, 181, 194, 144, 146, 150, 151, 162, 164, 166, 170, 172, 176,
196, 207, 223, 228, 230, 232, 249, 253, 255, 259, 195, 206, 219, 228, 233, 235, 236, 237, 238, 240,
260, 264, 279, 280, 281, 289, 295, 305, 306, 310, 241, 265, 305
319, 322 monkeys, 302, 312, 314
mitochondrial, 2, 4, 7, 10, 15, 32, 36, 38, 40, 42, 46, monoamine, 275
59, 76, 96, 100, 107, 108, 116, 120, 121, 123, monoamine oxidase, 275
130, 131, 132, 164, 171, 174, 182, 190, 197, 198, monocyte, 69, 73, 79, 84, 89, 90, 265
204, 208, 210, 211, 225, 227, 228, 230, 232, 233, monocyte chemoattractant protein, 69, 265
236, 241, 244, 245, 250, 251, 253, 260, 261, 262, monocyte chemotactic protein, 84
264, 268, 278, 279, 280, 281, 283, 286, 288, 290, monocytes, ix, 33, 42, 63, 69, 71, 73, 74, 77, 79, 89,
295, 296, 303, 304, 310, 311, 315, 318, 322 90, 120, 122, 141, 155, 206, 209, 221, 234, 237
mitochondrial abnormalities, 251, 253, 304 monolayer, 27, 36
mitochondrial damage, 10, 96, 131, 265, 278 monomeric, 288
mitochondrial DNA, 76, 107, 280, 290, 296 mononuclear cell, 42, 125
mitochondrial membrane, 10, 40, 76, 264 mononuclear cells, 42
mitogen, 33, 51, 212 Moon, 212
mitogen activated protein kinase, 33 morbidity, ix, x, 12, 91, 92, 111, 122, 126, 135, 136,
mitogen-activated protein kinase, 51, 212 149, 173, 194, 258, 259
mitogenesis, 23, 33 morphological, 119, 141, 226, 228
mitogenic, 17, 28, 31, 70, 85 morphology, 66, 200, 238, 246, 296, 313
mitotic, 260 morphometric, 129
ML, 18, 57, 86, 105, 131, 156, 177, 179, 181, 185, mortality, viii, ix, x, 12, 15, 46, 60, 63, 64, 65, 80,
247, 251, 293, 294, 318 90, 91, 111, 112, 126, 128, 135, 136, 160, 173,
MMP, 72, 146, 147 177, 190, 200, 258, 259
MMP-9, 72, 146, 147 mortality rate, 112, 160, 259
MnSOD, 40, 289 motion, 294
modalities, 105, 226 motor function, 258, 266
Index 343
motor skills, 261 myofibroblasts, 237, 252

mouse, 30, 56, 73, 84, 163, 168, 171, 185, 200, 201, myoglobin, 122, 210
204, 213, 217, 218, 232, 241, 266, 288, 289, 290, Myoglobin, 122, 130
292 myometrium, 139
mouse model, 30, 163, 168, 204, 217, 241, 266, 288, myosin, 72
292 myricetin, 320
movement, 113, 226, 234
MPTP, 278, 279, 281, 282, 295, 296
N
MRI, 269
mRNA, 31, 84, 124, 140, 142, 144, 153, 155, 200,
NA, 30, 34, 47, 54, 86, 247, 316
201, 204, 207
Na+, 116, 129, 152
MS, 14, 19, 51, 82, 83, 87, 88, 129, 130, 132, 150,
N-acety, 22, 43, 47, 59, 61, 62, 92, 99, 101, 105,
153, 155, 178, 183, 220, 286, 290, 294, 319
125, 133, 180, 189, 201, 205, 239, 241, 243, 244,
mtDNA, 96, 268
254, 311
MTHFR, 184
NAD, 22, 23, 39, 41, 51, 52, 53, 56, 58, 75, 83, 87,
multiple factors, 16
101, 106, 109, 152, 155, 171, 183, 187, 190, 218,
multiple sclerosis, 302, 305
219, 290, 292
multiplication, 71
NADH, 18, 38, 74, 75, 76, 83, 87, 228
murine model, 168, 206, 264, 267
NAS, 231
murine models, 168, 206, 264
Nash, 128
muscle, 13, 19, 21, 23, 26, 27, 28, 29, 31, 32, 33, 34,
National Health Interview Survey, 190
36, 37, 42, 49, 51, 52, 53, 56, 58, 66, 72, 76, 82,
natural, 7, 78, 142, 174, 182, 215, 241, 247, 261,
83, 84, 85, 86, 87, 88, 89, 94, 95, 100, 107, 113,
307, 319, 321
120, 122, 148, 152, 161, 162, 178, 194, 196, 201,
natural food, 174
210, 214, 305
NC, 57, 60, 288, 289
muscle cells, 13, 21, 23, 27, 28, 31, 32, 36, 51, 53,
ND, 23, 258, 261, 264, 266, 267, 276, 280
58, 66, 72, 76, 83, 84, 86, 87, 88, 94, 120, 148,
neck, 162
152, 162, 178, 201, 305
necrosis, ix, 91, 92, 93, 96, 97, 102, 114, 116, 117,
muscle contraction, 95
122, 126, 128, 129, 141, 180, 182, 188, 196, 234,
muscle tissue, 122
236, 238, 252, 260, 290
muscles, 32, 255
neocortex, 263, 264, 272, 288
Muslim, 109, 156
neoplastic, 238
mutagenic, 8, 9
neovascularization, 204, 308
mutant, 266, 279, 289, 314
nephritis, 119, 130
mutation, 8, 21, 169
nephron, 17, 113, 115
mutations, 7, 8, 9, 21, 161, 162, 227, 261, 277, 278,
nephropathy, xi, 115, 123, 129, 133, 183, 193, 194,
279, 296
198, 204, 205, 207, 220
MV, 51, 156, 181, 190, 218, 251, 316
nephrosis, 115
myelin, 305, 306
nephrotoxic, 112, 117, 118, 123, 127, 131
myeloid, 72
nephrotoxic drugs, 123
myeloma, 119
nephrotoxicity, 123, 125, 129, 132, 133
myeloperoxidase, 20, 42, 84, 172
nerve, xiii, 58, 196, 206, 218, 277, 299, 301, 305,
myocardial infarction, 26, 43, 51, 59, 81, 102, 109,
306
110, 120, 177, 291
nerve cells, 196, 306
myocardial ischemia, 37
nerve conduction velocity, 206
myocardial tissue, 104
nerve fibers, 306
myocardium, 106, 107
nerves, 259
myocyte, 109, 122
nervous system, 33, 170, 211, 265, 292, 315
myocytes, 109, 162
Netherlands, 274
myofibrillar, 98, 106, 186
network, 93, 128, 237
344 Index
neural networks, 163 New Zealand, 81

neuritic plaques, 263 NFT, 262, 263, 264, 265, 267
neurobehavioral, 184 NF-κB, 235
neurobiology, 284 Ni, 23
neurodegeneration, 258, 259, 277, 278, 288, 309, nicotinamide, 38, 74, 86, 106, 207, 232, 245, 282,
312, 313, 321 308
neurodegenerative, xii, 15, 211, 257, 260, 261, 263, nicotine, 101
265, 268, 271, 274, 276, 277, 278, 279, 281, 283, Nielsen, 149
284, 285, 287, 290, 296, 300, 302, 309 nigrostriatal, 277
neurodegenerative disease, xii, 15, 257, 283, 285, nitrate, 10, 77
290, 296, 302, 309 nitric oxide, viii, ix, xi, xii, 2, 19, 20, 21, 22, 23, 25,
neurodegenerative diseases, xii, 257, 283, 290, 296, 26, 45, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63,
302, 309 69, 70, 76, 87, 88, 94, 101, 108, 109, 120, 126,
neurodegenerative disorders, 260, 261, 285, 287 131, 138, 141, 146, 152, 153, 155, 156, 159, 167,
neurodegenerative processes, 211, 271, 277, 278, 170, 184, 185, 188, 203, 218, 219, 223, 232, 250,
281, 283 290, 292, 295, 300, 303, 314, 315, 316, 317, 321
neurofibrillary tangles, 15, 262 nitric oxide (NO), viii, xii, 25, 26, 69, 184, 203, 223,
neuroimaging, 269 232, 300
neurological condition, 262 nitric oxide synthase, 19, 21, 22, 23, 45, 53, 54, 56,
neurological disease, 289, 321 57, 60, 61, 69, 70, 76, 87, 88, 94, 101, 108, 109,
neurological disorder, 276 120, 126, 138, 141, 152, 156, 219, 232, 250, 290,
neuromodulator, 266 292, 314, 317, 321
neuron death, 14, 15 nitric-oxide synthase, 54
neuronal apoptosis, 265, 285, 287 Nitrite, 77
neuronal cells, 272, 289 nitrogen, vii, viii, x, 1, 2, 19, 25, 39, 69, 84, 94, 111,
neuronal death, 263, 286 113, 120, 130, 131, 231, 232, 260, 266
neuronal degeneration, 272, 301 nitrosamines, 8
neuronal loss, 258, 262, 277, 279, 321 nitrosative stress, 130, 302
neuronal plasticity, 271 nitrous oxide, 279
neurons, 14, 15, 233, 251, 258, 260, 262, 263, 264, NK, 81, 152, 156, 252, 288
265, 276, 277, 280, 281, 282, 287, 296, 301, 310, NMDA, 266
314, 315 NMDA receptors, 266
neuropathologic changes, 292 N-methyl-D-aspartate, 167
neuropathological, 286 NO, viii, xii, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16,
neuropathy, xi, xiii, 193, 198, 206, 299, 300, 302, 19, 21, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 38,
305, 311, 312, 317 39, 41, 43, 44, 45, 48, 50, 57, 59, 61, 70, 71, 74,
neuroprotection, 281, 284, 303, 314, 319, 320, 322 76, 77, 80, 100, 120, 121, 126, 131, 145, 148,
neuroprotective, 263, 266, 281, 287, 297, 308, 322 166, 167, 171, 184, 200, 203, 204, 205, 213, 218,
neuropsychological assessment, 297 219, 223, 232, 269, 300, 302, 304, 309, 310, 315
neurotoxic, 263, 264, 278, 281, 282, 283, 288 NO synthase, viii, 5, 26, 29, 32, 34, 38, 49, 57, 59,
neurotoxicity, 264, 282, 288, 294, 295, 301, 302 204, 218, 232, 309, 310
neurotransmission, 302 NO synthases, 5
neurotransmitter, 33, 262, 265, 266, 279, 289, 301 nodules, 238
neurotransmitters, 33 non-alcoholic fatty liver, 180, 247, 248, 249, 251
neutralization, 240, 316 non-enzymatic, 4, 10, 11, 12, 37, 104, 231
neutrophil, 3, 5, 6, 7, 20, 38, 42, 116, 153, 183, 212, non-immunological, 228, 233
252 non-institutionalized, 273
neutrophils, 7, 13, 71, 77, 116, 122, 129, 140, 141, non-insulin dependent diabetes, 203
198, 202, 234, 237 non-invasive, 180, 226
New York, iii, iv, 131 non-native, 230
Index 345
non-steroidal anti-inflammatory drugs, 117 185, 186, 190, 200, 223, 224, 225, 227, 231, 233,
non-vascular, 269 238, 239, 244, 246, 248, 249, 250, 252, 253
norepinephrine, 33, 118 obligate, 311
Norfolk, 187 observations, 160, 166, 168, 174, 196, 201, 305
normal, vii, viii, 1, 3, 4, 10, 13, 25, 28, 29, 30, 32, obstruction, 24, 114, 115, 116, 118, 119, 122
34, 46, 66, 67, 69, 73, 94, 95, 112, 113, 120, 125, occipital cortex, 262
137, 139, 140, 142, 144, 146, 147, 150, 151, 152, ocular diseases, 303, 307
153, 155, 161, 165, 166, 177, 182, 202, 217, 232, ODS, 187
233, 236, 238, 255, 258, 260, 261, 263, 264, 266, oil, 48, 49, 61, 149, 173, 185, 189, 255
277, 280, 285, 287, 290, 301, 310, 314, 317, 320 older adults, 190, 294
normal aging, 258, 277, 290 oligomers, 263, 277, 287
normal conditions, vii, 3, 13, 28, 32, 166, 233 oligonucleotides, 8, 20
normalization, 123, 162, 242 olive, 48, 49, 173, 189
North Africa, 279 olive oil, 48, 49, 173, 189
North America, 224, 273 Omega-3, 311
NOS, 5, 6, 13, 29, 34, 35, 36, 37, 39, 48, 70, 76, 94, Oncogene, 211
101, 120, 138, 200, 204, 269, 279, 302, 308, 309 oncogenes, 9
Nrf2, 280 open angle glaucoma, 16, 300, 311, 312, 313
NS, 77, 106, 181, 209, 232, 260 ophthalmic, 310, 322
NSAIDs, 117, 119, 165 optic nerve, xiii, 16, 299, 300, 301, 305, 306, 311,
N-terminal, 279, 287 313, 314, 317, 318
nuclear, xi, 31, 98, 107, 138, 159, 170, 178, 194, optic neuritis, 320
210, 232, 268, 272, 290 oral, 44, 47, 48, 59, 60, 90, 103, 124, 125, 133, 187,
Nuclear factor, 235 203, 204, 210, 218, 244, 245, 255, 277, 310, 311
Nuclear factor kappa, 235 organ, viii, 15, 16, 17, 25, 26, 27, 39, 47, 104, 112,
nuclear magnetic resonance, 210 115, 121, 137, 140, 164, 197, 206, 207, 271, 280
Nuclear magnetic resonance, 18 organelle, 96, 100, 228, 230, 264, 279
nuclear receptors, 178, 272 organelles, xii, 96, 128, 223, 229
nuclei, 277 organic, 4, 6, 11, 96, 118, 119, 125
nucleic acid, 119, 197 organic compounds, 4
nucleotides, 39 organic peroxides, 11
nucleus, 4, 301, 315 organic solvent, 118
nulliparous, 145 organic solvents, 118
nutrient, 75, 208, 284 organism, vii, 17, 94, 235, 318
nutrients, x, 135, 152, 155, 202, 258, 264, 271, 292, osteopontin, 172
293, 307 osteoporosis, 185
nutrition, 57, 186, 190, 271 ototoxicity, 123, 125, 133
nutritional supplements, 273 ovariectomized, 188
nuts, 49 ovariectomized rat, 188
ovariectomy, 171
ovary, 189
O
overload, xii, 133, 180, 223, 228, 260, 296
overnutrition, 164
oat, 29
overproduction, 172, 175, 265
obese, 162, 163, 164, 165, 169, 170, 173, 179, 182,
overweight, 190, 215, 249, 252
184, 185, 186, 187, 189, 216, 224, 242, 244, 248,
oxalate, 119
253
oxidants, 2, 69, 73, 74, 84, 87, 88, 89, 119, 122, 153,
Obese, 231
199, 302
obese patients, 162, 165, 182
oxidation, ix, xii, 1, 5, 6, 7, 8, 9, 10, 11, 14, 21, 34,
obesity, xii, 65, 136, 160, 161, 162, 163, 164, 166,
39, 41, 44, 46, 48, 63, 68, 69, 75, 76, 77, 79, 82,
168, 169, 170, 172, 176, 177, 178, 179, 180, 183,
346 Index
83, 84, 89, 90, 94, 95, 102, 112, 119, 123, 125, paradox, 3, 57, 305
138, 153, 160, 164, 166, 171, 172, 173, 175, 179, paradoxical, 28, 29
184, 189, 194, 196, 198, 199, 203, 216, 223, 226, parameter, 14, 242, 311
228, 230, 231, 233, 243, 250, 254, 255, 260, 265, parenchyma, 237
266, 267, 268, 269, 278, 279, 280, 281, 285, 290, parenchymal, 234, 236, 237, 252
308, 309 parenchymal cell, 234, 252
oxidative damage, vii, viii, xi, xiii, 1, 15, 16, 20, 25, parenteral, 125
46, 96, 100, 102, 109, 159, 160, 171, 181, 194, parietal lobe, 268
199, 203, 212, 233, 241, 244, 246, 264, 265, 270, Parkinson, vii, xii, 15, 257, 261, 266, 276, 277, 278,
272, 276, 285, 289, 290, 292, 293, 296, 299, 300, 280, 281, 283, 284, 292, 294, 295, 296, 297
310 Parkinson disease, vii, 266, 280, 295, 296
oxidative reaction, 241 Parkinsonism, 276, 282, 296, 297
oxide, ix, xi, 2, 5, 13, 19, 20, 21, 22, 23, 29, 33, 41, particles, 57, 67, 68, 82, 166, 189, 226, 236, 238
45, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, paternity, 149
69, 70, 76, 77, 85, 87, 88, 94, 101, 108, 109, 120, pathogenic, xi, xiii, 16, 92, 93, 120, 159, 160, 165,
126, 131, 138, 141, 146, 152, 153, 155, 156, 159, 182, 189, 276, 278, 295, 299, 300, 304
167, 170, 181, 184, 185, 188, 218, 219, 232, 250, pathogens, 2, 6, 48, 234, 235
275, 279, 289, 290, 292, 295, 302, 304, 312, 314, pathology, 16, 27, 64, 76, 92, 136, 225, 228, 231,
315, 316, 317, 321 240, 241, 243, 254, 258, 264, 267, 271, 277, 287,
oxygen, vii, viii, x, 1, 2, 3, 5, 6, 11, 12, 17, 18, 21, 289, 292, 293, 294
22, 25, 26, 33, 37, 38, 39, 40, 41, 51, 55, 57, 58, pathophysiological, viii, xiii, 2, 4, 7, 10, 12, 14, 17,
63, 64, 74, 75, 76, 77, 84, 86, 90, 92, 94, 96, 102, 25, 26, 28, 36, 37, 42, 64, 75, 76, 87, 92, 94, 113,
103, 111, 115, 116, 117, 120, 121, 122, 123, 126, 114, 115, 117, 137, 140, 143, 146, 148, 206, 257,
128, 130, 131, 132, 137, 139, 142, 146, 152, 163, 302
175, 201, 228, 229, 230, 238, 249, 259, 260, 303, Pathophysiological, 7, 155
304, 306, 308, 310, 319 pathophysiological mechanisms, xiii, 143, 257
Oxygen, 3, 24, 130, 132, 150 pathophysiology, vii, ix, x, xii, xiii, 27, 30, 41, 50,
oxygen consumption, 94, 115, 128, 260 51, 55, 56, 64, 68, 70, 73, 74, 75, 78, 81, 91, 92,
oxygenation, 21 105, 111, 112, 116, 122, 123, 126, 127, 136, 137,
oxyhemoglobin, 121 140, 141, 149, 150, 160, 168, 178, 186, 224, 225,
oxyradicals, 20 250, 251, 257, 276, 288, 299, 300, 301, 303, 310,
312
pathways, xi, xii, xiii, 4, 7, 12, 14, 16, 17, 28, 36, 45,
P
46, 55, 58, 94, 96, 103, 109, 123, 132, 156, 159,
161, 165, 166, 167, 176, 199, 200, 206, 207, 208,
p53, 9, 21, 235, 305
210, 212, 220, 223, 224, 225, 226, 227, 229, 230,
PA, 87, 108, 151, 157, 178, 182, 185, 188, 208, 252,
231, 234, 246, 253, 268, 269, 278, 299, 306, 311
286, 291, 293, 295, 315, 318
pattern recognition, 71
pacemaker, 93
PCR, 96
pacemakers, 93
PD, 15, 60, 85, 130, 133, 180, 276, 277, 278, 279,
pacing, 102, 103, 105
280, 281, 282, 283, 284, 296
PAI-1, 123, 143, 144
PE, x, 83, 129, 131, 135, 136, 137, 138, 139, 140,
pain, 137
141, 142, 143, 144, 145, 146, 147, 148, 149, 179,
pancreas, 32, 203, 227
290
pancreatic, xi, 161, 176, 184, 193, 194, 195, 196,
pediatric, 242, 253
199, 201, 205, 207, 209, 210, 211, 212, 213, 214,
pelvis, 114
217, 250
Pennsylvania, 273
pancreatic acinar cell, 184
pentamidine, 118
pancreatic islet, 195, 199, 209, 213, 217
peptidase, 228, 241
pantothenic acid, 243
paracrine, 29, 33, 235, 237
Index 347
peptide, 15, 30, 32, 36, 49, 113, 261, 263, 265, 287, phenotypic, 73, 169, 185
309 phenytoin, 119
peptides, 28, 31, 259, 262, 263, 264, 272, 283 Philadelphia, 128
perceptions, 105 phosphatases, 120
perfusion, 15, 16, 114, 115, 132, 137, 139, 140, 141, phosphate, 32, 38, 41, 74, 86, 101, 106, 113, 197,
143, 288, 304, 305, 312, 317 206, 207, 308
pericarditis, 114 Phosphate, 322
pericytes, 237, 305 phosphatidic acid, 33
perinatal, 136, 137, 156 phosphatidylcholine, 33, 244, 245
Peripheral, 285, 296 phospholipase C, 29, 51
peripheral blood, 196, 267, 268, 280 phospholipids, 9, 68, 83, 116, 120, 142
peripheral nervous system, 258 phosphorylation, 10, 29, 76, 95, 97, 120, 121, 123,
peripheral neuropathy, 114 145, 155, 177, 182, 187, 196, 210, 264, 272, 287,
peripheral vascular disease, 22 310
peritoneal, 84, 137 photoreceptor, 308
peritoneal cavity, 137 photoreceptor cells, 308
permeability, 67, 117, 136, 148, 164, 165, 166, 182, photoreceptors, 309, 320
234, 264, 301, 310 physical activity, 242
peroxidation, x, 9, 10, 14, 21, 43, 58, 116, 135, 142, physical properties, 255
144, 152, 164, 174, 198, 203, 208, 213, 226, 231, physicians, 176, 248
241, 266, 303, 307, 308, 312 Physicians, 259
peroxide, 4, 41, 94, 132, 152, 173, 194, 199, 304, physiological, vii, viii, 1, 2, 3, 7, 12, 13, 25, 27, 28,
316 31, 33, 36, 50, 89, 94, 115, 121, 137, 139, 140,
peroxisomes, 40, 132, 164, 181 146, 148, 172, 217, 227, 230, 232, 233, 237, 245,
peroxynitrite, ix, 2, 15, 21, 22, 35, 37, 38, 39, 42, 46, 277, 285, 310
54, 59, 63, 69, 70, 76, 85, 88, 103, 105, 121, 141, physiology, 55, 56, 112, 168, 263, 280, 285, 288
142, 205, 217, 232, 270, 302, 304, 315 phytoestrogens, 245
perturbation, 38, 171 PI3K, 252
perturbations, 160, 163 pig, 109, 145, 289, 313
PF, 18, 115, 132, 291 pigments, 79, 202
PG, 105, 149, 220, 295, 311, 318 pigs, 102
pH, 152 pilot study, 105, 189, 216, 221, 242, 243, 247, 253,
phagocyte, 52 256, 282, 317
phagocytic, 38, 74, 120, 122 pineal, 272
phagocytosis, 236, 238 pioglitazone, 242, 247
pharmaceutical, 203 PKC, 31, 320
pharmacodynamics, 215 PL, 20, 105, 108, 153, 216, 219, 250, 284, 312, 314,
pharmacokinetics, 44, 59 315
pharmacological, 12, 31, 92, 125, 200, 205, 208, placebo, 47, 59, 60, 79, 99, 103, 105, 125, 146, 156,
240, 245, 311 190, 203, 242, 243, 244, 245, 247, 255, 256, 271,
pharmacological treatment, 92, 311 272, 275, 276, 281
pharmacology, 48, 60, 156, 250, 285 placenta, 16, 137, 138, 139, 140, 141, 142, 145, 146,
pharmacotherapy, 33, 178 147, 150, 151, 153, 154, 156
phenol, 107, 320 placental, x, 135, 136, 137, 138, 139, 140, 141, 142,
phenolic, 48, 173, 308, 309 143, 144, 145, 146, 148, 150, 151, 152, 153, 154,
phenolic acids, 48 155, 156
phenolic compounds, 308 plants, 48, 174, 240
phenotype, 73, 86, 139, 150, 161, 169, 183, 266, plaque, 64, 66, 67, 72, 73, 74, 263, 267, 271, 272,
271, 272, 276, 279, 290 289, 292
phenotypes, 169
348 Index
plaques, 67, 69, 72, 99, 166, 198, 259, 263, 265, 283, Polyphenols, 48, 320
301 polyunsaturated fat, 9, 11, 21, 59, 173, 190, 227,
plasma, x, 11, 14, 32, 34, 37, 42, 44, 46, 55, 58, 59, 249, 260, 284, 308
67, 68, 69, 79, 83, 84, 112, 113, 115, 116, 118, polyunsaturated fatty acid, 9, 11, 21, 59, 173, 190,
119, 122, 130, 132, 135, 142, 143, 144, 152, 153, 227, 249, 260, 284, 308
155, 160, 163, 165, 169, 170, 171, 173, 174, 182, polyunsaturated fatty acids, 11, 21, 59, 173, 190,
183, 187, 188, 196, 202, 203, 215, 216, 232, 233, 227, 260, 284
234, 241, 244, 245, 267, 268, 269, 291, 292, 305, pomegranate, 185, 216
306, 315, 317, 320, 322 pools, 66
plasma levels, 14, 142, 144, 155, 165, 182, 188, 245, poor, x, 92, 93, 104, 135, 143, 148, 263, 271, 273
267, 269, 291, 317, 322 population, ix, xii, xiii, 10, 12, 50, 62, 92, 104, 111,
plasma membrane, 34, 116, 232, 306, 315 146, 149, 160, 179, 186, 223, 224, 248, 257, 258,
plasmids, 9 261, 269, 270, 274, 276, 280, 282, 286, 300, 307
plasminogen, 72, 150 pores, 236
plastic, 266 positive correlation, 101, 202
plasticity, 263, 271, 291 positive feedback, 7, 96, 229
platelet, 13, 27, 33, 58, 67, 69, 70, 71, 72, 73, 79, 86, postmenopausal, 189
89, 100, 137, 145, 166, 238, 310, 321 postmenopausal women, 189
Platelet, 137 postmortem, 278
platelet aggregation, 13, 33, 67, 70, 71, 73, 100, 145, postoperative, ix, 91, 92, 97, 104, 105, 107, 108
166, 310, 321 postoperative outcome, 105
platelet count, 137 post-transcriptional regulation, 95
platelet derived growth factor, 69 post-translational, 145, 214
platelets, 42, 67, 73, 150 potassium, 49, 114, 117
play, viii, xi, xiii, 11, 15, 16, 17, 25, 26, 32, 33, 36, potatoes, 49
39, 48, 71, 78, 94, 102, 104, 112, 120, 121, 124, powder, 271
126, 144, 145, 147, 163, 172, 193, 198, 202, 207, power, 9, 56, 92, 215, 305
228, 236, 238, 241, 268, 270, 280, 299, 300, 303, PPP, 46, 60
311 precipitation, 118, 230
plethysmography, 70 preclinical, xi, 193, 207, 208
plexus, 301 preconditioning, vii, 106
PM, 55, 60, 83, 86, 87, 128, 132, 149, 152, 180, 182, prediction, 65, 81, 86, 149, 154
183, 214, 290, 291, 296 predictors, x, 135
PN, 157, 217 preeclampsia, vii, 1, 12, 15, 16, 24, 31, 52, 60, 149,
pneumonia, 118, 259 150, 151, 152, 153, 154, 155, 156, 157, 268
PO, 213, 214 pre-eclampsia, 136, 138, 140, 146i, 147, 149, 150,
POAG, 16, 300, 303, 304, 306, 311, 322 151, 152, 153, 154, 155, 156
poisoning, 119 pre-existing, 118, 136, 143
Poland, 209 pregnancy, x, 15, 16, 28, 135, 136, 137, 139, 140,
polarity, 116, 117, 128 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
polycyclic aromatic hydrocarbon, 231 153, 154, 155, 244
polygenic, 168 pregnant, 136, 140, 143, 145, 146, 148, 152, 153,
polymerization, 262 154, 156
polymorphism, 184, 270, 292 pregnant women, 136, 140, 145, 146, 148, 152, 153,
polymorphisms, 303 154, 156
polymorphonuclear, 37, 42, 120 presenilin 1, 266
polymorphonuclear cells, 37 pressure, viii, xiii, 3, 13, 16, 25, 26, 27, 28, 30, 31,
polyphenolic compounds, 49, 174, 308 33, 34, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 50,
polyphenols, 48, 61, 62, 126, 172, 190, 282, 308, 52, 56, 58, 59, 60, 61, 62, 65, 74, 81, 109, 113,
309, 319, 320, 321 115, 116, 136, 168, 170, 171, 172, 175, 176, 237,
Index 349
259, 299, 300, 301, 303, 304, 306, 308, 310, 311, protective role, 28, 71, 79, 80, 207, 281
312, 313, 314, 315, 317, 318, 319, 320, 322 proteic, 4
prevention, x, 15, 16, 27, 43, 46, 49, 50, 52, 57, 58, protein, 2, 7, 9, 10, 11, 14, 15, 18, 21, 31, 32, 34, 41,
59, 60, 64, 65, 80, 89, 90, 99, 102, 103, 105, 108, 58, 68, 69, 72, 77, 82, 83, 84, 89, 95, 96, 97, 112,
110, 111, 112, 123, 124, 125, 126, 135, 143, 144, 113, 119, 123, 132, 141, 142, 144, 145, 153, 155,
145, 148, 149, 155, 171, 172, 175, 176, 201, 214, 165, 171, 173, 179, 181, 182, 184, 185, 194, 195,
215, 240, 241, 246, 254, 261, 269, 270, 271, 301, 198, 202, 204, 206, 214, 217, 227, 230, 232, 233,
310, 312 234, 235, 236, 255, 260, 261, 262, 263, 265, 266,
preventive, x, 47, 104, 111, 126, 190, 194, 202 267, 268, 270, 278, 279, 280, 285, 287, 290, 305,
primary open-angle glaucoma, 312, 313, 315, 316, 307, 310, 314
317 protein disulfide isomerase, 230
primary open-angle glaucoma (POAG), 313 protein folding, 230
primates, 53, 281 protein kinase C, 31, 89, 95, 123, 194, 206
pro-apoptotic protein, 235 protein kinase C (PKC), 31
probability, 34, 70, 275, 305 protein kinases, 230
procoagulant, 148, 160 protein misfolding, 231
producers, 33, 120 protein oxidation, 7, 10, 69, 83, 112, 153, 255, 265,
pro-fibrotic, 93, 97 266, 267, 278, 279, 285, 290
progenitor cells, 125 protein synthesis, 230
progenitors, 313 proteinase, 252
prognosis, 13, 149, 286 proteins, xi, 4, 8, 10, 15, 17, 20, 21, 27, 28, 40, 41,
program, 147, 206 44, 48, 60, 71, 72, 76, 77, 86, 95, 96, 97, 104,
proinflammatory, xi, 37, 41, 96, 193, 195, 200, 206, 116, 119, 123, 130, 136, 140, 141, 142, 168, 172,
209, 225, 234 177, 184, 193, 194, 197, 198, 203, 230, 235, 260,
pro-inflammatory, 26, 168, 235, 236, 237, 259, 265, 261, 262, 265, 268, 271, 272, 277, 280, 281, 285,
269 287, 289, 290, 306, 310, 313, 315
pro-inflammatory response, 265 Proteins, 267
proliferation, ix, 13, 23, 28, 29, 33, 37, 51, 63, 64, proteinuria, x, 119, 135, 136, 138, 148, 154, 205
67, 70, 71, 73, 74, 77, 79, 80, 85, 89, 145, 148, proteoglycans, 67, 72, 73
201, 232, 238, 245, 252, 260, 262, 320 proteolysis, 40, 263
promoter, 200, 210, 227 proteolytic enzyme, 263
pro-oxidant, vii, 2, 35, 96, 125, 133, 141, 233, 260, proteomics, 290
261, 278, 304, 316 protocol, 169, 307
propagation, 9, 41, 92, 160, 234, 236, 240, 242 protocols, 8, 104
propane, 142 protons, 6
property, 100, 166, 219, 244 proximal tubule cells, 116
prophylactic, ix, 91, 126, 156 pseudo, 147
prophylaxis, 104, 129, 155, 174, 203, 216 PT, 82, 154, 156, 181
prostaglandin, 7, 21, 31, 35, 45, 51, 143, 171, 199, public, xiii, 175, 194, 299
266, 268, 289, 317 public health, xiii, 175, 194, 299
prostaglandins, 28, 113, 234 PUFA, 9, 11, 14, 17, 260, 268, 271, 308, 311
prostanoids, 59, 101, 109, 201 pulmonary arteries, 93
protease inhibitors, 162 pulse, 46, 70
proteases, 72, 73, 75, 116, 120, 263 purification, 2
Proteasome, 279, 295 purines, 5, 75
protection, ix, xi, 18, 19, 34, 49, 63, 84, 108, 124, pyrene, 202
125, 159, 169, 171, 181, 193, 204, 208, 214, 217, pyridoxamine, 206, 220
219, 254, 260, 265, 268, 280, 282, 303, 307, 313, pyrimidine, 7
316, 319, 322
protective mechanisms, 208
350 Index
reactive oxygen, vii, ix, x, xi, xii, xiii, 1, 2, 21, 26,

Q
36, 53, 55, 61, 78, 91, 92, 98, 104, 111, 112, 127,
132, 137, 138, 140, 147, 151, 170, 171, 179, 183,
quail, 302, 314
184, 193, 210, 211, 212, 223, 251, 259, 285, 294,
quality control, 278
299, 318
quality of life, 261
reactive oxygen species, vii, ix, xi, xii, xiii, 1, 2, 21,
quercetin, 48, 62, 107
26, 36, 53, 55, 61, 78, 91, 92, 98, 104, 112, 127,
Quercetin, 62, 185
132, 137, 138, 140, 147, 151, 170, 171, 179, 183,
quinine, 119
184, 193, 210, 211, 212, 223, 251, 259, 285, 294,
quinone, 41, 100, 108
299, 318
quinones, 278, 281
Reactive Oxygen Species, 7, 12, 37, 141, 264, 278
reactive oxygen species (ROS), vii, ix, xi, xii, xiii, 2,
R 26, 78, 91, 92, 104, 112, 127, 137, 140, 171, 193,
223, 299
RA, 22, 54, 57, 59, 60, 61, 83, 90, 106, 128, 130, reactivity, 4, 16, 35, 42, 43, 69, 79, 138, 142, 184,
131, 132, 156, 178, 179, 210, 213, 215, 249, 256, 218, 241, 254
272, 288, 290, 313, 314, 321 reagents, 163
race, 89, 273 reality, 97, 106
radiation, 2, 40, 48, 303 reasoning, 261
radical formation, 39, 119, 123 receptive field, 319
radical mechanism, 21 receptor agonist, 52, 152
radiologists, 304 receptors, xi, 13, 29, 30, 31, 33, 45, 52, 53, 68, 69,
RAGE, 197, 198, 206, 207, 208, 211, 212 71, 73, 83, 141, 178, 182, 193, 197, 201, 206,
rail, 21 237, 238, 252, 258, 265, 266, 272, 302, 314
Ramadan, 177 recognition, 21, 71, 83, 195, 259
Ramipril, 214 reconcile, 50
random, 217, 275 recoverin, 18
random assignment, 275 recovery, 311
range, 9, 18, 27, 33, 71, 73, 205, 206, 208, 231, 238, recurrence, 108, 109, 149
244, 261 recycling, 100, 166, 302, 303
RAS, 30 red blood cell, 113, 199, 212
rat, 22, 23, 32, 52, 53, 56, 62, 73, 83, 102, 108, 109, red blood cells, 212
121, 124, 126, 131, 132, 134, 169, 171, 174, 177, red meat, 50
180, 182, 184, 185, 186, 187, 188, 200, 204, 205, red wine, 24, 48, 50, 61, 62, 126, 133, 171, 309, 320,
212, 219, 220, 221, 233, 245, 252, 253, 254, 255, 321
289, 302, 306, 312, 313, 314, 315, 318, 319, 321 redistribution, 128
rating scale, 262, 282 redox, viii, xi, xii, 1, 18, 25, 28, 31, 35, 37, 43, 44,
rats, 20, 23, 30, 39, 43, 47, 48, 51, 54, 56, 58, 60, 61, 46, 54, 55, 58, 87, 96, 106, 107, 159, 164, 168,
62, 102, 108, 109, 125, 126, 131, 133, 134, 145, 171, 174, 177, 188, 212, 224, 227, 232, 235, 250,
148, 155, 156, 169, 170, 172, 180, 181, 184, 185, 261, 265, 266, 268, 289, 290, 303, 316
186, 187, 188, 190, 200, 201, 204, 206, 213, 214, Redox, 19, 20, 51, 54, 55, 58, 85, 86, 108, 208, 212,
217, 218, 219, 221, 243, 249, 251, 253, 254, 255, 250, 290, 295
269, 281, 302, 307, 314, 315, 316, 319, 322 reducing sugars, 197, 198
RB, 81, 105, 182, 190, 216, 284, 285, 291, 295 redundancy, 221
RC, 18, 133, 210, 215, 286, 288, 294, 297, 316, 321 reflection, 96, 103
reactant, 14, 203 refractoriness, 164
reactants, 72 refractory, 93, 95, 98, 103
reaction rate, 44 regenerate, 44, 244
reactive nitrogen, viii, 25, 120, 130, 232 regeneration, 96, 177, 229, 234, 235, 237, 313
regional, 115, 150, 290, 306
Index 351
Registry, 297 reperfusion, 3, 7, 22, 92, 94, 95, 102, 103, 104, 110,
regression, 49 120, 121, 124, 126, 128, 130, 131, 132, 134, 137,
regular, 185 146, 151, 181, 182, 184, 285, 303, 305, 306, 310,
regulation, viii, x, 6, 22, 23, 24, 26, 27, 28, 30, 34, 315, 317
35, 37, 40, 43, 44, 45, 46, 53, 54, 55, 56, 58, 59, replication, 8, 9
66, 70, 71, 85, 86, 88, 92, 95, 96, 100, 101, 109, reproduction, 283
113, 131, 136, 141, 145, 146, 147, 148, 150, 152, reserves, 225, 241
155, 160, 163, 168, 170, 173, 174, 180, 181, 182, reservoirs, 279
183, 187, 200, 201, 205, 207, 212, 219, 227, 232, residues, 9, 10, 32, 40, 77, 197
237, 249, 250, 258, 265, 266, 301, 306, 312, 315, resistance, xi, xii, xiii, 14, 15, 37, 42, 53, 58, 61,
316, 318 136, 139, 143, 159, 160, 161, 162, 163, 164, 166,
reinforcement, ix, 18, 46, 91, 101, 104 168, 169, 170, 172, 176, 178, 179, 180, 182, 185,
rejection, 121 186, 188, 189, 190, 196, 202, 203, 208, 210, 215,
relationship, vii, 1, 13, 23, 44, 166, 170, 215, 265, 216, 223, 224, 225, 227, 233, 237, 238, 239, 240,
271, 274, 281, 283, 304 246, 249, 250, 251, 252, 270, 299, 301, 304, 306,
relationships, 306 320
relaxation, 19, 29, 31, 48, 53, 54, 70, 85, 95, 131, resistivity, 310
204, 213, 235 resolution, 54, 139
relevance, xii, 17, 22, 65, 66, 87, 103, 104, 113, 123, resources, xiii, 143, 257
126, 196, 208, 223, 277, 300, 306 respiration, 2, 7, 10, 17, 123, 228
reliability, 143 respiratory, 4, 6, 7, 32, 41, 42, 87, 100, 120, 155,
remethylation, 167 174, 228, 229, 279
remodeling, viii, ix, x, xiii, 16, 25, 28, 29, 30, 32, 34, responsiveness, 36, 51, 161, 201, 214
37, 43, 49, 53, 61, 74, 91, 92, 93, 94, 95, 96, 97, Resveratrol, 49, 134, 188, 321
102, 104, 105, 106, 107, 108, 109, 135, 139, 144, retention, 82, 84, 94, 164, 206
146, 157, 164, 299, 308, 311 reticulum, xii, 49, 95, 106, 168, 184, 186, 223, 228,
remodelling, 54, 106 250
renal, vii, ix, x, 11, 12, 13, 17, 22, 23, 24, 30, 32, 41, retina, 198, 301, 303, 305, 306, 309, 310, 313, 314,
44, 47, 53, 58, 60, 61, 81, 94, 111, 112, 113, 114, 316, 317, 318, 321
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, retinal disease, 309
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, retinoic acid, 283
137, 169, 172, 198, 202, 204, 205, 206, 208, 211, retinol, 143, 154, 203
213, 217, 218, 220, 221, 267, 269 retinopathy, 194, 205, 207, 220
renal disease, 17, 22, 24, 61, 81, 114, 119, 123, 124, RF, 19, 53, 252, 313, 315, 321
125, 130, 202, 220 rhabdomyolysis, 17, 122, 126, 129, 134
renal dysfunction, 47, 61, 117, 118, 122, 130 Rhabdomyolysis, 119, 122, 130, 131
renal epithelial cells, 128, 130 rhythm, ix, 91, 92, 93, 96, 97, 104
renal failure, ix, 111, 112, 114, 118, 119, 120, 126, rigidity, 237
127, 128, 129, 130, 133, 211 risk, ix, x, xi, xiii, 12, 13, 14, 17, 26, 44, 51, 58, 64,
renal function, ix, 111, 112, 113, 117, 122, 126, 128, 65, 68, 70, 72, 74, 79, 80, 81, 85, 86, 88, 92, 94,
129, 131, 137, 170, 204, 217, 269 103, 105, 107, 108, 111, 112, 117, 126, 128, 135,
renal hemodynamics, 32, 113 137, 140, 143, 144, 145, 146, 149, 154, 156, 159,
renal medulla, 129 160, 161, 162, 164, 170, 172, 173, 174, 175, 176,
renal replacement therapy, 112 177, 180, 186, 189, 190, 199, 200, 209, 216, 247,
renin, 30, 47, 49, 94, 106, 113, 166, 233, 237, 238 255, 258, 261, 268, 269, 270, 271, 273, 274, 275,
renin-angiotensin system, 94, 233, 237 280, 282, 283, 291, 292, 293, 294, 296, 297, 299,
rennin angiotensin system, 220 300, 305, 306, 308, 320, 321
reoxygenation, 75, 121, 131 risk assessment, 149, 154
repair, 2, 13, 21, 211, 230, 251, 265, 289
reparation, 9, 229, 230, 234, 235
352 Index
risk factors, ix, x, xi, 13, 51, 64, 65, 70, 74, 81, 85, selenium, 187, 188, 190, 202, 215, 273
92, 94, 111, 128, 135, 143, 159, 160, 164, 173, self-report, 273
174, 176, 180, 186, 190, 209, 247, 270, 273, 300 senescence, 12
risks, 48, 156, 216, 291 senile, 15, 262
RNA, 52, 260, 265 senile plaques, 15, 262
rodent, 125, 266, 306 sensing, 179, 265
rodents, 163, 170, 231 sensitivity, 97, 162, 169, 170, 176, 179, 187, 211,
rosiglitazone, 215 219, 259, 267, 280, 289, 295
RP, 52, 72, 150, 151, 152, 153, 178, 180, 209, 213, sensors, xi, 159
214, 220, 291 sepsis, 20, 22, 112, 117, 118, 259, 267
rural, 273, 293 series, 76, 81, 86, 142, 170, 195, 207, 209, 235
Rutherford, 149 serine, 279
serotonin, 35
serum, 19, 23, 57, 94, 97, 99, 113, 125, 126, 136,
S
137, 142, 143, 149, 152, 153, 154, 155, 160, 163,
164, 165, 172, 174, 202, 203, 204, 206, 216, 243,
SA, 24, 53, 82, 85, 107, 154, 157, 184, 190, 215,
245, 273, 280
250, 253, 291, 293, 320
serum albumin, 19, 202
Saccharomyces cerevisiae, 188
services, iv
safety, 220, 255, 322
severity, xiii, 16, 66, 72, 142, 145, 196, 263, 275,
saline, 118, 129, 306, 307
278, 299
salt, 32, 47, 56, 58, 61, 118, 206
sex, 273, 292
sample, 153, 202, 273, 293
SH, 9, 11, 107, 124, 125, 176, 181, 182, 200, 219,
saponin, 48
230, 244, 247, 251, 254, 282, 293
sarcoidosis, 119
shape, 313
saturated fat, 228, 233
shares, 69, 266
saturated fatty acids, 228
sharing, 275, 276, 278
scavenger, 10, 68, 69, 71, 73, 83, 100, 121, 125, 126,
shear, 13, 26, 27, 28, 32, 34, 39, 56, 69, 70, 71, 73,
174, 204, 205, 206, 219, 265, 288, 320
75, 83, 87
schema, 101
shock, 20, 72, 86, 115, 129, 306, 315
Schiff, 197
short period, 199
Schiff base, 197
short-term, 28, 44, 47, 202, 204, 242, 312
schizophrenia, 266
Short-term, 48
Schmid, 55, 56
shoulder, 67, 162
scientific community, 258
SI, 215
sclerosis, 204, 218
sign, 113
scores, 274, 281
signal transduction, 46, 164, 196, 199, 232, 258
scurvy, 29, 79
signaling, vii, viii, 2, 7, 20, 23, 25, 26, 29, 30, 35,
SD, 84, 109, 186
36, 37, 43, 52, 55, 58, 62, 87, 106, 109, 184, 186,
SE, 52, 83, 85, 154, 251, 288, 292, 296
194, 196, 206, 208, 210, 212, 214, 230, 250, 252,
seafood, 174
269, 288, 304, 314, 320
search, 92, 140
signaling pathway, 2, 7, 30, 109, 194, 196, 206, 208,
searching, 273
210, 212, 214, 320
secrete, 146, 157, 161, 226, 236, 237, 238
signaling pathways, 2, 7, 30, 109, 194, 196, 206,
secretion, xi, 29, 32, 72, 73, 86, 113, 142, 145, 150,
210, 212
162, 163, 178, 181, 184, 193, 194, 197, 200, 208,
signalling, 51, 107, 289, 291, 316
211, 219, 226, 227, 233, 236, 243, 315
signals, 18, 26, 38, 105, 199, 236, 237
security, 259
signs, 114, 148, 196, 277, 305
seed, 185
Singapore, 297
seeds, 49
sinus, 93, 96
selectivity, 184
Index 353
sinus rhythm, 96 specificity, 39, 77, 259, 267

siRNA, 30 spectrum, xii, 11, 20, 69, 223, 224, 247, 248
sites, 4, 7, 9, 30, 41, 67, 73, 74, 116, 125, 160, 162, speed, 17
164, 198, 265, 268, 275, 276, 289 sphingolipids, 232
skeletal muscle, 161, 178 sphingosine, 232
skills, 261 spin, 18, 183, 200
skin, 320 spinach, 174
sleep, 306, 318 spinal cord, 33, 266
sleep apnea, 306, 318 sporadic, 15, 258, 261, 270, 277, 279, 280, 283
small intestine, 7 sprain, 202
smoke, 211 SR, 19, 20, 21, 56, 88, 95, 177, 178, 211, 220, 221,
smokers, 13, 39, 57, 79, 80, 90, 198, 211, 274 248, 314
smoking, 65, 70, 89, 90, 107, 176 stability, 21, 99, 267
smooth muscle, 13, 19, 21, 23, 26, 27, 28, 29, 31, 32, stabilization, 103
33, 34, 36, 37, 42, 49, 51, 52, 53, 56, 58, 66, 72, stages, x, xiii, 3, 4, 9, 12, 43, 80, 83, 135, 143, 147,
76, 82, 83, 84, 85, 86, 87, 88, 89, 94, 100, 120, 180, 237, 257, 259, 261, 264, 265, 266, 267, 268,
148, 152, 178, 201, 214, 305 321
smooth muscle cells, 13, 21, 23, 27, 28, 31, 32, 36, standard deviation, 262
51, 53, 58, 66, 72, 76, 83, 84, 86, 87, 88, 94, 120, standards, 275
148, 152, 178, 201, 305 statin, 105, 271
SNpc, 277, 278, 279, 280, 281 statins, 65, 81, 92, 99, 107, 119, 205, 240, 270, 292
social security, 259 Statins, 99, 108, 270
SOD, 2, 3, 11, 15, 16, 40, 42, 44, 75, 101, 121, 124, statistics, 224
125, 132, 142, 144, 204, 232, 265, 267, 269, 271, Steatosis, 163, 226, 227
272, 280, 306 stellate cells, 182, 228, 237, 238, 251, 252
SOD1, 204 Stellate cells, 238
sodium, 30, 49, 94, 102, 109, 114, 115, 116, 117, steroid, 231, 306, 318
118, 129, 172, 309, 320 Steroid, 306
soil, 189 steroids, 171
solvents, 231 stiffness, xi, 60, 193, 198, 321
somatostatin, 32 stimulant, 212
sorbitol, 194, 203, 216 stimulus, 38, 70, 120, 141, 161, 234
sounds, 283 storage, xi, 125, 159, 161, 162, 168, 173
soy, 203, 245 strain, 168, 169, 316
soybean, 255 strains, 169, 170, 185
soybeans, 241, 245 strategies, ix, xii, 37, 43, 91, 92, 112, 123, 125, 144,
SP, 19, 22, 130, 183, 186, 262, 263, 265, 267, 272, 176, 183, 208, 218, 224, 240, 246, 259, 261
290, 316 strength, 148, 261
spasticity, 277 stress-related, vii, xi, 14, 47, 75, 193, 202, 243, 251
spatial, 166, 269, 272, 291 striatum, 266
spatial learning, 269, 272, 291 stroke, 12, 13, 37, 43, 64, 92, 137, 170, 173, 190,
specialized cells, 230 315
species, vii, viii, ix, x, xi, xii, xiii, 1, 2, 4, 5, 9, 11, structural changes, 41
12, 14, 17, 21, 25, 26, 36, 44, 51, 53, 55, 57, 58, structural protein, 141
61, 63, 64, 69, 78, 79, 86, 91, 92, 98, 104, 111, Subcellular, 131
112, 120, 123, 126, 127, 130, 132, 137, 138, 140, substances, xii, 10, 14, 27, 31, 42, 114, 118, 120,
147, 151, 163, 170, 171, 175, 179, 183, 184, 193, 140, 187, 203, 208, 224, 234, 236, 240, 241, 246,
210, 211, 212, 223, 229, 232, 233, 235, 237, 238, 260, 282, 305
240, 241, 246, 249, 251, 259, 260, 263, 266, 285, substantia nigra pars compacta, (SNpc), 277, 269
294, 299, 306, 308, 318, 319 substrates, 77, 231
354 Index
sucrose, 180, 190 synergistic, viii, 26, 50, 100, 121

suffering, 68, 309 synergistic effect, 50
sugar, 7, 9, 41, 165 synovial fluid, 19
sugars, 7, 49, 197, 198, 210 synthesis, viii, 6, 13, 14, 25, 26, 28, 29, 30, 32, 49,
suicide, 8, 316 60, 62, 94, 96, 100, 101, 108, 113, 121, 126, 143,
sulfonamides, 118, 119 145, 146, 170, 173, 176, 177, 187, 196, 199, 204,
sulphate, 137 205, 212, 224, 226, 227, 228, 230, 231, 232, 237,
Sun, 59, 186, 249 239, 244, 245, 250, 264, 304, 309, 311, 315
superoxide, viii, xiii, 2, 4, 5, 11, 18, 19, 22, 23, 25, systemic circulation, 122, 165
26, 28, 30, 32, 35, 37, 38, 39, 40, 42, 43, 44, 46, systems, vii, 1, 2, 9, 12, 16, 17, 24, 27, 36, 41, 43,
47, 48, 50, 55, 56, 57, 58, 59, 62, 69, 75, 83, 87, 46, 54, 137, 141, 145, 190, 215, 226, 233, 246,
92, 94, 95, 100, 103, 120, 121, 124, 130, 132, 262, 306, 310
139, 140, 141, 142, 145, 152, 153, 172, 176, 183, systolic blood pressure, 44, 47, 50, 59, 61, 74, 81
200, 201, 203, 205, 210, 217, 228, 229, 231, 232,
234, 254, 260, 265, 279, 288, 290, 293, 299, 306,
T
308, 314, 316
Superoxide, 11, 18, 19, 35, 37, 39, 40, 42, 59, 60,
T cell, 71, 72, 195, 209, 236
106, 123, 131, 132, 183, 189
T cells, 72, 195
superoxide dismutase, xiii, 2, 18, 19, 40, 75, 92, 121,
T lymphocyte, 177, 195
124, 130, 139, 142, 152, 153, 172, 200, 203, 205,
T lymphocytes, 177, 195
217, 232, 234, 265, 288, 293, 299, 316
tachycardia, 99, 106, 108, 109
supplemental, 47, 274
tacrolimus, 118, 119
supplements, 11, 44, 46, 59, 90, 173, 203, 216, 248,
tangles, 263
273, 274, 283, 284, 293, 294, 310
tannins, 48
supply, 117, 208, 264
targets, 95, 97, 141, 183, 234, 267, 292
suppression, 70, 194, 196, 199, 210, 219
tau, 279, 287, 288, 293
suppressor, 9, 21
tau pathology, 293
surgery, ix, 91, 92, 94, 95, 99, 103, 104, 105, 106,
T-cell, 72, 209, 315
107, 108, 117, 125, 128, 131, 133, 162, 165, 240
TE, 83, 212, 253, 254, 294
Surgery, 130, 178
tea, 282, 293, 308, 319, 320
surgical, 92, 121, 311
Tea, 308, 321
surgical intervention, 121
temporal, 150, 262, 269
survival, ix, 33, 91, 112, 188, 207, 217, 224, 230,
temporal lobe, 269
272, 279, 287, 297, 305, 316, 318, 320
tensile, 28
susceptibility, ix, 24, 79, 89, 91, 97, 116, 125, 133,
tensile stress, 28
165, 169, 179, 195, 214, 306, 309
tension, 115, 121, 131, 139, 142, 152, 314, 317
swallowing, 259
terminals, 263, 277
sweets, 49
ternary complex, 6
switching, 13
TF, 87, 88, 132, 227, 232
sympathetic, 33, 35, 58, 172
TGF, 123, 139, 148, 150, 238
sympathetic nervous system, 33, 172
Thai, 152, 212
symptomatic treatment, 255
thalamus, 321
symptoms, x, 114, 135, 139, 149, 261, 277, 283, 300
therapeutic agents, 132, 241, 247
synapse, 263
therapeutic approaches, 64, 126, 186, 271, 283, 319
synapses, 258, 262, 263, 301
therapeutic interventions, xii, xiii, 80, 224, 257, 283
synaptic plasticity, 263, 291
therapeutic targets, 292
syndrome, x, xi, 16, 112, 117, 119, 135, 137, 140,
therapeutics, 284
142, 147, 148, 149, 150, 159, 160, 161, 162, 164,
therapy, xii, 43, 46, 47, 50, 54, 81, 87, 89, 97, 99,
168, 169, 170, 171, 172, 173, 174, 175, 176, 178,
104, 105, 114, 125, 126, 130, 132, 133, 144, 145,
180, 185, 188, 189, 270, 278, 288, 305, 316, 318
146, 148, 156, 169, 173, 174, 188, 199, 204, 205,
Index 355
206, 215, 221, 224, 240, 241, 242, 244, 245, 246, TNF-α, 69, 72, 75, 163, 164, 195, 203, 232, 233,
254, 255, 272, 281, 282, 288, 292, 300, 309, 310, 235, 236, 237, 243, 245
319, 320, 322 tobacco, 198
thiamin, 271, 309 tobacco smoke, 198
thiamin deficiency, 271 Tocopherol, 79, 108, 109, 156, 282, 297
Thiamine, 292, 309 tocopherols, 46, 79, 154, 166, 241, 274
thiazide, 119 tocotrienols, 79, 241
thiazide diuretics, 119 tolerance, 163, 253
thiobarbituric acid, 14, 42, 203, 234 toll-like, 71, 209
thioredoxin, 55, 58, 203, 217, 234, 251 Toll-like, 71, 196
Thomson, 182 total cholesterol, 65, 200
thoracic, 32, 33 total energy, 226
threat, 235 toxic, x, 2, 3, 4, 5, 10, 38, 42, 111, 114, 118, 120,
threonine, 279 122, 130, 131, 133, 142, 167, 199, 211, 231, 233,
threshold, 44, 201 234, 263, 266, 301
thrombin, 28, 32, 34, 35, 38, 75 toxic effect, 199, 263, 266
thrombocytopenia, x, 135, 148 toxic products, 168
thrombolytic therapy, 103 toxic substances, 114, 234
thrombomodulin, 138 toxicity, xi, 4, 19, 22, 116, 125, 129, 193, 194, 199,
thrombosis, 33, 42, 64, 66, 72, 88 200, 203, 205, 207, 208, 209, 213, 214, 217, 231,
thrombotic, 73 232, 241, 248, 250, 252, 264, 272, 281, 287, 302,
thromboxane, 28, 35, 45, 266 316
thymine, 7 toxicology, 250
thyroid, 179 toxins, 72, 114, 117, 118, 165
tight junction, 27, 116 trabeculae, 33
time, 12, 19, 34, 47, 70, 73, 74, 75, 79, 92, 93, 94, traits, 169, 277
96, 112, 114, 124, 139, 146, 195, 199, 201, 202, trans, 231
204, 235, 238, 260, 264, 272, 275, 276, 282, 311 transcription, 8, 85, 96, 97, 173, 200, 214, 227, 232,
timing, 145, 148, 307 235, 249, 265, 272, 280
tissue, x, xiii, 3, 4, 5, 10, 15, 17, 21, 22, 28, 29, 31, transcription factor, 200, 214, 227, 249, 272, 280
33, 34, 36, 47, 55, 66, 67, 69, 71, 76, 93, 94, 95, transcription factors, 200, 227, 249
96, 97, 103, 104, 120, 121, 122, 124, 126, 131, transcriptional, 13, 95, 96, 97, 100, 101, 104, 124,
132, 135, 136, 142, 161, 162, 163, 165, 168, 169, 132, 145
170, 171, 176, 181, 183, 185, 196, 199, 204, 207, transduction, 182
210, 220, 226, 233, 234, 235, 236, 237, 238, 240, transection, 314
247, 251, 260, 264, 276, 278, 299, 301, 305, 306, transfer, 35, 39, 41, 76, 173, 183, 184, 218, 228, 229,
308, 309, 312, 315, 317 265, 267
tissue plasminogen activator, 71 transference, 140
TJ, 22, 24, 52, 53, 57, 62, 88, 106, 129, 131, 132, transferrin, 153
183, 213, 216, 289, 292 transformation, 73, 234, 238
TLR, 209 transforming growth factor, 93, 97, 139, 194, 238,
TLR4, 209 253
T-lymphocytes, 67, 71 transgenic, 30, 52, 132, 168, 213, 217, 232, 251,
TM, xiii, 16, 61, 62, 105, 109, 138, 217, 221, 250, 264, 266, 267, 271, 287, 289, 293
254, 295, 299, 300, 301, 303, 304, 306, 307, 308, Transgenic, 184
311, 317 transgenic mice, 30, 52, 213, 232, 266, 267, 271, 293
TNF, 69, 72, 75, 145, 155, 163, 164, 165, 182, 187, transgenic mouse, 217, 289, 293
195, 196, 203, 232, 233, 235, 236, 237, 243, 245, transition, 4, 8, 225, 260, 264
252 transition metal, 4, 260, 264
TNF-alpha, 165, 182 translational, 13, 75
356 Index
translocation, 145, 155, 198, 270 UK, 284

transmembrane, 27, 263, 287, 315, 322 ulceration, 319
transmembrane glycoprotein, 263 ultrasound, 155, 245, 319
transmission, 265 ultraviolet, 48, 303, 308, 320
transplant, 124 ultraviolet light, 320
transplantation, xii, 104, 121, 165, 223 UN, 113, 153, 188
transport, 7, 34, 41, 54, 76, 96, 115, 116, 117, 129, uncoupling proteins, 281
130, 174, 179, 196, 198, 207, 210, 228, 232, 236, underlying mechanisms, 97, 112, 172, 283
258, 263, 264, 281, 310, 321, 322 unfolded, 230, 250
transsulfuration, 167 unfolded protein response, 230, 250
trauma, 259 uniform, 66
tremor, 277, 281 United States, xiii, 22, 216, 224, 257, 261, 275, 276,
triacylglycerols, 236 284
trial, 50, 59, 60, 62, 79, 99, 103, 104, 108, 125, 144, unstable angina, 26, 166
145, 154, 156, 173, 175, 183, 189, 190, 202, 203, UP, 83, 84
216, 220, 231, 242, 243, 244, 245, 247, 255, 269, urban population, 186
271, 273, 275, 276, 281, 282, 294, 297, 309, 310 urea, 112, 113, 114, 115, 126, 137
triggers, 32, 116, 117, 156, 163, 164, 228, 230, 236, urethra, 114
263 uric acid, 5, 7, 11, 39, 87, 112, 118, 120, 137, 142,
triglyceride, 162, 173, 185, 201, 243 144, 280, 296
triglycerides, 68, 152, 224, 226, 245 uric acid levels, 137, 296
tripeptide, 11, 265 urinary, 107, 113, 114, 140, 144, 155, 173, 214
trophoblast, x, 135, 137, 139, 141, 142, 144, 145, urinary tract, 114
146, 147, 148, 150, 153, 156, 157 urine, 17, 32, 37, 113, 114, 116, 118, 122, 136, 168,
trust, 280 280
tryptophan, 10 uterus, 150
TT, 290 UV, 303, 316
tubular, x, 111, 113, 114, 115, 116, 117, 118, 120, UV radiation, 303
121, 122, 127, 128, 129, 130, 131, 183, 279
tumor, 9, 21, 93, 97, 196, 202, 252, 290
V
tumor necrosis factor, 93, 97, 196, 252, 290
tumors, 9
Valencia, 62, 155
tumour, 182, 215
validation, 295
turnover, 32, 95, 164, 187, 271, 272, 295
validity, 267
TXA2, 138
values, 14, 242
type 1 diabetes, 161, 183, 195, 198, 199, 201, 202,
vancomycin, 117, 119
203, 204, 209, 210, 212, 213, 216, 217, 220
variables, 64, 143, 154, 164
type 2 diabetes, 12, 14, 23, 46, 48, 55, 60, 61, 161,
variation, 80
162, 170, 173, 179, 183, 184, 189, 191, 196, 199,
vascular cell adhesion molecule, 85, 89, 212, 236
200, 202, 204, 208, 212, 215, 216, 219, 220, 221,
vascular disease, 22, 29, 37, 52, 54, 86, 88, 174, 178,
225, 250
182, 194, 196, 200, 220, 291, 302
type 2 diabetes mellitus, 23, 161, 162, 179, 184, 191,
vascular diseases, 54, 174, 302
202, 208, 212, 215, 219, 225
vascular endothelial growth factor, 75, 138, 147, 309
type II diabetes, 23, 178, 179, 210, 212, 233
vascular endothelial growth factor (VEGF), 75
tyrosine, 6, 9, 10, 21, 29, 35, 54, 154, 195, 234
vascular inflammation, 42, 55, 212
tyrosyl radical, 6
vascular occlusion, 73
vascular reactions, 28
U vascular risk factors, 174, 273
vascular surgery, 117
ubiquitin, 15 vascular system, 43, 234
Index 357
vascular wall, viii, 5, 12, 26, 27, 28, 30, 36, 38, 44, vitamin supplementation, 242, 282, 307, 310
47, 50, 63, 64, 69, 72, 74, 80, 145 vitamins, viii, x, xi, 17, 25, 46, 47, 58, 60, 99, 100,
vascularization, 103, 104 101, 102, 103, 104, 108, 109, 125, 136, 141, 144,
vasculature, xi, 26, 28, 31, 33, 37, 38, 39, 42, 57, 70, 145, 146, 147, 148, 155, 156, 159, 166, 170, 171,
86, 137, 138, 193, 204, 237 172, 183, 187, 189, 201, 204, 216, 218, 241, 273,
vasculogenesis, 143, 147, 148 275, 285, 291, 294, 321
vasoconstriction, viii, 13, 25, 26, 28, 29, 30, 31, 37, vitreous, 302, 303, 314, 315
52, 70, 94, 115, 116, 118, 121, 122, 148 VLDL, 73, 162, 226
vasoconstrictor, 7, 26, 28, 31, 32, 33, 74, 201, 233 VO, 180, 184, 248
vasodilatation, 28, 36, 61, 62, 136, 145, 169, 171, vulnerability, 104, 126, 165, 306, 318
204, 205, 302
vasodilation, 13, 26, 27, 29, 31, 35, 37, 38, 41, 44,
W
51, 56, 70, 77, 79, 120, 155, 168, 173, 183, 189,
234, 303, 309, 321
waste products, 112
vasodilator, viii, 25, 26, 29, 32, 33, 35, 36, 37, 41,
wastes, 115
46, 53, 59, 71, 89, 101, 118, 264
water, ix, 3, 11, 32, 41, 44, 90, 100, 111, 112, 113,
vasomotor, viii, 25, 26, 27, 35, 83, 85, 115, 140
117, 118, 231, 244, 311, 312
vasospasm, 15, 16, 29
water-soluble, 11, 44, 100, 244, 311
VC, 289
wavelets, 93, 94
VCAM, 69, 71, 72, 73, 85, 236
web, 97
vegetables, 11, 49, 50, 79, 124, 173
weight gain, 178
VEGF, 75, 138, 147, 148
weight loss, 174, 176, 179, 225, 239, 240, 242
vein, 19, 89, 105, 115, 131, 307
weight reduction, 252
velocity, 206
Weinberg, 130
venous pressure, 301
Western countries, 224
ventricles, 93
WG, 105, 247, 291
ventricular fibrillation, 105
wheat, 174
ventricular tachycardia, 106
wheat germ, 174
vessels, 28, 29, 32, 61, 65, 70, 71, 200, 264
white blood cell count, 107
veterinarians, 170
white blood cells, 107
victims, 261, 268, 270
white matter, 291
VIP, 156
WHO, 22
viral hepatitis, 235
wild type, 272, 279
virus, 8, 162
wine, 48, 50, 62, 126, 134, 309, 320, 321
visceral adiposity, 162, 164, 170, 179
Wisconsin, 184
viscosity, 109
Wistar rats, 185
vision, 311
WM, 62, 88, 129, 188, 220, 253, 289, 290, 297, 315,
visual field, 300, 304, 312
319
vitamin A, 273
Wnt signaling, 288
vitamin B1, 206, 269, 271, 291
women, x, 31, 44, 135, 136, 140, 141, 142, 143, 144,
vitamin B12, 269, 271, 291
145, 146, 148, 152, 153, 154, 155, 156, 171, 182,
vitamin B6, 206, 269, 291
187, 189, 190, 216, 274, 276, 293, 294
vitamin C deficiency, 124
World Health Organization, 81
vitamin E, viii, 11, 24, 25, 41, 42, 43, 45, 48, 50, 59,
wound healing, 252
60, 78, 79, 89, 90, 92, 99, 100, 101, 102, 108,
WP, 81, 87, 180, 215, 296, 297
125, 126, 133, 143, 145, 154, 156, 166, 171, 172,
174, 187, 188, 190, 202, 203, 216, 217, 239, 241,
242, 244, 247, 253, 255, 267, 271, 272, 273, 274, X
275, 276, 290, 293, 294, 303, 309, 310, 311, 315,
322 xenobiotic, 231, 233
358 Index
xenobiotics, 231
xenografts, 321
yang, 320
yeast, 21
yield, 39
yin, 320
yogurt, 49
young adults, 203
zebrafish, 313
zinc, 187, 195, 202, 273, 275, 294
Zn, 124, 203, 217, 265

Oxidative Stress and Antioxidants - Their Role in Human Disease-Nova Biomedical Books (2009)

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Oxidative Stress and Antioxidants - Their Role in Human Disease-Nova Biomedical Books (2009)

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OXIDATIVE STRESS

AND ANTIOXIDANTS: THEIR ROLE

Nova Biomedical Books

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc.  New York

more serious condition, known as nonalcoholic steatohepatitis (NASH), in which fat

Oxidative Stress: Basic Overview

Joaquin Toro1 and Ramón Rodrigo2

1. The discovery of McCord and Fridovich in 1968-1969 described the enzymatic

2.2. Generation of Reactive Oxygen and Nitrogen Species

O2 + 4H+ + 4e– → 2H2O

1. Diffusion to neighbor cells. The presence of an unpaired electron on its molecule

In mammalian cells ROS might be formed through different pathways, either

Fe(II) + H2O2 → Fe(III) + •OH + −OH) [25].

O2•− + H2O2 → O2 + •OH + OH−

Fe(III) + O2•− → Fe(II) + O2 [26].

xanthine + H2O + O2 → uric acid + O2•−

f. Mieloperoxidase: The mieloperoxidase enzyme (MPO) produces hypochlorous acid

RH + O2 + 2H+ + 2e– → ROH + H2O

2.3. Pathophysiological Conditions

In pathophysiological conditions, the main sources of ROS include the mitochondrial

2.4. Reactive Oxygen Species as Mediators of Cell Damage

2.4.1. DNA Damage

A. Direct Damage to DNA through Reactive Nitrogen Species:

• Endogenous formation of carcinogenic N-nitrosamine molecules:

RNH2 + N2O3 → RNHO + NO2–

RNHNO → RNNOH → ROH + N2

Endogenous production of these compounds has been demonstrated in immortalized

RNN+ + H2O → ROH + N2 + H+

In cultured cells, protocols on activated macrophages show oxidative and deamination

B. Indirect Modifying DNA Sequence by Reactive Nitrogen Species

2.4.2. Lipid Peroxidation

is a minor lipid peroxidation product generated by heating of endoperoxides derived from

2.4.3. Protein Oxidation

2.4.4. Other Damage

2.5. Antioxidant Defense System

Table 1-1. Functional characteristics of the antioxidant enzymes

Antioxidant Chemical Scavenged General characteristics

3. Reactive Oxygen Species as Factors

3.1. Cardiovascular Disease

endothelial cells. This is characterized by a detachment of endothelial cells or part of the

3.1.2. Stroke and Atherosclerosis

3.2. Metabolic Syndrome and Diabetes

The metabolic syndrome (MS) includes a variety of cardio-metabolic abnormalities

3.3. Neurodegenerative Diseases

3.3.1. Alzheimer's Disease

4. Diminished energy metabolism and decreased cytochrome c oxidase levels are

3.3.2. Parkinson’s Disease

Pre-eclampsia is a human pregnancy-specific disorder that adversely affects the mother,

a Resistance to aqueous humor outflow is increased by H2O2, which induces TM

3.6. Aging and Cancer

3.7. Renal Damage

4. Conclusions and Perspectives

[29] Schulz E, Jansen T, Wenzel P, Daiber A, Münzel T. Nitric oxide, tetrahydrobiopterin,

[47] Tretyakova NY, Wishnok JS, Tannenbaum SR. Peroxynitrite-induced secondary

a matter still to be debated. Some studies reported a potential antihypertensive effect,

2.1. Role of the Vascular Wall Components

endothelium modulates the balance between opposing mechanisms that are

2.1.2. Vascular Smooth Muscle Cells

migration, apoptosis and secretion of inflammatory cytokines, as well as physiological

2.2. Role of Vascular Active Hormones and Factors

elevation of blood pressure. Accordingly, as an example seen in diabetes mellitus, a setting

Published by Nova Science Publishers, Inc.  New York