Essentials

of physiology and
clinical biochemistry

___________________________

Alexandra M. Crăciun

2013
 Colecţia Universitaria

__________ ۩۩۩ __________

Concepţie grafică: Andra M. Crăciun

Descrierea CIP a Bibliotecii Naţionale a României

CRĂCIUN, ALEXANDRA M.

2
 Essentials
of physiology and
clinical biochemistry

Alexandra M. Crăciun

Editura MEGA
Cluj-Napoca
Financial support by Medist company towards the publication of this study guide is
gratefully acknowledged.

4
 Abbreviations

5’-NT 5’nucleotidase
A1AT Alpha1 Anti Trypsin
Ab Antibody
ABCA1 ATP Binding Cassette protein A1
ACAT Acyl CoA Cholesterol Acyltransferase
ACTH Adrenocorticotrophic Hormone
AD Autosomal Dominant
ADH Antidiuretic Hormone
ADP Adenozin 5’-Diphosphate
AFP Alpha-Fetoprotein
Ag Antigen
ALAT Alanin Amino Transferase
AMI Acute Myocardial Infarction
AMPc Adenozin Monophospate cyclic
Apo A Apolipoprotein A
Apo B Apolipoprotein B
Apo C Apolipoprotein C
Apo E Apolipoprotein E
APR Acute Phase Reaction
APTT Activated Partial Thromboplastin Time
AR Autosomal Recessive
ASAT Aspartat Amino Transferase
ASLO Anti Streptolisin O Antibody
ATP Adenozin 5’-Triphosphate
ATS Atherosclerosis
BAP Bone Alkaline Phosphatase
BMD Bone Mineral Density
BSP Bromsulphtalein
CE Cholesteril Ester
CEA Carcinoembrionar Antigen
CETP Cholesteryl Ester Transfer Proteins
Che Cholinesterase
CIRD Chronic Inflammatory Rheumatic Disease
CK Creatinkinase
CK-MB Creatinkinase, Isoenzyme MB
CLIA Chemiluminiscence Immunoassay
CLL Chronic Lymphocytic Leukemia
CM Mature Chylomicron
CMR Chylomicron remnants
CMV Cyto Megalo Virus
CNS Central Nervous System
CRP C Reactive Protein
CSR Corticosuprarenal gland
CTX C terminal Crosslinks of colagen I
DCT Distal Convoluted Tubule
DEXA Dual Energy X ray Absorptiometry
DIVC Disseminated Intravascular Coagulation
DM Diabetes Mellitus
DOPA 3,4-dihydroxyphenylalanine
ECV Extracellular Volume

5
 EDTA Ethylene-diamine-tetra-acetate (anticoagulant)
ELISA Enzyme-Linked Immunosorbent Assay
ESR Erythrocyte Sedimentation Rate
F I-XIII Coagulation factors I-XIII
FC Free Cholesterol
FSH Follicle Stimulating Hormone
FT4 Free Thyroxine
GABA Gamma-Aminobutyrate
GGT Gamma Glutamyl Transpeptidase
GLA Gamma-Carboxyglutamic acid
GLDH Glutamate-dehydrogenase
Glu Glutamic Acid
GOT Glutamate Oxalate Transaminase
GPT Glutamate Pyruvic Transaminase
GR Granulocytes
Hb Hemoglobin
HDL High Density Lipoprotein
HLA Human Leukocyte-associated Antigen (major complex of
histocompatibility) - A, B, C, DR
HMG-CoA Hydoxy Methyl Glutaryl CoA
HTA Arterial hypertension
IDL Intermediary Density Lipoprotein
IFG Impaired Fasting Glycemia
Ig A Imunglobulin type A
Ig D Imunglobulin type D
Ig E Imunglobulin type E
Ig G Imunglobulin type G
Ig M Imunglobulin type M
IGT Impaired Glucose Tolerance
IL Interleukin
im intra muscular
IM Infectious Mononucleosis
INF Interferon
INR International Normalised Ratio
IPF Insulin Promoter Factor
ISI International Sensitivity Index
LCAT Lecithin Cholesterol Acyl Transferase
LDL Low Density Lipoprotein
LDLR receptors for LDL
LH Luteinizing Hormone
LMW Low Molecular Weight
LPL Lipoprotein lipase
LP-X X-Lipoprotein
MAO Monoamine Oxidase
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Concentration
MCV Mean Corpuscular Volume
mEq mili Equivalents
MI Myocardial Infarction
MID Monocytes and Eosinophyles
MO Monocytes
MPV Mean Platelet Volume
MT Mucosal Transferin
MTTP Microsomal Triglyceride Transfer Protein
nkat nanokatal (10-9 katal)

6
NACB National Academy of Clinical Biochemistry
NSCLC Non-Small Cell Lung Cancer
NTX N terminal Crosslinks of colagen I
OGTT Oral Glucose Tolerance Test
OPG Osteoprotegerin
PAI 1,2 Plasminogen Activator Inhibitors type 1, 2
PAR Protease Activated Receptors
PBG Porphobilinogen
PG Platelet Glycoprotein
PICP Procollagen type I carboxy-terminal propeptide
PINP Procollagen type I amino-terminal propeptide
PIP Phosphatidylinositol 4- Phosphate
PIVKA Protein Induced by Vitamin K Absence
PLT Platelets
PLTP Phospholipid Transfer Proteins
posm osmotic pressure
PPAR-γ Peroxizome Proliferators Activated Receptors γ
PPi inorganic pyrophosphate
PPTA Plasmatic Precursor of Thromboplastin
proGRP proGastrin Releasing Peptide
PSA Prostate Specific Antigen
PSP Phenol-Sulphon-Phtaleina
PT Prothrombin Time
PTH Parathyroid Hormone
PTT Partial Thromboplastin Time
RA Rheumatoid Arthritis
RBC Red Blood Cells
RDW Red Blood Distribution Width
SGA Small for Gestational Age
SIADH Syndrome of inappropriate antidiuretic hormonal release
SLE Systemic Lupus Erythematosus
T 1/2 Half life-time
T3 Triiodothyronine
T4 Thyroxin
TAP Total Alkaline Phosphatase
TG Triglyceride
TnC Troponin C
TNFα Tumor necrosis factor
TnI Troponin I
TnT Troponin T
tPA tisse Plasminogen Activator
TQ Quick Time
TSH Thyroid Stimulating Hormone
ULN Upper Limit of Normal
UV Ultraviolet light
VHA Viral hepatitis type A
VHB Viral hepatitis type B
VHC Viral hepatitis type C
VLDL Very Low Density Lipoprotein
vWF von Willebrand Factor
WBC White Blood Cells
WHO World Health Organization
δ ALA delta-Amino-Levulinic acid

7
8
 Foreword

This first textbook serves the purpose of preparing medical

students for the Clinical biochemistry exam. The overall aims

were to use physiological and biochemistry knowledge as

fundament in approaching pathological aspects of diseases and

to underline the importance of biochemical makers in diagnostic

evaluation. It does not seek to be a comprehensive textbook of

physiology or metabolic biochemistry, more detailed books being

available and recommended for those seeking further information

or specialization, but intends to give medical students a brief

orientation in paraclinical diagnosis.

Hoping that this textbook will be a good learning guide, we

consider continuing with a new edition, as the numbers of

lectures and lab hours will increase in student’s curricula.

Cluj-Napoca, 2013 Dr. Alexandra M. Crăciun

9
10
 Content

Chapter 1: Proteins .................................................................................... 13

Chapter 2: Enzymes.................................................................................... 23

Chapter 3: Lipides ...................................................................................... 33

Chapter 4: Diabetes mellitus and hypoglycemia ........................................ 41

Chapter 5: Exploration of liver diseases ..................................................... 45

Jaundice.................................................................................... 52

Pancreas and gastrointestinal tube ......................................... 57

Chapter 6: Exploration of iron and haem metabolism................................. 59

Porphyria .................................................................................. 62

Chapter 7: Exploring metabolic bone diseases .......................................... 69

Calcium, magnesium, phosphorus ........................................... 73

Chapter 8: Reference values for routine lab tests .............. ....................... 83

11
12
 Proteins

Proteins

Proteins in human plasma

- About 100 proteins are isolated and characterized
- Highly heterogeneous, ranging in their concentrations from g/l (albumins) to ng/l
(osteocalcin)
- Many are glycoproteins (containing till 10-25% carbohydrates) - albumins, α-
amylase; CRP are not glycosylated
- Most of the plasma proteins have the isoelectric point in the acidic pH range. They
migrate towards anode when the migration in the electrical field takes place in
alkaline environment.

Structure of plasmatic proteins

Proteins are made of aminoacids, bound together by peptide bonds.
To each protein may be described a structural organization form:
• primary structure - aminoacids sequence in the polypeptide chain; include the N
and C terminal ends of the molecule.
• secondary structure - alpha-helix, beta-sheet, a local conformation stabilized by
hydrogen bonds
• tertiary structure - the overall shape of a single protein molecule; the protein
domain is a specific structural region of a protein, that confers a specific function of
the protein (ex. enzyme)
• quaternary structure - the final shape of a protein, formed by several protein
molecules, usually called protein subunits (ex. hemoglobin – α 2 β 2 subunits)

Functional classification
 Transport proteins - haptoglobin, transferrin, ceruloplasmin, apolipoproteins
 Acute phase proteins – CRP, amyloid protein A, haptoglobin, fibrinogen
 Complement and coagulation factors
 Enzymes – amylase, lipase, cholinesterase
 Proteinase inhibitors – α1-antitripsin, antithrombin III
 Hormones (proteohormones) – calcitonin, PTH, insulin

13
Chapter 1

 Immunoglobulins – IgG, IgA, IgM, IgE, IgD

Role of plasmatic proteins

 Common:
o to maintain the coloidosmotic pressure (mainly albumin)
o are an important source for aminoacids
o buffering system
 Specific:
o Transportation of:
 lipids (apoproteins A, B, C, E),
 bilirubin (albumin),
 calcium (calbindin),
 iron (hepcidin, transferrin, ferritin),
 hormones,
 drugs (cisplatine uses copper transport channels),
 trace elements (copper - ceruloplasmin)
o enzymatic activity (transaminases, alkaline phosphatases, etc.)
o immunity - antibodies (Ig), complement
o haemostasis: balance between coagulation and fibrinolysis
o heme metabolism.

Where and how are proteins produced?

Where? Many are produced in the liver, with exception of immunoglobulins that are
produced by plasmocytes.

How? Stages of protein synthesis:

1. transcription of the DNA of the gene encoding the protein into mRNA.
2. translation of the sequence of aminoacids encoded in the codons of the
mRNA into the novo polypeptide; recognition of complementary base pairs
between the tRNA anticodon and mRNA codon is essential.
3. functional groups are finally added (ex. carboxyl to coagulation factors) or
whole molecules (ex. carbohydrates -glucose, galactose, mannose) to the
polypeptide

14
 Proteins

4. elimination of the functional protein in the Golgi complex and in

extracellular environment

Importance of the half-life time (T 1/2 )

- is the time within which a protein reaches its half original concentration
- is independent of the baseline concentration
- the shorter the T 1/2 is, the more frequently blood test should be performed in
monitoring the effect of the treatment in one patient. This is particularly
important in monitoring anticoagulant treatment or tumor markers.

Catabolism of proteins – pathways:

 Denaturation (acid in the stomach)
 Hydrolysis (proteases) – resulting aminoacids and further NH 3 and CO 2
and H 2 O. NH 3 is toxic and therefore is transformed into urea, in the
liver, in the ureogenesis cycle.
Some amino acids are used for protein biosynthesis, while others are converted to
glucose through gluconeogenesis, or enter into citric acid cycle.
The use of protein as a fuel is particularly important under starvation conditions
allowing the body's own proteins to be used to support life, particularly those found in
muscle. In hyper pyretic state of diseases, especially in children, ketone bodies
appear in urine (from degradation of triglycerides).

Total protein concentration in serum reflects distribution between intravascular

and extravascular fluid compartments, biosynthesis and elimination (degradation,
catabolism, losses). Reference values: 6-8 g/dl.
 Hyperproteinemia is mainly associated with hyperglobulinemia
o Apparent increase due to dehydration
o Stasis during venepuncture
o Increased protein synthesis (paraproteinemia)
 Hypoproteinemia is mainly associated with hypoalbuminemia
o Apparent decrease due to low synthesis (albumin, immunoglobulin) or
loss (mainly albumins)
o Relative water excess (over hydration), excessive infusion therapy or
considerably reduced urine production (anuria).

15
Chapter 1

ELECTROPHORESIS OF SERUM PROTEINS has as principle the separation of

proteins based on differences in the electrical charges and in the sizes of proteins.
This method is used to identify which proteic fraction is modified from normal and to
orientate toward a syndrome.
Serum proteins can be separated in five major fractions:
1. albumin: 52-60%
2. alpha-1 globulins: 3-5%
3. alpha-2 globulins: 7-9%
4. beta globulins: 11-14%
5. gamma globulins: 16-20%

Figure 1: Electrophoretic separation of seric proteins. If plasma is used instead of

serum, beta fraction is increased, due to fibrinogen, which is a beta protein.

Albumin – synthesized in the liver and filtered by the kidney glomeruls.

 hyperalbuminemia: caused by water depletion
 hypoalbuminemia: caused by liver disease, malnutrition and malabsorption,
external loss (nephritic syndrome), gastrointestinal enteropathy, burns, ascitic
collection, increased catabolism (cancer, infectious diseases).
o Edema or transudate - albumins concentration < 20g/l
o Exudate – proteins concentration >30g/l.

16
 Proteins

Globulins – most represented by immunoglobulins (Ig).

 IgA – role in natural protection barrier of skin and mucosal surfaces (digestive,
respiratory, lachrymal, genital); increase in: Crohn’s disease, celiac disease,
dermatomyositis.
 IgG – role in secondary defense and protection of tissue spaces; increase in:
SLE, chronic active hepatitis
 IgM – role in primary defense and bloodstream protection; increase in primary
biliary cirrhosis, perinatal infection,
 IgE – involvement in anaphylactic reactions (allergies); increase in: asthma,
allergies, parasitic infestation; IgE increase will not lead to
hypergammaglobulinaemia
 IgD – role in the activation of the B cells, of the immune defense system

Paraproteins
 Are Ig or Ig fragments produced by a family of plasma cells which has been
developed from one type of plasmatic cell (monoclonal).
 They lack antibody function but structurally are same as functional
immunoglobulins.
 They may consist of light chains (kappa or lambda) or of heavy chains
fragments.

DYSPROTEINEMIA – refers to qualitative and quantitative changes in the plasmatic

proteins. It can be the result of an increase or of a decrease of a certain protein type
or group of proteins, leading to a change in the albumin/ globulins ratio (normally >1).
In the past were used some indicative tests to identify dysproteinemia, pointing which
proteic fraction may be increased (Tymol test for beta and gamma increase, Kunkel
test and ZnSO 4 test for gamma increase).

Classification of dysproteinemia:
I. With normal total protein (TP) concentration: 6-8 g/dl
o Acute phase reaction – alpha-1 and alpha-2 peaks of proteinogram
are increased due to increase of CRP, amyloid A protein, haptoglobin,

17
Chapter 1

as result ESR increases. Etiology: acute inflammatory diseases

(pneumonia), surgical interventions, cancer, burns, etc.

Figure 2: Proteinogram in acute phase reaction. Alpha-1 and alpha-2 fractions are increased.
Albumin may be slightly decreased, due to its delayed synthesis in comparison with acute
phase reactants.

o Chronic inflammation – is associated with a broad gamma peak on

proteinogram, named polyclonal gammopathy (Figure 3). At least two Ig
are increased. Appears in chronic diseases: tuberculosis, chronic
compensated hepatopathy, SLE, RA.

Figure 3: Electrophoretic comparison between monoclonal and polyclonal gammopathy.

18
 Proteins

II. With decreased TP concentration: < 6 g/dl

o Nephritic syndrome – alpha-2 globulins are increased; albumins are
significantly decreased, due to loss at kidney level
o Liver cirrhosis – albumins are decreased due to deficient albumin
synthesis; alcohol may induce increase of Ig A that will lead to a
common block beta and gamma globulins.
o Exudative enteropathy – all proteic fractions are decreased.
III. With increased TP concentration: > 8 g/dl
o Multiple myeloma – gamma peak is increased and sharp, due to
increase of only one type of Ig (IgG 60%, IgA 25%, IgD 1.5%);
increased ESR (100 mm/h);erythrocytes rulleaux (like money stacks),
urinary Bence-Jones protein, specific X ray appearance at large bones
(hip, calvaria) and vertebrae, and characteristic bone marrow findings
(increase of plasmocytes). Evolution of the disease leads to kidney
insufficiency.
o Waldenström disease – gamma peak is increased due to increase of
Ig M. Anemia and lymphadenopathy are present, skeletal changes are
rare. Disease evolution leads to hyperviscosity of blood and increased
risk for internal hemorrhages.
o Light chains disease – kappa and lambda fragments from Ig may lead
to their deposition in the kidney leading to amyloidosis.

Hypogammaglobulinemia is a disorder that is caused by a lack of B-lymphocytes

and a resulting low level of immunoglobulins (antibodies) in the blood.
o Transient – babies around 6 months old; transient
hypogammaglobulinemia of infancy
o Primary
 IgA deficiency (frequent respiratory and digestive infections
during childhood);
 X-linked Agammaglobulinemia (Bruton Disease)- the genetic
defect leads to blocked maturation of B cells
o Secondary – chronic lymphocytic leukemia (CLL), protein losing
enteropathy, immunosuppressive therapy (drugs, irradiation).

19
Chapter 1

Some tests that indicate hypogammaglobulinemia include:

o Low serum immunoglobulins and B lymphocytes
o Missing specific antibodies to any vaccines the child has received
o Absence of antibodies to A and B blood group antigens

SOME PLASMA PROTEINS ARE MEASURED IN URINE AND CEREBROSPINAL

FLUID (CSF)
- albuminuria, microalbuminuria, α 1 -microglobulin, α 1 -glycoprotein, α 1 -
antitrypsin, α 1 -microglobulin, transferrin, α 1 -microglobulin α 2 -macroglobulin,
β 2 -microglobulin, etc - in nephritis
- 80% of proteins in CSF are of plasmatic origin (albumin, IgG, α 1 -antitrypsin,
α 1 -glycoprotein, transferrin), while 20% are produced by the central
nervous system.

Urine protein – proteinuria of more than 150 mg/ 24 hours.

o Pre-renal – Bence-Jones protein, hemoglobin, myoglobin
o Renal – glomerulonephritis;
o glomerular proteinuria (>3g proteins/ 24h loss);
o tubular proteinuria (< 1g proteins/ 24h loss)
o Post-renal – calculi, malignancy, infection.

CSF protein – increase in bacterial or viral meningitis, intracranial neoplasm,

mechanical obstruction. Increased Ig in CSF may appear in multiple sclerosis,
tuberculosis and neurosyphilis.
Ratio of Ig/ Albumin in CSF makes possible to differentiate between blood and local
synthesis of proteins. It results a diagram = Reiber index. Values below a defined line
indicate passive transfer (blood origin), while values above this line indicate local
synthesis of Ig by central nervous system.

20
 Proteins

DEFICIENCY OF SPECIFIC PROTEINS:

o Alpha-1 antitrypsin is responsible for the inhibition of proteases present in
serum. It’s deficiency is a genetic disorder caused by defective production of
alpha 1-antitrypsin (A1AT), leading to decreased A1AT activity in the blood
and lungs, and deposition of excessive abnormal A1AT protein in liver cells.
Severe A1A deficiency causes panacinar emphysema and/or chronic
obstructive pulmonary disease (COPD) in adult life in many people with the
condition (especially if they are exposed to cigarette smoke), as well as
various liver diseases in a minority of children and adults, and occasionally
more unusual problems.
o Antithrombin III deficiency is a rare hereditary disorder that is diagnosticated
when a patient suffers recurrent venous thrombosis and pulmonary embolism.
Inheritance is usually autosomal dominant (AD). In nephritic syndrome,
antithrombin is lost in the urine, leading to a higher activity of coagulation
factors II and X increasing the risk for a thrombotic event.

21
Chapter 1

22
 Enzymes

Enzymes

General information
- Enzymes are biological molecules that are responsible for chemical reactions
in vivo.
- Most enzymes are proteins, have a three dimensional structure, and are
associated with organic (vitamin K for carboxylase) and inorganic (Mg2+)
cofactors in the catalytic process.
- Enzyme couples to a substrate (specific or relatively specific) to form an
enzyme-substrate complex, finally resulting the product of reaction.
- One enzyme may be used in vivo several times (hundreds) before is degraded
by proteases.

Factors that influence the enzymatic activity

- Optimum pH (acid -for pepsin, acid phosphatases; alkaline – for chemotripsin,
alkaline phosphatase)
- temperature (optimum activity at 37°C)
- if the substrate is in sufficient concentration, enzymatic activity depends on the
enzyme quantity (concentration).

Expressing enzymatic activity

- U/l (Unit/l) = micromoles per liter of substrate transformed by the enzyme in
one minute. 1 U corresponds to 16.67 nkat.
- Kat (katal) = micromoles of substrate converted by the enzyme per second.

23
Chapter 2

Isoenzymes – are different structural forms of the same enzyme. They differ in
structural parts that indicate where it was produced, while the structure of the domain
(that confers its function) is the same in all, all Isoenzymes catalyzing the same
chemical reaction. Example of isoenzymes: CK-MM, CK-BB, CK-MB; LDH 1-5; ALP
(liver, bone, intestinal, kidney, placental).

Mechanism of enzyme changes in serum

a. Enzymes are secreted actively in plasma
- Liver enzymes are synthesized by liver and secreted actively in plasma, where they
act on specific substrates. Example: LCAT, cholinesterase, coagulation factors.
b. Decreased activity reflect an insufficient production by the cell.
Example: suppression by drugs (statins, intoxication with organophosphorics)
c. Enzymes of exocrine origin – are produced by glandular tissues and poured in
excretory ducts. Their increases reflect the obstruction of the ducts, or destructions of
the gland. Example: amylase (salivary or pancreatic), acid phosphatases (prostate).
d. Intracellular enzymes -their increase reflect a suffering tissue, from alteration of
membrane permeability to cellular necrosis. The amplitude of change (increase in
plasma) depends on the number of destroyed cells and of other environment factors
– vascularization or the organ and half-lifetime of each involved enzyme.
Membrane permeability may be increased by:
- Genetic defect (muscular dystrophy)
- Hypoxia
- Decrease in glucose transport to the cell
- Distention of tissue (rapid installed hepatomegaly)
- Bacterial toxins or viruses
- Drugs or chemicals
e. Enzyme increases unrelated to specific pathology
- Age and gender
- Pregnancy and lactation
- Hemolysis of blood during sample collection or preparation
- Drugs: antibiotics, green medicine
- Incorrect preparation of patient before blood sampling (non fasting state)

24
 Enzymes

Enzymes in disease
Certain tissue cells contain characteristic enzymes which enter the blood only when
the cells to which they are confined are damaged or destroyed.
The presence in the blood of significant quantities of these specific enzymes
indicates the probable site of tissue damage.

Distribution of enzymes in the body – mostly in metabolic active tissues:

- Heart: AST, ALT, CK-MB, LDH 1
- Muscles: AST, CK-MM, aldolase, LDH 5
- Liver: ALT, CE, GGT, ALP, LDH 5
- Bone: alkaline phosphatase, acid phosphatase
- Pancreas: alpha-amylase, lipase, ALAT, ASAT, LDH 4
- Brain: CK-BB
- Thyroid: ALAT, ASAT, CK
- Parotid gland: amylase
- Prostate: acid phosphatase
- Lungs: LDH 3
- Kidney: 1α-hydroxylase; 17-,18-,21-hydroxylases

A. ENZYMES INVOLVED IN CARDIAC PATHOLOGY

Myocardial infarction (MI), commonly known as heart attack, results from partial or
total hypoxia of the heart tissue due to an arterial partial or total blockage with a
thrombus.
Patients are accusing a precodial pain with pain irradiation in the left arm.
During this event enzymes CK, AST and LDH will increase.
In principle, the larger the necrosis, the higher increase of enzymes is associated.
CK catalyzes the reversible transfer of phosphate groups between creatine and
phosphocreatine as well as between ATP and ADP. CK total: 90 – 180 U/l; CK-MB: <
24 U/l. If CK-MB does not exceed 6% of total CK, there is no destruction of
myocardium.

25
Chapter 2

Indication of CK and CK-MB:

1. MI diagnostic
2. post-MI evolution, for secondary infarctions
3. differential diagnostic with pulmonary embolia and shock
4. post-surgical control in heart operations
5. exclusion of a MI in thoracic trauma
6. exclusion of myocardial destructions in patients with severe intoxications

 CK increases 6 h after MI, reaching a maximum peak 18-29h after the

event,
 increases maximum 10 times upper normal limit (UNL)
 in some cases CK-MB precedes rises of total CK
 multiple MI are both reflected by increase of CK-MB and CK total, but in
some cases only CK-MB detects a new infarct, but not CK total
 efficient thrombolytic therapy may lead in the first stage of treatment to
an increase of the enzymes, which are “washed away” from the place
of necrosis.

Because in many cases the exact time of MI event is not known, CK and CK-MB
must be repeated after 4h, if the results are not conclusive (in association with EKG,
clinical features).

Differential diagnostic:
- Intramuscular injection may cause increase of total CK, indicating a secondary
MI; similarly in patients with abscess, after MI – but these increases are lower
than in MI
- in pulmonary embolia CK total is increased, but CK-MB is normal (< 24 U/l)
- in Duchenne muscular dystrophy CK may increase to 100 times ULN in
severe forms and 10 times in mild forms.

Other non-enzymatic markers increased in MI:

o Troponin (T, I, C) – increases more dramatically than CK. Increases in MI,
after cardiac surgery and after thrombolytic treatment.

26
 Enzymes

o Myoglobin – not specific, useful when measured in dynamics (before and

after, in time)
o Albumin, modified by ischemia (not by cellular necrosis). It has a diagnostic
value in differentiation of the cardiac pain from other chest pain.

Value of enzymatic diagnostic in comparison with EKG:

o Usually the EKG modifications are preceding the enzymatic increases; the
latest have the role to confirm the diagnostic
o enzymatic determinations are useful in multiple micro infarcts, hypertrophy of
the myocardium, heart blocks
o enzymes often detect secondary MI

B. ENZYMES INVOLVED IN MUSCULAR PATHOLOGY

o one third of body weight is represented by the muscles
o muscles contain important enzymes: CK-MM, LDH 5 , ASAT and aldolase
o the degree of change in these enzymes is dependent on the blood distribution
and muscular activity of the tissue.
o physical effort in excess may lead to hypoxia, till shock (due to generalized
hypoxia)
o im injections with Penicillin, Tetracycline, Diazepam and antiaritmics may lead
to muscular local damage (cytolysis of skeletal muscle cell = rhabdomyolysis).

Dermatomyositis – is a connective-tissue disease that is characterized by

inflammation of the muscles and of the skin. In acute phases are increasing
ASAT, LDH, CK.
Inflammation of the connective tissues typically produces a reddish-purple
(violaceous) rash of the skin which was exposed to sun. The rash is named after the
tendency of plants to grow toward the sun (heliotropic) and is characteristic in
dermatomyositis.

27
Chapter 2

Progressive muscular dystrophy is characterized by progressive skeletal muscle

weakness, associated with progressive death of muscle cells and tissue, that will be
finally be replaced by conjunctive, fibrous tissue.
It is a group of inherited non inflammatory muscle disorders without a central or
peripheral nerve abnormality.
Duchenne progressive muscular dystrophy is a genetic disease related to
chromosome X. CK and aldolase in caring women bearing the mutant gene will show
increased values.
CK level may be up to 100 times the upper limit of normality in severe forms and 10
times in mild forms. AST and LDH increase in severe forms.
CK determination is used for:
o detection of carriers (women)
o diagnostic of risk before clinical signs are present (for Duchenne only, in Limb-
girdle dystrophy CK may fail to find carriers but is increased in 70% in clinical
manifest disease).

Polymyositis - inflammatory disease that comprise many groups of muscles.

o CK is elevated in 70% of patients; in children elevations are higher than in
adults.
o Therapeutic test with cortisone may distinguish between a progressive
muscular dystrophy and a polymyositis.

Malignant Hyperthermia (1:50 000 anesthesia) is caused by halothane,

succinilcolin, xiline, diazepam, barbiturate.
o Mechanism: in some subjects a rapid efflux of calcium is triggered from the
intracellular deposits into myoplasma, leading to sudden and sustained
muscular contraction. It may lead to metabolic acidosis (increased production
of lactic acid).
o It is associated with tachycardia, tachypnea, muscular rigidity and
hyperthermia (41-42 ° C).
o Increased CK and ASAT confirm the diagnostic.
o Treatment: calcium antagonists.

28
 Enzymes

C. ENZYMES INVOLVED IN HAEMATOLOGICAL DISEASES

 Lactate dehydrogenase (LDH) is involved in glucose metabolism.
 LDH catalyzes the reversible reaction between pyruvic and lactic acids.
 The richest tissues in LDH are: cardiac muscle, skeletal muscle, liver, kidney
and red blood cells.
 LDH isoenzymes are tetramers (4 subunits), and by electrophoresis 5
fractions may be separated, with different origins:
o LDH1 – heart muscle
o LDH2 – red blood cells
o LDH3 – lungs
o LDH4 – kidney, pancreas, placenta
o LDH5 – liver and skeletal muscles

Diagnostic:
o LDH2/LDH1 ratio is >1 in healthy subjects, <1 in myocardial infarction;
o One of the most important diagnostic uses for the LDH isoenzymes test is in
the differential diagnosis of myocardial infarction or heart attack. The total LDH
level rises within 24-48 hours after a heart attack, peaks in two to three days,
and returns to normal in approximately five to ten days.
o LDH5 increases in liver diseases

Increase of LDH:
o Myocardial infraction (LDH1)
o Megaloblastic anemia (LDH2), myelodysplasic syndrome, hemolysis, leukemia
o Pulmonary infarction (LDH3)
o Pancreatitis, kidney diseases (LDH4)
o Hepatocellular damage (viral infections – CMV, Epstein Barr, hepatitis B and
C; intoxication with drugs), muscular diseases (LDH5)
o Hypothyroidism (LDH1)

LDH in Megaloblastic anemia:

o LDH increases more than 10 times UNL;
o low reticulocytes; normochromatic and megaloblastic RBC.

29
Chapter 2

o therapeutic test with vitamin B12, administered im will normalize the

parameters within three weeks.
LDH in hemolytic anemia:
o LDH increases slightly, 2-5 times upper normal reference
o AST may slightly increase
LDH in leukemia:
o LDH (LDH2 and LDH3) is increased, no response to the therapeutic test
(vitamin B12/ folic acid)
o Associated with low cholesterol may indicate a bad prognostic for the patient.

D. ENZYMES INVOLVED IN BONE PATHOLOGY

Two important physiological processes are occurring during life related to bone
metabolism: bone modeling (till peak bone mass is achieved) and bone remodeling,
a process that is active during the whole life span, and which is responsible for
maintaining the normal bone structure.
Two types of cells are completing these processes:
 osteoblats (for bone formation) – young bone cells producing collagen type I,
osteocalcin and bone alkaline phosphatase;
 osteoclasts (for bone resorption) – multinucleate cells producing acid
phosphatases

Changes of Alkaline phosphatase (ALP) and bone isoenzyme (BAP) in serum:

o Osteoporosis – decreased, normal or increased ALP
o Osteomalacia/ Rickets – increased due to secondary hyperthyroidism.
Children and old subjects may have physiological increased ALP levels. In
rickets ALP may be 2-3 times the upper physiological value.
o Hyperparathyroidism – increased bone turnover, increased osteoblastic
activity
o Paget disease – increased bone turnover, increased BAP, ALP
o tumoral processes:
 in processes associated with osteoformation ALP increases;
e.g.: Carcinoma metastasis: mammary, prostate: ALP increased.

30
 Enzymes

 metastatic prolipheration in osteoclast line is associated with

osteolysis – bone acid phosphatase is increased; e.g.:
osteolytic sarcoma (S. Ewing, reticulosarcoama): ALP normal
values.
o Decreases of alkaline phosphatase (ALP) in serum:
- Vitamin C deficiency (severe)
- Hypothyroidism installed in childhood
- after perfusion with EDTA administered therapeutically after Pb (lead)
intoxication
- dialyzed patients

Changes in serum acid phosphatase:

 Gaucher disease
 prostate adenoma/ cancer

31
Chapter 2

32
 Lipids

Lipids

General structure of lipoproteins

Fats are not soluble in blood and in order to be transported in blood, lipids are
associated with hydrosoluble compounds (ex. proteins), resulting lipoproteic
complexes.

Structure of lipoproteins:
- Outside layer: formed by apoproteins (A, B, C, E), free cholesterol,
phospholipids
- Core: consisted of cholesteryl esters, triglycerides.

Absorption and transport of exogenous lipids

 Biliary acids are preparing dietary lipids for absorption by emulsification, in the
small intestine.
 Pancreatic lipase transform triglycerides in free fatty acids, that will enter
passive or active in the lymph, at the level of the gut mucosa.
 In the gut cells apo B48 and chylomicrons are formed, named nascent
chylomicrons after they enter in the lymph.
 Chylomicrons contain triglycerides (TG; >80%), cholesterol esters (CE; <15%)
and apoproteins (B48).
 From the lymph, nascent chylomicrons enter in the bloodstream via de ductus
thoracicus (above the liver) in about T (½) = 5-20 min.
 In the blood stream the nascent chylomicron receives apo C and apo E from
HDL, becoming a mature chylomicron (CM).
 Arriving at the tissular level, apo C II (cofactor) stimulates lipoprotein lipase
(LPL), an endothelial enzyme that will degrade CM in free fatty acids and
cholesterol that will be uptaken by the cells, and glycerol. The result is a
smaller lipid complex, remnant chylomicron, which is transported to the liver.
 The uptake of the remnant CM in the liver is dependent on apo E - apo B48
receptors. Genetic differences in apo E genotype may lead to differences in
the uptake of remnants, delayed uptake increasing the risk for cardiovascular
diseases.

33
Chapter 3

Transport of endogenous lipids

 By internal redistribution TG and CE are packed in the liver in VLDL (very low
density lipoproteins) and released directly in the bloodstream.
 VLDL complex contain apo B-100 and is transformed by LPL in remnant VLDL
(=IDL). The fate of IDL (Intermediary Density Lipoproteins):
 Uptaken by liver (via receptors apo E – apo B100);
 IDL (the majority) is transformed in LDL , that carries CE to
peripheral cells. Tissues have specific receptors for LDL that are
distributed on the surface of the cells.
 LDL represents 70% of total cholesterol; more than half is used
by liver and organs that need cholesterol (ovary, testes,
adrenals);

Reverse cholesterol transport

 the peripheral tissue excess of cholesterol is uptaken in the FC form, by ABC1
proteins, resulting the nascent HDL.
 Before uptake, CE (intracellular deposit form of cholesterol) is transformed in
FC.
 In plasma, the nascent HDL containing FC is converted to CE by the enzyme
lecithin-cholesterol-acyl transferase (LCAT), resulting mature HDL.
 The uptake of HDL by the liver is mediated by scavenger receptors SR-BI.
 In the liver CE is transformed in FC, which is excreted in the bile.
 In blood, much of CE appear to transfer to chylomicron remnants, LDL, VLDL
by CETP (Cholesterol Esters Transfer Proteins) and PLTP (Phospholipid
Transfer Proteins). Theoretically, inhibition or blocking CETP will direct more
cholesterol to the liver.

The role of cholesterol in:

 Synthesizing of biomembranes;
 Synthesizing of steroid hormones: adrenal and gonadal
 Synthesizing of biliary acids
 Synthesizing of vitamin D

34
 Lipids

Mechanisms that regulate cholesterol levels in blood:

 Our body produces and assimilates cholesterol from diet. Intestinal
absorption of cholesterol is adjusted, dependent on the intake. A high intake
of cholesterol will limit the intestinal absorption level and a low intake of
cholesterol will increase its absorption.
 Cholesterol enters in the cells after LDL couples with specific receptors
distributed on the surface of the cell. In the cell, LDL enters in lysosomes,
where the complex is degraded to final products: CE is transformed in free
cholesterol (FC); TG in free fatty acids; proteins in aminoacids. The excess
is of cholesterol is deposited in the form of CE, after esterification by the
enzyme Acyl CoA Cholesterol Acyltransferase (ACAT).
 The endogenous production of cholesterol may be suppressed by blocking
the transcription for LDL receptors and by blocking the key enzyme in the
synthesis of cholesterol: HMGCoA-reductase. Statins, drugs used to lower
cholesterol, are blocking HMGCoA-reductase.

Effect of diet and exercise on serum lipids:

 LDL and VLDL decrease by a diet in low cholesterol and low fat
 A controlled diet with low intake of carbohydrates may decrease VLDL, TG
 Regular exercise decrease LDL and increase HDL levels

Risk factors for coronary artery disease:

 Inflammation – increased hsCRP
 Hyperlipemia – increased cholesterol, triglycerides, and LDL cholesterol,
oxidized LDL.
 Glucose intolerance – OGTT modified
 Cigarette smoking (free radicals, NO), stress

Assessment of lipid levels – after at least 12 hours fasting:

 Creamy layer on top and clear serum (after overnight at 2-8 ˚C) suggest
increase of chylomicrons.
 Turbid samples suggest increased VLDL

35
Chapter 3

 HDL and LDL are small and do not cause turbidity in samples if they are
increased.
 Reference intervals:
Total lipids: 500 – 800 mg/dl
Cholesterol: 110 - 200 mg/dl (< 5.0 mmol/L)
Triglycerides: 50-150 mg/dl (< 2.0 mmol/L)
HDL-chol: > 35; > 45 mg/dl (> 1.55 mmol/L)
LDL-chol: 70 – 130 mg/dl (3.5-4,4 mmol/L)
Apo B: Males: 0.63-1.88 g/l; Females: 0.56-1.82 g/l
Apo A1: Males: 1.09-1.84 g/l; Females: 0.60-2.28 g/l
 Reference intervals may vary, dependent on geographical region, dietary
habits and genetic profile of a population. In the healthy subjects serum
cholesterol and triglycerides are increasing with age, and are different in
men and women, so ideally the reference interval should be age/gender
related.

 Formulas used for calculations:

LDL Chol [mg/dl] = Total Chol – (VLDL + HDL); VLDL= TG/5
Formula is valid only for TG < 400 mg/dl.
Total lipids [mg/dl] = 2.25 x Total Chol + TG + 90

HYPERLIPEMIAS
Hyperlipidemia refers to an increase of cholesterol (hypercholesterolemia),
triglycerides (hypertriglyceridemia), or mixed hyperlipemia (both cholesterol and
triglycerides are increased).

Causes:
Secondary (more frequent):
 Diabetes mellitus
 Hypothyroidism - LDL, IDL increased

36
 Lipids

 Nephritic syndrome - increased LDL, probably due to increased VLDL

production
 Liver diseases:
o Cholestase: extrahepatic/ intrahepatic obstruction (tumors).
o Abnormal lipoproteins
 Lp –X = cholesterol and lecithin.
 Lp-Y = triglycerides and apo B.
 Pregnancy
 Alcohol excess
 Oral anticonception
 Diet rich in cholesterol and lipids
Symptoms: xanthoma, glucose-intolerance, early fat diseases

Primary (inherited)
o The WHO classification of hyperlipemia based on Frederickson
classification (1971) is presented in Table 1.
o Inheritance and prevalence of primary hyperlipidemias vary:
 familial hypercholesterolemia (type IIa) – AD; 1:500
 familial combined hyperlipidemia (type IIb, IV, V) – AD; 1:300
 familial lipoprotein lipase deficiency/ cofactor apo CII deficiency
(type I) – AR; 1:1 000000
 Remnant hyperlipidemia (type III) – 1:3 000

Familial deficiency of LPL - Type I

- Clinic: acute pancreatitis, hepato-, splenomegaly, not overweight, xanthoma,
abdominal pain, lipemia retinalis
- Triglycerides are 10 times UNL
- No increased risk for coronary artery disease
- Risk for acute pancreatitis

37
Chapter 3

Table 1: Classification of primary hyperlipidemias:

WHO classif. CHOLESTEROL TRIGLYCERIDES Modified Aspect of serum
fraction

Type I < 260 mg/ dl >1000 mg/ dl Chylomicrons Clear with creamy
layer

Type II a > 300 mg/ dl < 150 mg/ dl LDL Clear

Type II b < 350 mg/ dl 150-300 mg/dl LDL and VLDL Turbid

Type III < 350 mg/ dl 350–500 mg/ dl Chylom.Remn. Turbid with
creamy layer

Type IV < 260 mg/ dl 200-1000 mg/dl VLDL Turbid

Type V > 300 mg/ dl >1000 mg/ dl VLDL and Turbid with
Chylom. creamy layer

Familial hypercholesterolemia - Type IIa

- Increased LDL-Cholesterol
- Abnormality in the gene encoding receptors for apoB100-LDL-Chol receptors
- Clinic: tendon xanthoma, xantelasma
- High risk for cardiovascular diseases: 85% of heterozygotes will have an MI till
60 years old.
Familial Combined Hyperlipemia - Type IIb, IV, V
- Overproduction of B-100 associated with VLDL
- without tendon xanthoma!
- Type IIb: VLDL and LDL increased
- Type IV: VLDL increased; familial ligand defect apo B, due to mutations in
ligand domain of apo B100. TG overproduction.
- Type V: VLDL and chylomicrons increased
- Moderate risk for cardiovascular diseases

38
 Lipids

Familial dys-beta-lipoproteinemia – Type III

- Cause: isoforms of apo E (E2/E2) which are poor ligand; E2/E2 in 1% of the
population;
- Increased remnants of Chylomicrons and VLDL (IDL) in circulation
- Xanthoma, obesity, fat deposition in palmary creases
- Cardiovascular diseases frequent

TREATMENT OF HYPERLIPIDEMIAS:
- Change diet and life style; exercise
- Statins: HMG-CoA reductase inhibitors; increasing LDL receptors will
increase LDL clearance and decrease of endogenous cholesterol synthesis
- Fibrates: stimulate LPL, increasing the lipoproteic clearance of complexes
rich in triglycerides
- Niacin: decreases VLDL synthesis in the liver and increases HDL synthesis
- Estrogens: increase the tissular LDL receptors, and increase HDL
- Cholestyramine: is the safest drug for treatment of hypercholesterolemia. It
binds to the bile salts interfering with their re-absorption in the small intestine.
Because bile salts are synthesized from cholesterol, it helps by lowering the
serum LDL cholesterol.

HYPOLIPEMIAS
Secondary hypolipoproteinemia
- Chronic cachexia (cancer)
- Rapid use of LDL: myeloprolipherative diseases
- Intestinal malabsorption
- Immunoglobulins disorders: cryoglobulines, globulins from myeloma by
forming complexes of Ig-lipoproteins
- protein loss enteropathy

Primary hypolipoproteinemia:
Hypo-alpha-lipoproteinemia:
- Tangier disease causes cholesterol deposition in macrophages throughout
the body due to impairment of cellular efflux and the absence of HDL

39
Chapter 3

- < 50% HDL, apo A-I and apo A-II

- mutations in ABCA1 blocks the efflux of cholesterol from peripheral tissues
- Clinic: opaque cornea in young age
- FC (unesterified) and phospholipids accumulate in the renal microcirculation,
proteinuria follows
- Abnormal HDL: bilayer disks and small spherical particles.
- Big orange-colored, fat tonsils (accumulated CE)
- Neuropathy, splenomegaly, corneal infiltration
Hypo-beta-lipoproteinemia:
o a-beta-lipoproteinemia – refers to apo B deficiency:
 Mutations in MTTP (microsomal triglyceride transfer protein)
 Homozygous only have problems: no CM, VLDL and LDL in
plasma
 Triglycerides < 11 mg/dl (<0.12 mmol/L)
 Total Cholesterol < 90 mg/dl (2.3 mmol/L)
 Presence of acantocytes (Chol/PL ratio high) and degeneration
of retina
 Children normal at birth, but later present steatorrhea and growth
delay
o Chylomicron retention
 In neonates: intestine does not produce Chylomicrons, apo B48
 LDL and VLDL are 50% of normal

40
 Diabetes and hypoglycemia

Diabetes mellitus and hypoglycemia

Diabetes mellitus (DM) is a syndrome characterized by chronic elevated glucose

levels in serum, associated with clinical features: polydipsia, polyuria.

Classification of DM:
I. Insulin deficient – Type I, with juvenile onset, HLA DR3 and DR4
II. Insulin resistant – Type II, obese and non-obese
III. Secondary, caused by:
i. Pancreatic disease – acute pancreatitis
ii. Hormonal disease (cortisol, hyperaldosteronism, growth
hormone excess, pheochromocytoma)
iii. Chemical agents (some drugs – diuretics, phenytoin,
cyclosporine)
iv. Genetic diseases (muscular dystrophy, cystic fibrosis,
glycogen type I storage disease).
Diagnostic criteria:
I. Random glucose levels in serum above 200 mg/dl
II. Fasting glucose levels in serum higher than 126 mg/dl
III. An oral glucose tolerance test (OGTT) is indicated when fasting or random
plasma glucose are not within upper reference range. This test is
recommended in pregnancy glycosuria as well.
IV. Clinical symptoms: polydipsia, polyuria, weight loss
V. Glucose positive in urine if glycemia is above 180 mg/dl
VI. Glycated hemoglobin (HbA1c) is > 6.3%. This test evaluates the mean
glycemic levels in the last 2 months. It is useful in emergency units as well,
for example in patients with acute MI due to an undiagnosed DM. This test
has its limitations in patients with anemia or increased RBC (people living at
high altitudes).

41
Chapter 4

Table 2: Diagnostic criteria of DM after American Diabetes Association (1997), adopted by

WHO in 1998. IGT= Impaired Glucose Tolerance, IFG = Impaired Fasting Glycemia
(Hyperglycemia at base line, but OGTT normal). IGT and IFG do not represent clinical
entities, but represent risk factors for cardiovascular and/or DM.

Glycemia [mg/dl]
NORMAL
- fasting < 100
- OGTT at 2 h < 140
DIABETES
- fasting ≥ 126
- OGTT at 2 h ≥ 200

IGT
- fasting < 125
- OGTT at 2 h > 140

IFG
- fasting 110 – 125
- OGTT at 2 h < 140

Metabolic changes in DM:

- Hyperglycemia
- Increased fatty free acid levels in plasma. If they are used as energetic fuel,
glucose utilization is inhibited.
- Reduced uptake of glucose in peripheral tissues, due to insulin deficiency or
resistance
- Increased triglycerides. Increased free fatty acid concentration will increase
synthesis of ketone bodies in the liver.

Complications of DM:
- Acute:
o Hypoglycemia – caused by insulin excess
o Ketoacidosis – increase of insulin antagonist hormones, due to
infections or inadequately treatment. Hyperglycemia associated with
sodium and water depletion may lead to hyperpotassemia (due to
acidosis and to deficiency of insulin which normally introduces K, Mg
and phosphorus in the cell) ---Increase of phosphorus and Mg

42
 Diabetes and hypoglycemia

depletion--- Increase of ketone in urine (metabolic acidosis) and

hyperventilation (Kussmaul, to compensate acidosis).
o Hyperosmolar coma – occurs mainly in type II DM. Ketoacidosis is
absent or mild. Water is mostly lost, hypersodemia, high plasma urea
and plasma osmolality.
o Lactic acidosis – related to use of biguanides in type II DM.
- Chronic:
o Retinopathy - refractive retinal arteries
o Nephropathy – proteinuria, microalbuminuria positive
o Neuropathy – diabetic foot, neural and vascular (ischemia) damages
are present
o Cardiovascular – risk for MI, due to ischemia of the blood vessel
(arteries) by atheroms.

Monitoring DM treatment:
- Urinary glucose testing; positive test indicate high glucose levels above 180
mg/dl
- Glucose in serum or capillary blood. Glucose values in capillary blood are
lower than those in serum or plasma. The method is measuring actually the
glucose dissolved in plasma, and if capillary blood is used, part of the volume
is occupied by red cells.
- Glycated hemoglobin (HbA1c) test is not influenced by diet and reflect
glycemic values over the last 2 months. Decrease with 1% may have a benefit
by reducing the risk for cardiovascular events, renal and neurological
complications by 5- 25%.

Hypoglycemia
- Newborns, especially small for gestational age (SGA) newborns
- Insulinoma - Fasting hypoglycemia with coexisting absolute or relative
hyperinsulinemia has become the basis for the diagnosis of insulinoma.
- Carcinoma tends to be associated with the highest percentages of proinsulin,
usually above 50%, and lower values signal a benign tumor.
- Hypopituitarism – LH, FSH, prolactin, TSH are decreased

43
Chapter 4

- Addison’s disease – adrenal insufficiency; test with ACTH administration and

measure cortisol in serum and urine 30 min and 60 min after injection.
- Insulin overdose
- Starvation

URIC ACID is the final oxidation (breakdown) product of purines (adenine, guanine)
metabolism and is excreted in urine.
- About 70% of daily uric acid disposal occurs at the level of kidney and in
impaired renal excretion leads to hyperuricemia.
- Serum accumulation of uric acid can lead to a type of arthritis known as gout:
a classic history of one or more episodes of monoarticular arthritis followed by
intercritical periods. Maximum inflammation evolves within 24 hours. Rapid
resolution of synovitis after colchicine therapy.
- Characteristic in gout:
- Unilateral first metatarsophalangeal joint attack,
- Hyperuricemia,
- Subcortical bone cysts visible on plain X ray radiographic film,
- sterile joint fluid obtained from an affected joint during an attack.

Hyperuricemia can result also from:

- High intake of purine-rich foods: venison (deer, wild pigs)
- High fructose intake
- Impaired excretion by the kidneys

Hypouricemia has been associated with:

- Multiple Sclerosis: patients in relapse averaging ~2.68 mg/dL in comparison
with healthy controls averaging ~4.87 mg/dL.
- Low dietary zinc intakes cause lower uric acid levels. This effect can be even
more pronounced in women taking oral contraceptive medication.
- Sevelamer, a drug indicated for prevention of hyperphosphataemia in patients
with chronic renal failure, can significantly reduce serum uric acid.

44
 Liver and gastrointestinal tract

Exploration of liver diseases

Role of the liver in:

- proteic synthesis
- carbohydrates metabolism
- lipid metabolism
- biliary secretion
- coagulation and fibrinolysis
- inactivation of some hormones
- detoxification (metabolites, drugs)

Morphopathological lesions developing in the liver:

Inflammation, Necrosis, Fibrosis, Steatosis, Cholestasis, Tumoral processes

I. Tests indicating an inflammatory process

Inflammation is a biological manifestation of vascular tissue with plasmocytes
infiltration in the tissue, as response to harmful stimuli, such as pathogens, damaged
cells, or irritants:
- Rubella, CMV, Epstein Barr
- Chemical poisoning – Chloroform, Pyrimidifen
- Hepatitis (A,-B,-C,-D,-E)
- Toxic mushrooms
- Yellow fever
- Gallstones
- Porphyry Cutanea Tarda
- Alcoholic hepatitis
Plasmocytes (B lymphocytes) infiltration in the tissue will produce Ig.
IgM is the first antibody to appear in response to initial exposure to antigen (primary
infection).
IgG is the antibody that confirms the immunity to a certain antigen and increases in
the secondary infection. Therefore is important which antibody is tested, IgM or IgG
in exploring an infectious disease.

45
Chapter 5

In some hepatic diseases are increased Ig:

- Chronic Hepatitis - IgG
- Biliary cirrhosis - IgM
- Alcoholic Hepatopathy - IgA

II. Tests indicating hepatocyte cytolysis

Cytolysis of the liver cell may be the result of:
 osmotic lyses, caused by the bursting or rupturing of cell
membrane when the cell can no longer contain the excessive inflow
of water,
 the degeneration of cell caused by the disruption of cell membrane.
Before cytolysis occurs, by influx of extracellular liquid, some tests may indicate the
increase of cell permeability:
1. ALAT, ASAT- ALAT is more abundant in cytoplasm while 40% of ASAT in the
cell is placed in mitochondrion. Therefore, ALAT will increase first when the
cell membrane expands. Smaller elevations of ALAT may occur in cholestasis.
2. LDH 5
3. OCT (ornithine carbamoyltransferase), SDH (succinate dehydrogenase), ICDH
(isocitrate dehydrogenase) – enzymes that are present in mitochondria, as
specific enzymes of the Krebs cycle.
4. GLDH (Glutamate Dehydrogenase) - present in mitochondria.
Degree of increase depends on:
- Number of involved cells and degree of cell damage
- Vascularization of the damaged tissue
- Existence of an inflammatory barrier
- Half-life time of the enzyme
- Viral acute hepatitis – increases of 10-50x; ALAT especially; transaminases
increase before hyperbilirubinemia occurs.
- Chronic hepatitis - increases in range of 5-20x
- Activation of chronic hepatitis - increase of ASAT, as sign of necrosis; de Rittis
coefficient (ASAT/ALAT=1.3) increases.
- Compensated cirrhosis - stable increases - 2-3x;
- Alcoholic Hepatopathy 5-10 x, especially ASAT
- Liver cirrhosis parenchymal uncompensated – normal values or slight increase

46
 Liver and gastrointestinal tract

- Tumoral processes- slight increase only; more ASAT (necrotic lesions)

- Acute Necrosis (intoxication with fungus, organophosphorics solvents) - high
increase of ASAT 100x. LDH increase is more specific and extensive than
ASAT in these intoxications in comparison with infectious inflammation.

III. Tests evaluating the hepatic synthesis of proteins

1. Che (Cholinesterase)- has a large reference interval for individual values;
decrease of Che reveals a decreased hepatic proteosinthesis
o decreased values may appear in severe anemia, malnutrition,
malabsorption, acute phase reaction, intoxication with
organophosphorics;
o increased values may appear in abdominal type of obesity (metabolic
syndrome), HLP type IIb, IV, V, nephritic syndrome.
2. PT (Prothrombin time) or Quick time (QT) - FVII has a short T 1/2 (6-8 hours). Is
useful in diagnostic of acute liver insufficiency.
3. Albumins are not useful in acute liver insufficiency. In liver cirrhosis with
ascites, parts of albumins pass in ascitic fluid (followed by its decrease in
blood).

IV. Tests indicating cholestasis

1. ALP (Alkaline phosphatase)
2. Serum biliary acids- sensitive test of hepatobiliary disease
3. GGT (Gamma glutamyl transpherase); Elevated serum levels occur in:
alcohol, phenobarbitone, phenytoin, rifampicin administration.
4. 5Nt (5 nucleotidasis) - unlike GGT, it is not affected by enzyme inducing
agents
5. Urinary bilirubin – indicate increase of direct bilirubin, present in cholestasis.

Causes of cholestasis:
 Biliary obstruction
 Hepatocellular damage
 Metabolic syndrome (liver steatosis)

47
Chapter 5

V. Tests indicating liver fibrosis - Fibrotest

FibroTest, known as FibroSure in the US, is a patented biomarker test that uses the
results of six blood serum tests to generate a score that is correlated with the degree
of liver damage in people with a variety of liver diseases.
FibroTest has the same prognostic value as a liver biopsy, considered the gold
standard method in stadialisation of liver fibrosis.
FibroTest has been evaluated in relation to liver biopsy in a large number of patients
with hepatitis C, hepatitis B, alcoholic liver disease, non-alcoholic fatty liver disease
and in the general population.

FibroTest score is calculated from the results of a six-parameter blood test:

1. Alpha-2-macroglobulin,
2. Haptoglobin,
3. Apolipoprotein A1,
4. Gamma-glutamyl transpeptidase (GGT),
5. Total bilirubin,
6. Alanine transaminases (ALT).

FibroTest derivatives:
- ActiTest: diagnostic of necrotic-inflammatory hepatitis;
- SteatoTest: diagnostic for liver steatosis;
- NashTest: diagnostic for NASH (Non-Alcoholic Steato Hepatitis) inflammation;
- AshTest: diagnostic for Alcoholic liver disease inflammation.

The tests are not applicable in 1 to 5% of cases:

- Acute hepatitis- e.g., acute viral hepatitis A, B, C, D, E; or drug-induced hepatitis
- Extrahepatic cholestasis, e.g., pancreatic cancer, gallstones
- Severe hemolysis, e.g. transfusion with incompatible blood group
- Gilbert's syndrome with high unconjugated hyperbilirubinemia
- Acute inflammatory syndrome
In these situations the blood test may be postponed till it is feasible.

48
 Liver and gastrointestinal tract

V. Other specific tests that are modified in hepatic diseases

o Hematological:
1. Target cells (hepatic disease),
2. megalocytes (alcohol)
3. Prothrombin time (PT) and activated partial thromboplastin time (APTT):
PT is testing extrinsic pathway– oral anticoagulant treatment (coumarone)
APTT testing intrinsic pathway (VIII; IX;XI;XII) – intravenous anticoagulant
treatment (heparins, HMWH= High Molecular Weight Heparins)
o Antibody titers:
1. Mithocondrial Ab – primary biliary cirrhosis
2. Antinuclear factor and Smooth muscle Ab- autoimmune disease of liver and
biliary channels
o Antigens and Antibodies for viral hepatitis: HVA Ab (for VHA), and HBs
Ag, HBe Ag, HBe Ab, HBs Ab for VHB, and HVC Ab and viremia for VHC.
 Hepatitis B surface antigens (HBsAg) appear 1 week to 2
months after exposure, and approximately 2 weeks to 2 months
before the onset of symptoms. HBsAg disappears during the
convalescent phase of acute disease and is an indicator of acute
infection or chronic infection with unresolved antigenemia.
 Hepatitis B surface antibodies (HBsAb) appear as the
antigens disappear. The presence of these antibodies indicates
recovery and immunity. After vaccination for VHB, HBs Ab
should be checked after five years, for evaluating the titer that
confers protection.
 Hepatitis Be antigen (HBe Ag) appears before the onset of
clinical disease, after HBsAg, and disappears within
approximately 2 weeks. The presence of the HBe Ag indicates
active viral replication, and most infectivity.
 Antibodies to hepatitis Be antigen (HBe Ab) appear shortly
after HBe Ag disappears. Their presence in patients with acute
hepatitis B suggests that the infection is resolving, or that there
is no complicating liver disease. HBe Ab may be positive in
chronic asymptomatic carriers.

49
Chapter 5

 Hepatitis B core antibodies (HBcAb) appear after the surface

antigens appear. HBcAb persist throughout life and are a marker
for previous infection.

VI. Tests indicating specific disorders affecting the liver

- Wilson disease - serum copper and ceruloplasmin are decreased
- Hemochromatosis - serum iron and ferritin are increased, saturation of
transferrin is >50%.
- Alpha-1 antitrypsin – deficiency of serum alpha-1 antitrypsin
- Alpha fetoprotein – synthesized by fetal liver till about 13 weeks; its synthesis
ceases after birth. Increases in primary liver cancer. Other non-malignant
diseases may produce mild increases: hepatitis, alcoholic cirrhosis, ulcerative
colitis. Increases as well in teratomas of testis or ovary, useful in monitoring
therapy.

Jaundice

Bilirubin results from heme degradation. Heme is a member of a family of

compounds called porphyrins. Many important proteins contain heme as a
prosthetic group.
Heme proteins:
- Hemoglobin (oxygen transport)
- Myoglobin (oxygen transport)
- Cytochromes (electron transport)
- Catalase (H 2 O 2 utilization)

HEMOGLOBIN DEGRADATION AND CONVERSION TO BILIRUBIN AND BILIARY

PIGMENTS
Most of the heme which is degraded comes from hemoglobin (Hb) in red blood
cells, which have a life span of about 120 days. There is thus a turnover of about 6
g/day of hemoglobin. Normally, senescent red blood cells and heme from other
sources are
engulfed by cells of the reticuloendothelial system.

50
 Liver and gastrointestinal tract

Vascular transport of hem after Hb degradation

- Haptoglobin: hemoglobin-haptoglobin complex is metabolized in the liver and
spleen, forming a complex iron-globin that prevents the loss of iron through
urine.
- Hemopexin: binds the free heme. The complex heme - hemopexin is
overtaken by the liver, iron being deposited in the form of ferritin.
- Methemalbumin: oxidized heme - albumin complex. The globin is recycled or
converted into amino acids. Heme is oxidized.

Hb catabolism
1) Conversion of heme to bilirubin takes place in the cells of the reticuloendothelial
system in spleen, liver and bone marrow. Heme ring is cleaved by a microsomal
heme oxygenase resulting biliverdin, which is transformed by biliverdin reductase in
unconjugated bilirubin (indirect).
The high lipid solubility of bilirubin determines its behavior and its further metabolism:
 that it must be transported in the blood by a physiological carrier - albumin
 that it is soluble in the lipid bilayer of cell membranes.
2) Conjugation of bilirubin with glucuronic acid: takes place in the hepatocyte.
This increases its water solubility, decreases its lipid solubility and eases its
excretion. Conjugation is accomplished by attaching two molecules of glucuronic acid
by uridine diphospho glucuronosyl transferase (UDP glucuronyl transferase,
bilirubin conjugation enzyme).
The major product is bilirubin diglucuronide, or conjugated bilirubin (direct
bilirubin). Bilirubin diglucuronide is excreted into the bile. It is subject to subsequent
transformations to other species by the intestinal bacteria.
3) Metabolism of bilirubin diglucuronide by intestinal bacteria.
In normal individuals, intestinal bilirubin is transformed by bacteria to produce the
final porphyrin products found in urine (urobilinogens and urobilins) and in faeces
(stercobilinogen and stercobilin). A small fraction of urobilinogens is reabsorbed into
the blood, extracted by the kidney, and excreted in the urine. Bilirubin and its
catabolic products are collectively known as the bile pigments.

51
Chapter 5

JAUNDICE is a yellowish pigmentation of the skin or of the conjunctive membranes

over the sclera and other mucous membranes caused by hyperbilirubinemia over 3
mg/dl. A hyperbilirubinemia under 2.5 mg/dl may not be associated with jaundice.

Bilirubin increase may be caused by many factors that lead to jaundice classification:
1. Pre-hepatic jaundice
a. Hemolysis – blood transfusion with incompatible blood group (ABO,
Rh), talassemia
2. Hepatocellular jaundice
a. Physiological neonatal jaundice
b. Infection: hepatitis, CMV, mononucleosis
c. Chemicals, drugs: alcohol, acetaminophen
d. Genetic error of bilirubin metabolism: Gilbert syndrome, Crigler-Najjar,
Rotor and Dubin-Johnson disease.
e. Genetic errors of specific proteins: alpha- antitrypsin deficiency,
Wilson’s disease
f. Autoimmune: chronic active hepatitis
3. Post-hepatic jaundice
a. Intrahepatic bile ducts: drugs, primary biliary cirrhosis, cholangitis
b. Extrahepatic bile ducts: gall stones, pancreatic tumors,
cholangiocarcinoma.

HOW TO APPROACH ELUCIDATION OF THE CAUSE OF JAUNDICE?

Jaundice is associated with positive bilirubin in urine?
YES HEPATOCELLULAR DAMAGE
POST-HEPATIC CHOLESTASIS
INTRAHEPATIC CHOLESTASIS
ROTOR/ DUBIN-JOHNSON SYNDROME

NO: PHYSIOLOGICAL JAUNDICE

GILBERT SYNDROME
HEMOLYSIS
GLUCURONYL TRANSFERASE DEFICIENCY

52
 Liver and gastrointestinal tract

Figure 4: Post-hepatic jaundice. Direct bilirubin increases in the blood stream due to an
obstacle at biliary canaliculus, accumulating in the hepatocyte “escaping” through reverse
pathway.

INHERITED DISORDERS OF BILIRUBIN METABOLISM

Are related to deficit of uptake (a), conjugation (b) and secretion (c) of bilirubin.
a. Gilbert syndrome caused by uptake deficiency of indirect bilirubin.
- Increased indirect bilirubin
- Normal hepatic function, including gamma GT
- Bilirubin negative in urine
- Hypocaloric test (400 kcal/24h) may induce jaundice in these patients.
b. Glucuronyl transferase deficiency - rare
- unconjugated bilirubin (indirect) is increased
- bilirubin negative in urine
- appear usually in newborns
- severe forms (type I= total absence), known under name Crigler-Najjar
syndrome are incompatible with life. Deficiency type II (to 10% of
normal) may be treated.
c. Dubin-Johnson and Rotor syndromes - rare
- causes: decreased excretion of direct bilirubin from hepatocyte to biliary
canaliculus.
- conjugated hyperbilirubinemia and bilirubin positive in urine.
- In Dubin-Johnson syndrome is present an accumulation of a black
pigment in the hepatocyte

53
Chapter 5

NEONATAL JAUNDICE
- appears more frequently in premature newborns
- appears in the first 10 days of life, usually in the 4th or 5th day
- causes: enzymatic immaturity of hepatocyte for bilirubin conjugation
- increases of indirect bilirubin are toxic in newborns. The indirect form of
bilirubin is hydrophobic, passes hemato-encefalic barrier and may lead to
nuclear jaundice (kernicterus).
- treatment: phototherapy with UV. In skin indirect bilirubin (insoluble) will be
transformed in hydrosoluble forms of bilirubin – nontoxic, which is eliminated
in urine.
- preventive administration of phenobarbital to mothers presenting early birth
risk. Phenobarbital passes the placenta and induces the synthesis of UDP
glucuronyl transferases.
- unsolved jaundice after 10 days, rises a pathological cause.

Pancreas exploration

Assessment of pancreatic function:

I. Exocrine function - performed by the acinar cells. These cells produce the
following enzymes:
a. Amylase which breaks down starch and glycogen and is used to diagnose
acute pancreatitis; Total amylase represent the sum of pancreatic and salivary
enzymes activity. It is measured in serum and in urine.
Increases in:
- Acute pancreatitis
- Salivary pathology: infections (with urlian virus– parotidites), tumors, calculus
- Neighborhood pathology (penetrating ulcer, mesenteric ischemia, acute
cholecistitis, intestinal occlusion)
- Gynecological pathology (extrauterin pregnancy, ovarian tumor)
- Opiates constrict the pancreatic sphincters, which causes false elevations of
amylases.

54
 Liver and gastrointestinal tract

b. Trypsin
- More specific than amylase because is produced mainly by pancreas
- is a proteolytic enzyme (functions in protein breakdown).

c. Lipase hydrolyzes fats to produce alcohols and fatty acids. Elevated levels are
present in people who have acute pancreatitis:
- usually parallels serum amylase levels, appearing later than amylase after the
onset of acute pancreatitis and remains elevated for 5 to 7 days (longer than
amylase)
- useful marker in more chronic pancreatic diseases (pancreatitis, pancreatic
tumors).
- As amylase is excreted by kidneys, increases in kidney failure, intestinal
infarction/ obstruction
- in nonpancreatic diseases elevations are less than 3 times upper limit of
normal (ULN), in comparison with acute pancreatitis when elevations are 5 to
10 times ULN.

II. Endocrine function is performed by the islets of Langerhans and are composed
of three types of cells:
o (1) α cells produce glucagon, which stimulates the conversion of
glycogen into glucose (glycogenolysis).
o (2) β cells are responsible for making insulin, which functions to
promote glycogenesis and thereby lowers glucose levels.
o (3) δ cells produce gastrin and somatostatin.

III. Functional tests:

- Endocrine:
o OGTT – for diagnostic of DM
- Exocrine:
o Secretin test (measure bicarbonate output)
o Lundth test (measure pancreatic digestive capacity)
o Sweat electrolytes are measured to diagnose cystic fibrosis.
Pilocarpine nitrate is used to stimulate sweating on skin which is
collected on a small disc. Sweat is eluted from the disc and analyzed

55
Chapter 5

for chloride and sodium content. Newborn screening programs and

genetic tests that assess the presence of genetic alterations in a
number of genes related to cystic fibrosis are also available.
o PABA test (oral administration of bentiromide, followed by
measurement of p-aminobenzoic acid in the urine or blood)
o Faecal fat

Acute Pancreatitis
- Amylase increases in the first 6 hours after onset and remain increased about
2 days; after that period of time amylase will be increased in urine only.
- In patients that develop acute kidney insufficiency, hyperamylasemia may be
longer increased.
- Associated with: alcoholism, biliary tract disease
- may be precipitated by: hyperchylomicronaemia (increased triglycerides above
400 mg/dl)
- Biochemical tests: serum amylase (increased 5-10x UNL) and increased
urinary amylase; serum trypsin; serum and urinary lipase
- Other parameters: glycemia (hyperglycemia), hypertriglyceridemia,
hyperbilirubinemia, renal function (urea, creatinine increased)
- Calcium – hypocalcaemia, due to possible mechanisms:
 Acute phase reaction drops albumin with a consequent
reduction of total calcium (ionic form is unchanged)
 Lipase induces hydrolysis of fat and will complexate
calcium in unabsorbable forms (insoluble soaps).

Chronic pancreatitis
Clinical features: severe epigastric pain, weight loss, and steatorrhea
Etiology:
o alcoholism, calcification of pancreas, recurrent acute pancreatitis
o pancreatic cancer
Laboratory investigation:
o increased or normal amylase and lipase values
o microscopic examination of digestion – faeces reveal insufficient digestion of
lipids, fibers, muscular fibers, glycogen

56
 Liver and gastrointestinal tract

o functional exocrine tests: Secretin, Lundth, PABA, OGTT

Gastrointestinal exploration
Functional Gastric exploration

o Basal and stimulated gastric acidity; normally gastric fluid has a pH<3
– low pH (achlorhydria) is indicative for pernicious anemia
– hypersecretion of gastric fluid may be caused by a secreting tumor (e.g.
Zollinger-Ellison syndrome)
– acid secretion in treatment of ulcers to be observed
– verify vagotomy (e.g. in treatment of ulcers)
o Infection with Helicobacter pylori (HPyl) –unease test, Ag HPyl (in faeces), Ab
HPyl (in blood) (IgG, IgM, IgA)

Biliary ducts exploration

Biliary drainage:
o macroscopic aspect (turbid, purulent): Bile A (intestinal), bile B
(vesicular), bile C (hepatic).
o microscopic examination- leucocytes, epithelial cells, cholesterol
crystals, tumoral cells, lamblia (giardia) cysts
Biliculture: in purulent cholecistitis

Intestinal exploration
o Occult Blood Test (Gregersen test) – detect presence of heme. Attention to
diet (blood sausages), drugs, gingival hemorrhages
o in case of digestive hemorrhages - complete hemogramme; may reveal
normochromatic, normocytic anemia
o microscopic examination of digestion – muscular fibers, lipid droplets, starch
granules
o Coproparasitologic examination - parasites, eggs of parasites
o Coproculture – Shigella, Salmonella, E.coli (enterotoxigenic,
enteropathogenic, enterohemoragic).

57
Chapter 5

Malabsorption
Clinical features: Diarrhea, abdominal discomfort, weight loss, nutritional deficiencies:
iron, vitamin D and K, folic acid, vitamin B 12
Causes:
o Maldigestion: pancreatic insufficiency, bile salts deficiency
o Malabsorption:
 Drugs affecting mobility (some antibiotics)
 Alcohol: induces vitamin B 12 / folic acid deficiency
 Cholestyramine (treatment of hyperlipemia) – binds biliary salts
o Malassimilation:
 Celiac disease
 Jejune resection, Small bowel stasis

58
 Iron and heme metabolism

Exploration of iron and heme metabolism

Most circulating iron is derived from the release of iron following RBC destruction.
Iron is contained in the porphyrin ring heme proteins (hemoglobin, myoglobin) and in
some enzymes that contain heme (catalase, cytochromes).

The reversible interaction of iron with oxygen and the ability of iron to function in
electron transfer reactions make iron physiologically important.

Absorption of iron occurs mainly in the in the ferrous form (Fe2+), in duodenum, an
important level where iron availability is controlled (mucosal transferrin/ mucosal
ferritin).
Iron transport involves a transport protein (free iron is toxic) called transferrin,
which binds Fe3+ (ferric form). The bound iron either moves to the mitochondria for
heme synthesis or is stored in cells as ferritin.
Iron storage includes soluble forms (60%) - ferritin (found in most cells) and
insoluble forms (30%) - hemosiderin. One third of iron is stored in the liver, one third
in the bone marrow, and one third in the spleen.
Iron elimination – in urine, in sweat and in faeces by physiological processes as cell
desquamation at intestinal level, biliary excretion or occult hemorrhages. Women (18-
55 yrs) lose 25 mg Fe with 50 ml blood, monthly.

Iron analysis involves the examination of three iron compartments:

o blood cells (hemoglobin),
o stored iron (ferritin),
o circulating iron:
 Serum iron is reflecting iron from transferrin
- is decreased during iron deficiency states, chronic
inflammation, and menstruation; following blood
loss;

59
Chapter 6

- is increased following overload, iron poisoning, and

hepatitis, and during the use of oral contraceptives.
 Iron Binding Capacity (IBC) is a measurement of the maximum
concentration of iron that transferrin can bind. False increases
may appear in treatment with chloramphenicol, fluoride.
 Transferrin levels are analyzed by immunoassays
 Serum ferritin is the most sensitive indicator of iron deficiency.
Ferritin levels decline early in anemia and increase early in
chronic diseases.

IRON DISEASE STATES include:

A. FERIPRIVE ANEMIA AND IRON DEFICIENCY
Etiology:
o caused by blood loss (menstrual cycle, ulcer, tumor)
o inadequate diet (flower and diary based diet)
o deficient absorption: stomach resection, small intestine resection, chronic
diarrhea, malabsorption syndrome; achlorhydria is a risk factor for iron
malabsorption
o genetic deficiency of transferrin: congenital atransferrinemia

Lab exploration:
o decreased ferritin, iron, hemoglobin, reticulocytes
o increased IBC
o Blood smear reveals: normochromatic and hypochromatic, microcytes,
anulocytes (in advanced stages of iron deficiency).

Before decrease of Hb, iron is used for Hb synthesis.

Iron deficiency is influences the activity of hemines:
o mitochondrial alpha-glycerophosphate oxidase: it’s deficiency leads to
increase of lactic acid in muscles (muscular fatigue)
o monoaminoxidase (MAO) is involved in catecholamine catabolism. It’s
deficiency explains irritability (delayed catabolism of stress hormones –
adrenaline, noradrenalin, dopamine)

60
 Iron and heme metabolism

o iron deficiency and exposure to cold: thyroid hormones will not increase
correspondingly, leading to disturbed thermogenesis (permanent sensation
of cold).
o prolonged iron deficiency leads to microcytic anemia associated with atrophy
of digestive mucosa: lingual, pharyngeal, esophageal. Sideropenic
dysphagia refers to the burn sensation associated with difficulty to swallow.
(Plummer-Vinson syndrome). Achlorhydria of gastric mucosa responds well
to the treatment with iron.

Congenital atransferrinemia is a hypochromic anemia, with microcytosis with very

low values of transferrin (0-40 mg/dl in comparison with 200-400 mg/dl in control).
o Is resistant to iron therapy.
o Remedies after perfusions with plasma or purified transferrin.
o Antibodies against transferrin receptors were described.

B. IRON OVERLOAD
Hemosiderosis (no tissue injury, iron in macrophages) – after blood transfusions
Hemochromatosis
 Hereditary hemochromatosis is an AR disease, in which iron is
deposited directly in the liver, heart, and kidney, leading to organ
failure.
- Increased iron and saturation of transferrin
- IBC is very low
- mutation or polymorphism most commonly
associated with hemochromatosis is p.C282Y.
Particularly males are at high risk of developing
hemochromatosis
 Sideroblastic anemia is caused by iron overload in
mitochondria, leading to repression of the translation of δ-
aminolevulinic acid (δ-ALA) synthase.
 Acquired hemochromatosis following acute events of
talassemia or lead poisoning. This disorder also occurs with
chronic excessive absorption of normal iron intake.

61
Chapter 6

 Aceruloplasminemia - is an AD disorder caused by mutations

in the ceruloplasmin gene. Iron accumulates in the pancreas,
liver and brain.

Porphyria

Heme is a member of a family of compounds called porphyrins.

Many important proteins contain heme as a prosthetic group:
- Hemoglobin (oxygen transport)
- Myoglobin (oxygen transport)
- Cytochromes (electron transport)
- Catalase (H 2 O 2 utilization)

HEME SYNTHESIS begins and ends in the mitochondria and takes place partially in
the cytoplasm (Figure 5).

Figure 5: Heme synthesis.

1) Delta-aminolevulinic acid synthase (δ-ALA synthase) - in the mitochondria

The substrates are succinyl-CoA and glycine.

62
 Iron and heme metabolism

The product is delta-aminolevulinic acid (δ ALA).

An essential cofactor is pyridoxal phosphate (vit B-6).
This is the rate-limiting reaction of heme synthesis in all tissues, and it is therefore
tightly regulated.
2) ALA synthase - in the mitochondria
The substrates are two molecules of ALA.
The product is porphobilinogen, the first pyrrole.
It is very susceptible to inhibition by lead.
3) Uroporphyrinogen I synthase and uroporphyrinogen III cosynthase
The substrates are four molecules of porphobilinogen. Production of uroporphyrin III
requires two enzymatic reactions in the cytoplasm.
4) Uroporphyrinogen decarboxylase - in the cytoplasm, catalyses the formation of
coproporphyrinogen III.
5) Coproporphyrinogen III oxidase in the mitochondria, will lead to formation of
protoporphyrinogen IX that is found in mitochondria.
6) Protoporphyrinogen IX oxidase - in the mitochondria
Protoporphyrinogen IX oxidase converts the methylene bridges between the pyrrole
rings to methenyl bridges, leading to protoporphyrin IX.
7) Ferrochelatase - in the mitochondria
Ferrochelatase adds Fe2+ to protoporphyrin IX, forming heme.
• The enzyme requires Fe2+, ascorbic acid and cysteine (as reducing agents).
• Ferrochelatase is inhibited by lead.

Regulation of heme synthesis

o The main sites of porphyrin synthesis are bone marrow cells and liver cells.
o 85% from heme synthesis takes place in the erythroblasts and is finalized in
RBC. In bone marrow synthesis is regulated at the level of enzymes
ferrochelatase and porphobilinogen deaminase.
o In the liver the rate of synthesis is controlled through regulation of the
enzyme δ-aminolevulinic acid (δ-ALA) synthase by the hem (negative
feedback)
o Certain drugs and steroids can increase heme synthesis via increased rate
production of the enzyme ALA synthase (doping in sport).

63
Chapter 6

Summary
Porphyrin is synthesized from four pyrrole rings, which side chains are substituted for
the eight hydrogen atoms.
A variety of substances make up the side chains (A-acetic acid; M-methyl; P-
propionic acid; V-vinyl) (Figure 6).
Names of Porphyrins:
The names of the porphyrins consist of a word and a number, e.g. uroporphyrin III.
The word denotes the kinds of substituents found on the ring, and the number refers
how they are arranged.
There are three important words:
• uroporphyrin contains A and P only
• coproporphyrin contains M and P only (A has been changed to M)
• protoporphyrin contains M and P and V (some P has been changed to V)
There are two important numbered series, I and III. Series II and IV do not occur in
natural systems. In series I the substituents repeat in a regular manner, e.g.
APAPAPAP (starting with ring I). In series III the order of substituents in ring IV is
reversed: APAPAPPA. (Figure 6).

Figure 6: Structure and nomenclature of porphyrins.

64
 Iron and heme metabolism

Which porphyrins can be measured?

Solubility determines routes of excretion, and that depends on the number of
carboxylated groups (-COO-):
o uroporphyrins, 8 carboxylates (more soluble, is excreted in the urine)
o coproporphyrins, 4 carboxylates (is excreted in both urine and faeces)
o protoporphyrins, 2 carboxylates (less soluble, is excreted in the faeces)
o porphobilinogen (PBG) and ALA are precursors of porphyrins and are
typically found in the urine in acute porphyria patients.
o free erythrocyte porphyrins (FEP) are porphyrins that can be extracted from
RBCs (protoporphyrin). FEP are increased in persons with lead poisoning and
iron deficiency anemia.

Qualitative tests include tests for:

(1) Urine porphobilinogen, which is a screening test
(2) Urine porphyrins, which is a screening test for uroporphyrin and coproporphyrin

Quantitative tests include a variety of procedures:

1. Urine tests for presence of porphobilinogen, uroporphyrin, coproporphyrin,
and ALA
2. Blood tests include analysis of ALA dehydratase (an enzyme in the blood that
breaks down ALA) and whole blood or erythrocyte protoporphyrin
3. Fecal tests for coproporphyrin and protoporphyrin

CLASSIFICATION OF PORPHYRIAS:
Based on the signs and symptoms manifested by the patient, neurologic versus
cutaneous:

A. THE NEUROLOGIC PORPHYRIAS include the symptoms of abdominal pain,

psychotic behavior, and neuromuscular difficulties.
All three neurologic porphyries have in common increased urinary ALA and
porphobilinogen levels.
o Acute intermittent porphyria (AIP) (most common) - increased
uroporphyrin.

65
Chapter 6

 caused by porphobilinogen deaminase deficiency, involved in

the conversion of porphobilinogen in uroporphyrinogen III.
 ALA synthase activity is increased because deficiency of
enzyme porphobilinogen deaminase does not succeed to
increase heme production. Result: massive increase of
intermediary products/ porphyrin precursors.
 Clinic: abdominal pain, vomit, constipation, depression with
 neurologic dysfunctions: peripheral neuritis, respiratory paralysis,
death.
 Lab: uroporphyrin that changes in time the color of urine
exposed to light (becomes darker); urinary ALA and PBG
increased. Skin lesions do not occur in AIP because in that
condition only porphyrin precursors accumulate, not porphyrins.
o Variegate porphyria (rare) - increased protoporphyrin and
coproporphyrin.
o Coproporphyria (rare) - increased coproporphyrin levels.

B. THE CUTANEOUS PORPHYRIAS are induced by the presence of excess of

porphyrin precursors in the skin, which generate under light exposure oxygen free
radicals that attack cells and produce photosensitivity and skin lesions.
The three cutaneous porphyries have in common normal urinary ALA and
porphobilinogen levels.
o Congenital erythropoietic porphyria (rare) - increased uroporphyrin and
coproporphyrin.
o Protoporphyria (rare) - increased protoporphyrin and increased free
erythrocyte protoporphyrin levels.
o Porphyria cutanea tarda (most common) appears in adults following liver
disease or excessive alcohol intake. It has a clinical presentation of increased
levels of uroporphyrin.

C. COPROPORPHYRINURIA is a moderate elevation of urine coproporphyrin

secondary to a number of disorders including:
o pregnancy,
o neoplasia, intoxication,

66
 Iron and heme metabolism

o liver disease.
D. PORPHYRINEMIA is a moderate elevation in erythrocyte protoporphyrin
secondary to a number of disorders, including:
o Iron deficiency, caused by poor nutrition, malabsorption, poor iron transport, or
blood loss
o Anemia (hemolytic, iron-deficiency, sideroblastic)
o Lead poisoning

67
Chapter 6

68
 Calcium, magnesium, phosphorus and bone

Exploring metabolic bone diseases

Bone is a metabolic active tissue, every year 9% of trabecular and 2% of cortical

bone being replaced by new bone tissue. Babies are born with about 300 soft bones,
and at adulthood the skeleton has 206 hard bones.

Two physiological processes are occurring in bone tissue:

o Bone modeling – determines the size and shape of the bone, active in all
growing vertebrates.
o Bone remodeling – essential for the maintenance of normal bone
structure. The balance between bone formation and resorption is
accomplished by osteoclasts (involved in bone resorption) and
osteoblats (involved in bone formation).

The bone turnover represents the rate at which the existing bone is replaced by an
equal amount of newly formed bone. Formation and resorption are closely coupled
processes that are in balance in healthy people.
The net result of remodeling varies with the following factors:
o The age – during development bone turnover is in favor of bone formation;
in adulthood bone deposition and resorption are in balance. As the aging
process continues the rate of bone deposition declines.
o Physical stress – heavily stressed bones become thicker and stronger.
Gravity is as well an important physical factor contributing to bone’s
structural maintenance. Lack of gravity for astronauts is a major
impediment for long flights, due to bone loss.
o Hormones levels: growth hormone, thyroid hormones, parathormone, sex
hormones, calcitonin and calcitriol (active vitamin D, considered hormone)
o Vitamins D, K, C, A
o Rates of calcium and phosphate absorption and excretion
o Genetic factors (Marfan syndrome, achondroplasia)
o local factors (cytokines)

69
Chapter 7

o glucocorticoids act as direct inhibitors on the proliferation and function of

osteoblasts

Bone formation markers:

o tP1NP – reflects the matrix proliferation, is released during collagen type I
synthesis.
o Bone Alkaline Phosphatase (BAP)- reflects matrix maturation
o Osteocalcin (OC) – reflects matrix mineralization process

.
Figure 7: Remodeling cycle and bone formation markers.

Bone resorption markers:

o Urinary calcium (historical value) – used to distinguish between a fast and
normal bone loser
o Urinary hydroxyproline (influenced by diet)
o C or N Telopeptide of collagen type I (urine, serum)
o Pyridinoline and deoxypyridinoline (in urine)
o Beta Cross Laps (CTX, crosslinks from C terminal fragment from the triple
helix of collagen type I) – most used
Diagnostic of osteoporosis is put with precision by Dual-Energy X-ray Absorptiometry
(DEXA), but biochemical bone markers should be selected, when a therapeutically
response is followed. A positive therapeutically effect will appear in maximum 3

70
 Calcium, magnesium, phosphorus and bone

months after starting the treatment. DEXA may reveal a positive effect one year after
the treatment.

Metabolic bone diseases:

o Osteoporosis (OP) – the mineral and organic mass are reduced; in early
stages of OP normal calcium, phosphate and TAP may be found.
 Primary OP is postmenopausal
 Secondary OP occurs with:
 Cushing syndrome
 Hyperthyroidism
 Multiple myeloma and bone metastases associated with
increased bone resorption
 Malabsorption
o Osteomalacia/ rickets – the mineral component is reduced
 Vitamin D deficiency
 Malabsorption, dietary deficiency, rare sun exposure
 Vitamin D resistance:
a. impaired production of vitamin D metabolites:
 Chronic renal failure – low 1,25 vit D
 Chronic liver disease – low 25 vit D
 Deficiency of 1-hydroxylation by the kidneys
b. resistance to vitamin D metabolites:
 Phosphaturic rickets, AD inheritance, hypophosphatemia
and high phosphaturia
 Acid-base disturbances – renal tubular acidosis
 Fanconi syndrome - There is a defect in the reabsorption
of glucose, amino acids, phosphate and potassium.
Aminoaciduria associated with rickets, due to failure of the
proximal renal tubules or to dysfunction of the
deamination process.
o Paget disease – known as osteitis deformans, is a chronic disorder that
affects part of the whole of one or many bones. The excessive breakdown and
formation of bone tissue can cause bone to weaken, resulting in bone pain,

71
Chapter 7

arthritis, deformities, and fractures.

 ALP very high, rapidly increasing values may indicate
development of osteogenic sarcoma
 calcium and phosphate are normal.
o Renal osteodistrophy
 ALP increased
o Hyperparathyroidism
 PTH increased, hypocalcaemia, hypophosphatemia,
 Hyperphosphaturia
 Steroids suppression test: in hyperparathyroidism,
hypercalcemia will not suppress when steroids are given for 10
days, 40 mg/day

Calcium, magnesium, phosphorus

90% of calcium, 85% of phosphorus and 60% of magnesium in whole body are
stored in bone.

CALCIUM - 90% in bone, 9% intracellular, 1% blood

o Calcium in bone:
 60-70% bound to hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 –calcium
phosphate,
 Bone matrix is formed by:
 90% collagen (contain hydroxyproline and hydroxylysine –
specific aminoacids of collagen)
 10% noncollagenous proteins (OC and OPG).
o Calcium in cell:
 stored in deposits (sarco/endoplasmic reticulum)
 calcium depletion stops the cell in G0 stage
o Calcium in serum:
 8 - 11 mg/dl ( 2.25-2.75 mmol/l)

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 Calcium, magnesium, phosphorus and bone

 50% Ca 2+ and 50% is protein bound and not diffusible

 the ionized form has important functions (PTH direct stimulation
and role in coagulation, neuromuscular transmission,
maintenance of the cardiac rhythm)
 Ca 2+ > 50% in acidosis and < 50% is ionized in alkalosis
 Total Ca decreases in hypoalbuminemia without decreasing
ionic Ca2+
 Decrease of Ca2+ is followed by tetany and neuromuscular
convulsions. Decrease of Ca2+ stimulates directly PTH
 Ionic calcium (Ca2+ ) may be measured directly (selective
electrodes) or may be calculated by the formula:

Ca 2+ = (6 x Ca total – TP : 3) : (TP + 6)
where TP= total proteins (g/dl); Ca total = total calcium (mg/dl); Ca2+(mg/dl): 4 = Ca2+ (mmol/l)

Bioavailability
Sources – dairy products, egg yolk, nuts
Absorption – fibres, oxalates, free fatty acids in excess (steatorrhea) interfere with
calcium absorption
Requirements:
• 600 -800 mg/day – adult
• 800-1200 mg/day - children
• 1200-1300 mg/day pregnancy, lactation
• 1600-1800 g/day older
Losses: 100 -200 mg/day in faeces and urine

Factors that regulate calcium in serum:

1. PTH:
o in bone stimulates bone resorption and Ca release,
o increases calcium absorption at intestinal level,
o increases calcium reabsorption at kidney proximal contort tubes (PCT).
2. Calcitonin – decreases calcium in serum by increasing calcium clearance rate at
kidney level.

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Chapter 7

3. Vitamin D – increases calcium absorption at intestinal level;

o increases osteoblats activity, increases reabsorption of calcium and
phosphate at kidney level
o D 2 ergocalciferol – of vegetal origin
o D 3 cholecalciferol – of animal origin (milk, egg yolk, liver)
4. Estrogens and androgens inhibit the osteoclasts,
o hyperfunction of adrenal cortex or of the thyroid may induce hypercalcemia
and calcium bone depletion.
5. Carbohydrates increase Ca absorption and lactose increases absorption and
retention of calcium.

PROTEINS DEPENDENT ON CALCIUM METABOLISM

Calcium sensing receptors:
o Calmodulin -binds calcium, involved in inflammation, metabolism, apoptosis
o Calnexin- Ca binding protein in cytoplasm, chaperone molecule, controlling
protein folding
o Gelsolin- actin binding protein
o Neuronal:
 Hippocalcin- neuronal calcium-binding protein
 Neurocalcin- neuronal calcium-binding protein
 Recoverin- calcium-binding protein detected in the
photoreceptors of the eye. It plays a key role in the inhibition of
rhodopsin kinase, a molecule which regulates the
phosphorylation of rhodopsin. This ultimately controls the ability
of the eye to adapt to, and recover from, exposure to the
presence of light.

Calcium binding proteins:

o Coagulation factors – vitamin K dependent (FII, FVII, FIX, FX)
o Calbindin- mediates the transport of calcium across the enterocytes from the
apical side, where entry is regulated by the calcium channel TRPV6
o Matrix Gla Protein (MGP) – a calcification inhibitor that needs vitamin K to
prevent vascular calcification

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 Calcium, magnesium, phosphorus and bone

o Osteocalcin (OC) – produced by osteoblasts, has a role in correct calcium

deposition in bones, binding to hydroxyapatite
o Osteonectin – bone specific protein, binds selectively to hydroxyapatite and
to collagen
o S100 - are involved in regulation of protein phosphorylation, transcription
factors, in calcium homeostasis and in inflammation.
o Troponin C - is a part of the troponin complex, it binds calcium having an
important role in regulation of muscular contraction.

HYPERCALCEMIA
Clinical features:
- decreased neuromuscular excitability
- disturbance of cardiac rhythm
- kidney stones
- articular calcifications
- bone pain
- abdominal pain, nausea and vomiting
- polyuria
- depression 30-40%, anxiety, cognitive dysfunction,
fatigue, insomnia, coma.
Primary hyperparathyroidism
o parathyroidian adenoma - autonomic synthesis of PTH
o in blood: hypercalcemia, hypophosphatemia, increase of PTH
o in urine: increase of phosphates and calcium
Intoxication with vitamin D
o prolonged administration of vitamin D
o clinical features: nausea, vomit, head pain, polydipsia, polyuria,
hypertension
Malignancies of bone
o primary tumors or metastasis – secretion of peptides PTH like
o increased bone turnover – prolonged immobility after bone fractions,
hyperthyroidism

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Chapter 7

o Chondrosarcoma - an X-ray may show an intramedullary osteolytic

lesion with non-uniform calcifications in the proximal tibia.
Diuretics may lead to hypercalcemia
o using an exchange ions resin in dialyzed patients in order to decrease
hyperpotassemia, may lead to hypercalcemia.
Sarcoidosis
o chronic inflammatory syndrome associated with formation of
granulomas (nodules) in different tissues (often located in lung and
lymph nodules).
o Hypercalcemia may be seen in 10% of patients, probable due to
production of calcitriol in macrophages and in the cells of granulomas.
Milkalkali syndrome (Burnett)
o caused by repeated ingestion of calcium and absorbable alkali (such
as calcium carbonate, or milk and sodium bicarbonate).
o untreated may lead to metastatic calcification and renal failure.
Calcinosis cutis
o deposition of calcium in soft tissues (conjunctive and muscular);
o it is associated with inflammation (dermatomyositis) and osteoporosis.

HYPOCALCEMIA
Clinical features:
- increased neuromuscular excitability - fibrillations,
fasciculations, muscular pain.
- positive Chvostek, Trousseau signs, tetany
Hypoparathyroidism
o surgical ablation for thyroid adenoma
o hypocalcaemia, hyperphosphataemia
o hypophosphatemia
Pseudohypoparathyrodism – no response of kidney to PTH
o PTH is normal or increased in serum
o administration of PTH (in pseudohypoparathyroidism do not increase
urinary phosphates)
Hypersecretion of calcitonin

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 Calcium, magnesium, phosphorus and bone

o thyroidal neoplasm
o pheochromocytoma - peptides calcitonin-like
Vitamin D deficiency (rickets, Osteomalacia)
o decreased vitamin D absorption
o decreased calcium in serum
o as response to hypocalcaemia secondary hyperparathyroidism is
triggered, resulting increase of PTH
o increase of PTH leads to increase of bone turnover reflected by
increase of ALP (BAP)
o lab: hypocalcaemia, hypophosphatemia, hyperphosphaturia, increased
PTH and ALP
Vitamin D resistant rickets – alfa-1 hydroxilase deficiency

Chronic kidney insufficiency

o increased urea and organic acids will fixate calcium in an unionisable
form
Acute Pancreatitis
o calcium with fatty acids (resulted from triglycerides) form saponines,
which are complexes from which calcium is not absorbable, leading to
hypocalcaemia
Osteomalcia – see pg 71

Ingestion of laxatives (Mg and P) may lead to hypocalcaemia.

BONE DISEASES THAT DO NOT MODIFY CALCIUM LEVELS IN SERUM

Hypophosphatasia is a rare inherited metabolic disease of decreased tissue

nonspecific alkaline phosphatase. The disease, transmitted AD or AR, comes in one
of five forms: perinatal, infantile, childhood, adult and odontohypophosphatasia.
Common symptoms include bone malformations and higher chance of bone fracture.
Total alkaline phosphatase is decreased.

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Chapter 7

Magnesium

Magnesium is an important element in metabolic processes:

o carbohydrates metabolism; ATP/ ADP production (source of energy)
o proteic synthesis
o nucleic acid synthesis
o muscular contractions
o together with calcium , Na, K is involved in neuromuscular irritability and
blood coagulation.

Distribution of magnesium in human body:

o 60% of total Mg is stored in skeleton,
o 39% of total Mg in skeletal muscle and myocardium,
o 1% of total Mg is present in extracellular environment, of which:
 35% is bound to albumin
 65% is free, in ionized form.

Biological ranges: 1.9 – 2.5 mg/dl ( 0.6 – 1.03 mmol/l)

Sources:
o green vegetables, fish, meat.

Absorption of Mg:
o passively and actively (stimulated by vitamin D)
o its absorption is limited by phosphates
o deficient in:
 diarrhea, steatorrhea
 intestinal surgery
 irradiation enteropathy
 DM.
Elimination:
o In urine, 100 mg daily, as much as absorbed (100 mg).

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 Calcium, magnesium, phosphorus and bone

o normally 95% of Mg filtered in glomeruls is reabsorbed in renal tubules.

When a deficiency of renal function is occurring, Mg increases in the
blood stream.

HYPERMAGNESEMIA
Magnesium is an effective calcium channel blocker. In addition, intracellular
magnesium blocks several cardiac potassium channels.
Clinical features:
- neuromuscular: loss of deep tendon reflexes,
somnolence, muscle paralysis
- cardiovascular: bradycardia and hypotension,
heart block and cardiac arrest may occur at a
plasma magnesium concentration above
18 mg/dl.
- Increased Mg may act as sedative, depressing
cardiac activity, neuromuscular activity.
Causes of hypermagnesiemia:
o Kidney Insufficiency
o diabetic acidosis
o hypothyroidism
o Addison disease
o antacids with Mg
o dehydration
o diuretics – Thiazide

HYPOMAGNESEMIA

In Mg deficiencies – urinary Mg decreases before seric Mg.

Ca and Mg are tightly connected: a magnesium deficiency may lead to calcium
release from bone, responsible of calcifications in aorta or in kidney.

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Chapter 7

Clinical features:
- tiredness, lack of concentration,
- dizziness
- palpitations
- muscular cramps: hyperflexion, Carpopedal spasm
- trembling of extremities
- Chvostek and Trousseau signs positive
- Tachycardia
- patients with these signs are very susceptible to
auditive and visual stimulus

Causes of hypomagnesaemia:
o chronic diarrhea
o after hem dialysis
o chronic kidney disease
o liver cirrhosis
o chronic pancreatitis
o hyperthyroidism and Hypoparathyroidism
o prolonged lactation
o malabsorption
o chronic alcoholism

Mg supplementation is recommended after heart surgery, after MI, or for prevention

of cardiac arrithmias.
Treatment: hypomagnesaemia is more efficient treated with Mg and vit B6, especially
in children.

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 Calcium, magnesium, phosphorus and bone

INTERFERING FACTORS:
False increased Mg:
o prolonged therapy with salicilates, Lithium
o if there are kidney lesions
o blood Hemolysis.: majority of Mg in blood is present in
RBC; Hemolysis leads to false increase values.
False decrease Mg:
o calcium gluconate interfere with the testing methods
o Drugs: amfotericin B, aldosteron, insulin, neomycin,
diuretics with mercury.
o treatment of diabetic coma leads to hypomagnesaemia.
After insulin administration, Mg together with K enters in
the cell.
o Mg deficiency causes inexplicably hypocalcaemia and
hyperpotassemia. Patients may have gastrointestinal and
neurologic signs. In some deficiencies Mg administration
may correct calcium deficiency.

Phosphorus

85% is combined with calcium in bones.

Phosphorus is found in chemical complexes: esters, phosphates.
Phosphorus enters the cell together with glucose.
Role of phosphorus in:
o bone metabolism,
o carbohydrates
o lipid metabolism
o acid-base balance
o energetic electron transphere.
Reference values: adults 0.87 – 1.45 mmol/ l

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Chapter 7

HYPERPHOSPHATEMIA:
o in kidney dysfunction and uremia
o kidney insufficiency
o hypoparathyroidism
o excess of alkaline substances
o excess of vitamin D
o bone tumors
o acromegaly.

HYPOPHOSPHATEMIA
o hyperparathyroidism
o rickets
o diabetic coma
o hyperinsulinemia - excess of insulin
Hypophosphatasia – refers to a bone disease associated with decreased total
alkaline phosphatase.
Diagnostic:
o quantification of urinary inorganic pyrophosphate
(PPi) levels, which are elevated in carriers.
o increased urinary levels of phosphoethanolamine
(PEA) are observed in most patients.

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 Reference values

Reference values for routine lab tests

I. Biochemistry

Serum markers Reference interval Units

ALAT/ TGP 5 - 40 mg/dl
ASAT/ TGO 5 - 37 mg/dl
Calcium – ionic form 1.17 – 1.29 mmol/l
Calcium - total 8.6 - 11 mg/dl
Chloride 97 - 111 mmol/l
Creatinine 0.6 – 1.3 mg/dl
Direct Bilirubin < 0.3 mg/dl
Electrophoresis of seric Albumin: 52 -65 %
proteins Alfa1: 3-5
Alfa 2: 7-9
Beta: 11 - 14
Gamma: 16 - 20
Gamma GT 5 - 50 U/l
Glycemia 70-105 mg/dl
HDL cholesterol M >35 F>45 mg/dl
Iron 50-175 μg/dl
LDL cholesterol 70 - 130 mg/dl
Magnesium 1.7 – 2.5 mg/dl
Potassium 3.5 – 5.1 mmol/l
Sodium/ Natrium 136 - 145 mmol/l
Total alkaline phosphatase 60 - 160 U/l
Total Bilirubin < 1.3 mg/dl
Total Cholesterol 110 - 200 mg/dl
Total proteins 6-8 g/dl
Triglycerides 50 - 150 mg/dl
Urea 20 - 50 mg/dl
Uric acid 2.5 - 7 mg/dl

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Chapter 8

II. Immunology

Reference interval Units

ASLO < 200 U/l
VDRL negative positive/ negative
RPR negative positive/ negative
Rheumatoid factor <8 U/l
C Reactive Protein <6 mg/l
IgA, seric 0.7 – 4 g/l
IgE seric < 100 U/l
IgM seric 0.4 – 2.4 g/l
IgG seric 7 – 16 g/l
Complement C3 0.9 – 2.1 g/l
Complement C4 0.1 – 0.4 g/l
Total Ab H. Pylori negative positive/ negative
HIV1+2 test negative positive/ negative
Anti VHC negative positive/ negative
Anti VHA IgM negative positive/ negative
Cortisol* 08:00 am: 6.7 – 22.6 µg/dl
Estrogens*# Ovulation:12.5 - 498 Pg/ml
free PSA* <1 ng/ml
FSH*# Follicular phase: 3.9 – 8.8 U/l
FT4* 0.61-1.12 Ng/dl
HBs Ag negative positive/ negative
LH*# Ovulation: 2.2 - 103 U/l
Progesterone*# Lutheal phase: 0.3 – 18.6 ng/ml
Prolactin* 3.4 – 26.8 ng/ml
PSA* 0-4 ng/ml
TSH* 0.34 – 5.6 mU/l
* Reference intervals are established on Access 2, Beckman Coulter (SUA) analyzer, based on CLIA
(chemiluminiscence enzyme immunoassay) method.
#
Reference intervals for women; only some intervals are presented for the menstrual cycle; for
menopausal and for men references are not indicated.

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 Reference values

III. Urinary determinations:

Reference interval Units

Complete urinary Urinary summary:
examination Proteins - negative
(summary and Leukocyte - negative
sediment) Nitrates - negative
Glucose - negative
Ketone bodies - negative
Urobilinogens - normal/ negative
Bilirubin - negative
Blood - negative
pH – 4.6 - 8
Density - 1005-1035

Urinary sediment:
- Leucocytes - 1 - 2 leukocyte/field
- RBC - absent
- Epithelial Cells – without atypical of
nucleus
- Squamous, renal, transitional cells
- Casts - absent
- Crystals - oxalate, triphosphate, cysteine
- Yeasts - absent
- Parasites-Trichomonas vaginalis absent
Urinary Glucose 0 - 15 mg/dl

Urinary Proteins 0 - 15 g/dl

24 - 141 mg/ 24h

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Chapter 8

Elements of the urinary sediment:

ERYTHROCYTES LEUKOCYTES CAST

GRANULAR CAST

LEUKOCYTES

YEAST

EPITHELIAL RENAL CELLS CRISTALS:

OXALATES PHOSPHATES CYSTINE

SQUAMOUS CELLS

CHOLESTEROL TYROSINE LEUCINE

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IV. Hematology:

Reference interval Units

Hemoleucogramme (CBC)*
Hemoglobin 12 - 17 g/ l
Hematocrit 35 - 52 %
RBC 4 – 5.10 x1012 /l
LEU 5 - 11 x 109 /l
PLT 150 000 – 400 000 / µl
RET 5 – 25 ‰
LEUKOCYTES %
Lymphocytes 1.2 - 3.4 x 109 /l
Monocytes 0.1-0.6 x 109 /l
Eosinophils 0.0-0.7 x 109 /l
Basophiles 0.0-0.1 x 109 /l
Granulocytes 2.5-8.0 x 109 /l
RBC indices
VEM 76 - 96 fl
HEM 27 - 32 pg
CHEM 30 - 38 g/dl
RDW 11-16 %

Cytology of blood smear - Segmented: 50 - 70 %

Unsegmented: 0-4
Leukocytes formula Lymphocytes: 25 - 40
Monocytes: 2-8
Eosinophils: 1-4
Basophiles: 0-1

ESR M: < 15 mm/h

F: < 20

Prothrombin Time 9 - 13 sec

Prothrombin activity 70 - 120 %
INR 0.80 – 1.20 -

Fibrinogen 200 - 400 mg/dl

APTT 29 - 35 sec
O (I), A(II), B(III),
Blood Group ABO O (I), A(II), B(III), AB(IV) AB(IV)

Blood Group Rh positive/ negative positive/ negative

* Reference values are established on automated hematology analyzer Excell 2280, Drew Scientific

(SUA).

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 V. Blood gases

Reference interval Units

pH 7.35 – 7.45 -

pCO 2 35 – 45 mmHg

Standard bicarbonate 22 – 26 mmol/l

pO 2 65 – 100 mmHg

88