Mini Review

Detection of F2-isoprostanes and F4-neuroprostanes in Clinical Studies

Hsiu-Chuan Yen
Graduate Institute of Medical Biotechnology
Department of Medical Biotechnology and Laboratory Science
Chang Gung University, Taoyuan, Taiwan

Detecting stable products of oxidative damage is the most reliable approach to access oxidative
stress in vivo. F2-isoprostanes (F2-IsoPs) and F4-neuroprostanes (F4-NPs) are the most specific
markers of lipid peroxidation, which is superior to other markers of oxidative damage for clinical
studies in many ways. F2-IsoPs is formed from peroxidation of arachidonic acid that is abundant in
all kind of cells, while F4-NPs is derived from docosahexaenoic acid enriched in neurons. Moreover,
F2-IsoPs is known to exhibit biological activities, such as vasoconstriction and platelet aggregation.
Gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS) is the
reference method and the method with highest sensitivity to quantify F2-IsoPs and F4-NPs. F2-IsoPs
is not only a widely used gold marker of lipid peroxidation detectable in all types of body fluids, but
also a cause of diseases due to its biological activities, a marker to evaluate severity or predict outcome of diseases, or a tool to monitor the effectiveness of antioxidant therapy. Clinical studies detecting F4-NPs are little because cerebrospinal fluid or brain tissues are needed, but it is more useful
than F2-IsoPs in selectively evaluating neuronal oxidative damage. This paper will discuss the above
issues with emphasis on the advantages and considerations in clinical studies.
Key words: Lipid peroxidation, Gas chromatography/negative-ion chemical-ionization mass
spectrometry, Body fluid, Neuron

Introduction
Increased production of reactive species, especially reactive oxygen species and reactive nitrogen species, or
decreased antioxidant capacity can result in the status of
oxidative stress, which has the tendency to cause oxidative damage. Oxidative damage to important macromolecules, DNA, lipid, or protein, may lead to disturbances of normal physiological functions, which plays
an important role in pathogenesis of various human diseases or mechanisms of toxicity induced by xenobiotics.
Many of these basic concepts can be found in the book
of Halliwell and Gutteridge [1]. There is increasing demand for accessing oxidative stress in humans, which
has become an important issue in the field of clinical
laboratory science. However, inappropriate markers and
nonspecific assays were often employed in the literature.

The best way to access oxidative stress in humans is

to detect specific and stable markers of oxidative damage using reliable methods. There have been many
markers of oxidative damage and various methods for
each marker with different advantages and disadvantages
[1]. However, since the discovery of F2-isoprostanes
(F2-IsoPs) as the most specific marker of lipid peroxdation by Jason Morrow and Jackson Roberts at Vanderbilt
University in 1990 in humans [2], F2-IsoPs analyzed by
mass spectrometry has been well recognized as the most
reliable marker of oxidative damag and widely applied
in various clinical studies [3,4]. Moreover, in addition to
F4-neuroprostanes (F4-NPs), various products of lipid
peroxidation related to or similar to formation of F2-IsoPs,
including F3-IsoPs, D/E form of IsoPs and NPs, A/J form
of IsoPs and NPs, isothromboxanes, isofurans and neurofurans, isoketals and neuroketals, F2-dihomo-IsoPs,
and urinary metabolites of 15-F2t-IsoP, are also generated
from different polyunsaturated fatty acids or via different

Received: March 9, 2010

Address for correspondence: Hsiu-Chuan Yen, Ph.D., Department of Medical Biotechnology and Laboratory Science,
Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan 333, Taiwan.
Tel: +886-3-2118800 ext. 5207, Fax: +886-3-2118692, Email: yen@mail.cgu.edu.tw

J Biomed Lab Sci 2010 Vol 22 No 1

Dectection of F2-isoprostanes and F4-neuroprostanes

mechanisms with different significance [5,6]. This review paper will only focus on F2-IsoPs and F4-NPs because they are more important in clinical studies. Clinical utilities of F2-IsoPs and F4-NPs will be discussed by
including our work on the studies of aneurysmal subarachnoid hemorrhage (aSAH) and traumatic brain injury (TBI) in humans [7-9].

Formation and Nomenclature of F2-IsoPs

and F4-NPs
F2-IsoPs is a group of prostaglandin (PG)F2-like compounds that are generated from arachidonic acid (AA,
C20:4 -6), an abundant polyunsaturated fatty acid present in all kinds of cell membranes, via free radicalcatalyzed lipid peroxidation. It was proved to be independent of action of cyclooxygenase (COX) because
human subjects receiving COX inhibitors did not have
lower levels of these compounds in body fluids. F2-IsoPs
was named because it has F-type prostane rings similar
to PGF2 [2,10,11]. At beginning, it was in fact an accidental discovery of Morrow et al. when plasma samples
stored at -20 for several months were subjected to
routine analysis by gas chromatography/negative-ion
chemical-ionization mass spectrometry (GC/NICI-MS)
for 9,11-PGF2, a metabolite of PGD2. Unknown
PGF2-like compounds, identified as peaks adjacent to
that of 9,11-PGF2 of GC chromatograms, were found
in those plasma samples at the levels that were approximately 50-fold higher than that in fresh plasma. Freezethaw cycles of plasma also increased the levels of those
compounds [10].
F2-IsoPs is initially generated on phospholipids on
tissues or lipoproteins in plasma as esterified form from
esterified fatty acids and can be released into surrounding body fluids or circulation as free form mediated by
phospholipase A2-like activities. Detection of F2-IsoPs in
body fluids therefore can reflect the levels of lipid peroxidation in tissues [11,12]. Stafforini et al. identified
that intracellular and plasma platelet-activating factor
acetylhydrolases (PAH-AH) could be responsible for the
hydrolysis of F2-IsoPs from phospholipids [13]. Moreover, there are four regioisomers of F2-IsoPs, which are
denoted as 5-, 12-, 8, and 15-series regioisomers based
on the number of carbon atom of side chains on which
the hydroxyl group is localized. Quantity of 5- and
15-series regioisomers is much higher than that of other
two regioisomers both in vitro and in vivo. Although
each regioisomer theoretically consists of eight racemic
diastereomers, 15-F2t-IsoP is the most abundant isomer
2

of F2-IsoPs [11,14]. On the other hand, enantiomer of

PGF2, ent-PGF2, could be generated from IsoP pathway and might account for most of PGF2 found in urine,
which was thought to be exclusively from the COX pathway. It should be noted that PGF2 and ent-PGF2 could
be differentiated under the analysis of special liquid
chromatography/mass spectrometry (LC/MS), but not
GC/NICI-MS [15].
The terminology for F2-IsoPs or 15-F2t-IsoP has
been confusing especially in the early years after the
discovery of F2-IsoPs. It was not more unified until the
nomenclature system of Taber et al. for IsoPs was approved by the Eicosanoid Nomenclature Committee,
which was also sanctioned by the International Union of
Pure and Applied Chemistry (IUPAC) [3,16]. This nomenclature system named four major regioisomers as 5-,
12-, 8, and 15-series regioisomers, which corresponded
to I-IV regioisomers initially categorized by Morrow et
al. [2] and provided principles to name different isomers.
However, many publications or companies still used
different old terms of 15-F2t-IsoP, such as 8-iso-PGF2
and 8-epi-PGF2, without updates in this aspect. The
chemical structure of 15-F2t-IsoP is almost identical to
that of PGF2 except the stereochemistry as shown by
Figure 1. Based on the system of Taber et al., 15-F2t-IsoP
should be pronounced as 15-F-2-transe-isoprostane
and it was designated in this way because two side
chains are oriented trans in respect to hydroxyl groups
on the cyclopentane ring [16]. On the other hand, the
group of FitzGerald has developed a different nomenclature system and kept using this system is their publications. That system was developed by Rokach et al and
named four regioisomers as types VI, V, IV, and III regioisomers [17], which corresponded to 5-, 12-, 8, and
15-series regioisomers of Taber et al. [16]. Moreover,
iPF2-III denotes 15-F2t-IsoP in the system of Rokach et
al. [17].
In 1998, Roberts et al. further proved the presence
of F4-NPs, which was derived from free radical-mediated
lipid peroxidation of docosahexaenoic acid (DHA, C22:6
-3), both in vitro and in vivo with the same principle as
the formation of F2-IsoPs from AA. Eight different regioisomers for F4-NPs, 4-, 7-, 10-, 11-, 13-, 14-, 17- and
20-series, were predicted [18]. In this paper, the authors
showed that F4-NPs was generated in a greater amount
than F2-IsoPs from equal amounts of AA and DHA, respectively. In addition, esterified F4-NPs was barely detectable in 1 ml of human plasma, while free F4-NPs in
normal human cerebrospinal fluid (CSF) could be detected and the levels were elevated in that of patients
with Alzheimers disease (AD). The nomenclature sysJ Biomed Lab Sci 2010 Vol 22 No 1

Fig. 1.

Chemical structure of PGF2, 15-F2t-IsoP, and the deuterium (D)-labeled 15-F2t-IsoP, [2H4]-15-F2t-IsoP.

tem for NPs has also been proposed by Taber and Roberts [19]. Furthermore, by using a unique LC/MS approach, Yin et al. indicated that 4- and 20-series NPs
were the major regioisomers among eight possible regioisomers of NPs both in vitro and in rat brain [20].
Because DHA is present in all kinds of neural cells
in the brain but most enriched in neurons, F4-NPs is
therefore considered as a more selective indicator for
oxidative damage to neurons or gray matter and is more
useful in studying neurodegenerative diseases [21,22].
As proposed by Roberts et al., phosphatidylserine with
esterified F4-NPs would be a very distorted molecules
and could significantly affect neuronal dysfunction [18].
As shown by many conditions of brain injury in animal
experiments, the extent in the elevation of F4-NPs was
greater than that of F2-IsoPs [23,24].

Analysis of F2-IsoPs and F4-NPs and Specimen Considerations

F2-IsoPs has been quantified by enzyme immunoassay
(EIA), gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS), and LC/MS or
liquid chromatography/tandem mass spectrometry
(LC/MS/MS), while F4-NPs has only be quantified by
GC/MS [4,18]. Among them, GC/NICI-MS is the most
sensitive method that is most suitable for routine quantification of F2-IsoPs and F4-NPs in human body fluids,
which is especially important for clinical studies using
J Biomed Lab Sci 2010 Vol 22 No 1

specimen with limited amount and low concentrations.

GC/NICI-MS is also the reference method for analyzing
F2-IsoPs that has been validated for human plasma and
urine by the groups of Morrow and Roberts [22,25,26].
NICI mode is a very uncommon technique that is hard to
manage. It is different from the commonly known electron impact ionization (EI) mode. In NICI mode, the
analyte derivatized with electron withdrawing groups
captures low-energy electron, which is produced by the
reaction of high-energy electrons and methane gas, and
becomes an unfragmented negative ion, which therefore
has higher abundance and sensitivity compared with the
fragmented target ion under EI mode. Although there are
commercial EIA kits available using polyclonal antibodies against 15-F2t-IsoP, EIA is an unreliable method with
low specificity, accuracy, and precision [25,27,28]. One
major problem of EIA is that the antibody is not specific
because there are numerous isomers in PGs metabolism
and IsoP pathways with similar structure to 15-F2t-IsoP.
The specificity tested by the manufacturers of the kits
was impossible to be sufficient. If SPE procedures are
performed to remove some of other compounds, the recovery cannot be normalized unless radioactive [3H]15-F2t-IsoP is used as the internal standard, which would
not be practical for clinical studies or regular laboratories [27]. Based on our previous experiences, EIA was
also prone to be interfered by impurities in reagents used
[29]. On the other hand, LC/MS method is superior to
GC/NICI-MS method in identifying different regioisomers and diastereomers of F2-IsoPs and F4-NPs [20,30].
3

Dectection of F2-isoprostanes and F4-neuroprostanes

However, because of its lower sensitivity compared with

GC/NICI-MS, LC/MS or LC/MS/MS can only be used
to reliably detect free form of F2-IsoPs levels in urine, in
which levels of F2-IsoPs are about 10 fold or higher than
that in plasma or CSF. Although few papers have reported low detection limit for plasma levels of F2-IsoPs
detected by LC/MS/MS, these studies in fact measured
total F2-IsoPs, the sum of free and esterified F2-IsoPs, in
plasma, not free form alone [31,32].
F2-IsoPs and F4-NPs can be analyzed as free form in
body fluids or esterified form on tissues or in plasma
depending on the specimen available or the questions to
be addressed. As stated before for the discovery of
F2-IsoPs, samples for F2-IsoPs and F4-NPs have to be
stored at -80, not -20, and cannot be subjected to
freeze-thaw cycles [10]. The fact that esterified form is
more susceptible to artifact caused by ex vivo oxidation
should be carefully deliberated. Tissues samples or
plasma samples for analyzing esterified form have to be
immediately frozen by dry ice or liquid nitrogen and
stored at -80 upon collection. Plasma for measuring
esterified form of F2-IsoPs requires the addition of antioxidants into plasma before freezing, which was not
required for detection of free form. Levels of free form
was not altered even plasma was left at room temperature for 2 hours. It was also unchanged in urine when
incubated at 37 for one week [2,11]. The detection of
esterified form is therefore more problematic than free
form in plasma for clinical studies. Another point should
be considered is that esterified F2-IsoPs in plasma should
only stand for lipid peroxidation in the blood system, not
whole body, while free form stands for whole-body levels or systemic levels of lipid peroxidation [3]. Furthermore, serum should be avoided because small amount of
F2-IsoPs could be released from platelet via COX pathway during platelet activation in vitro and levels of
F2-IsoPs in serum could be suppressed by administration
of aspirin in human subjects [33] even though it was not
a concern for the levels in plasma and urine in vivo
[4,11,34]. On the other hand, F2-IsoPs is detectable in all
kinds of body fluids so far investigated in the literature
by GC/NICI-MS method [11,35]. If possible, the body
fluids surrounds the tissues to be investigated should be
obtained to closely correlate status of oxidative damage
and tissue abnormalities, such as cerebrospinal fluid
(CSF) for brain dysfunction, bronchoalveolar lavage
fluid for pulmonary diseases, and synovial fluid for joint
problems. Although urine samples are often used because they are easy to be collected and have very high
concentrations of F2-IsoPs, urinary levels of F2-IsoPs
may not represent systemic levels because it can be
4

greatly affected by the kidney. Instead, major urinary

metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2tIsoP formed by the metabolism in the lung, should be
measured to represent systemic levels of oxidative damage
[13,11]. However, the internal standard for GC/NICI-MS
analysis of this metabolite is not commercially available
and therefore cannot be widely applied.
Detailed procedures for analyzing free and esterified
form of F2-IsoPs in body fluids or tissues by GC/NICIMS method have been described in details [25,26,36]. It
requires sophisticated sample processing and maintenance of GC/NICI-MS instrument. The working flow for
free form of F2-IsoPs consists of the following steps: the
mixing of a stable isotope-labeled internal standard,
[2H4]-15-F2t-IsoP, and samples; adjustment of pH to 3.0
to enrich nonionized form of F2-IsoPs; two steps of solid
phase extract (SPE) using C18 and silica SPE columns;
first derivatization to form pentafluorobenzyl esters for
later formation of negative ion in GC/MS; thin-layer
chromatography (TLC) purification; second derivatization to form trimethylsilyl (TMS) derivative to increase
volatility and prevent hydroxyl groups on F2-IsoPs from
reacting with glass components and columns; and injection of final analytes dissolved in undecane into GC/MS
to be analyzed under NICI mode. The structure of internal standard, [2H4]-15-F2t-IsoP, is shown in Figure 1.
Four hydrogen atoms on two carbons are replaced by
four deuterium atoms. The use of C18 and silica SPE
columns employed the principles of reverse phase and
normal phase chromatography to remove the majority of
unwanted compounds with the polarity very different
from F2-IsoPs. Moreover, TLC purification that recovers
a portion of compounds from TLC plates not only to
removes excessive reagents after first derivatization, but
also further narrows down the number of compounds,
among those compounds with similar chemical properties from eicosanoids metabolism, entering GC columns
with F2-IsoPs after SPE steps. Examples of GC chromatograms for analysis of F2-IsoPs in normal CSF, plasma,
and urine samples are illustrated by Figure 2. It should
be noted that the peak at m/z 569.4 (peak a) with the
same retention time of [2H4]-15-F2t-IsoP peak (peak c) is
used for quantification, but it includes other isomers of
F2-IsoPs, so total F2-IsoPs are measured by GC/NICIMS method [26]. Moreover, there are much more unwanted compounds in urine than CSF or plasma samples,
which often cause more problems to the maintenance of
GC/MS. On the other hand, as mentioned above, the
peak of PGF2 (peak b) might contain ent-PGF2 especially in urine [15].
Procedures for analyzing free form of F4-NPs were
J Biomed Lab Sci 2010 Vol 22 No 1

Fig. 2. GC chromatograms for F2-IsoPs analysis of a CSF (A), plasma (B), and urine sample from normal subjects. Peaks a, b, and c represents peaks of F2-IsoPs, PGF2, and [2H4]-15-F2t-IsoP, respectively. For this figure,
approximately 100 pg of [2H4]-15-F2t-IsoP was mixed with 0.5 ml of CSF (A) or plasma (B), while 500 pg of
[2H4]-15-F2t-IsoP was mixed with 0.2 ml of urine (C) before SPE steps. The concentrations of F2-IsoPs were 12
pg/ml, 30 pg/ml, and 1308 pg/ml in CSF (A), plasma (B), and urine (C), respectively. It was calculated by multiplying the exact amount of added [2H4]-15-F2t-IsoP with the ratio of peak a to peak b in peak height, which was then
divided by the volume of samples used.

first briefly indicated when first discovered by Roberts et

al. [18] and further described in details by Arneson and
Roberts [22]. The principles are almost the same except
that the scraping range on TLC plates for F4-NPs is
longer than that for F2-IsoPs and the wash solution for
silica SPE column is slightly different. However, as discussed in our recent paper, it was confusing that the cutting range for TLC was wider in the paper of Arneson
and Roberts [22] than that in Roberts et al. [18], but the
TLC range indicated by Roberts et al. should be followed. Moreover, unlike F2-IsoPs, F4-NPs are present as
multiple peaks over a range of retention time, the quantification requires the integration of peak area for multiple
peaks and is much more complicated. We have refined
the analysis of F4-NPs by analyzing oxidized DHA with
unknown samples for each run of analysis in order to
define the range of peaks to be integrated in samples [8].
As to internal standard used for F4-NPs analysis, although [2H4]-15-F2t-IsoP has been used, previously there
was a trend to use [18O2]-17-F4c-NP [37], which could
only be obtained from Jason Morrow, as an internal
standard. However, we addressed several problems with
this internal standard, especially the interference by the
presence of F2-dihomo-IsoPs in human CSF, and indicated that [2H4]-15-F2t-IsoP should be used [8]. GC
chromatograms for analysis of F4-NPs in a normal human CSF sample are demonstrated by Figure 3.
Esterified F2-IsoPs is abundant in all kinds of tissues, while esterified F4-NPs is usually conducted only
J Biomed Lab Sci 2010 Vol 22 No 1

for brain tissues. To analyze esterified form of F2-IsoPs

and F4-NPs, they need to be first converted to free form
after total lipids are extracted [22,25,26]. First, tissues
are homogenized in Folch solution containing antioxidant and reducing agent and extracted to obtain total
lipids. Second, after adding NaCl solution, the analyte in
lower organic layer is recovered. Third, KOH solution is
added to release esterified IsoPs or NPs on phospholipids
into free form by alkaline hydrolysis reaction after resuspension by methanol. Finally, the solution is neutralized
and diluted with water (pH 3.0). The same procedures
for free form described above are then proceeded.

Advantages of F2-IsoPs and F4-NPs as Markers of Oxidative Damage in Clinical Studies

As reviewed by Roberts and Morrow, there are six major
advantages of using F2-IsoPs as a marker to access oxidative damage in vivo [3]. First, F2-IsoPs is a very stable
compound. Second, F2-IsoPs is the most specific marker
of lipid peroxidation in vivo compared with other markers of lipid peroxidation, such as malondialdehyde (MDA)
or lipid hydroperoxide. Third, F2-IsoPs is readily detectable in all kinds of biological tissues and body fluids by
the GC/NICI-MS methods, which allows the establishment of normal ranges and is valuable for clinical studies under various conditions. Fourth, it has been well
demonstrated that F2-IsoPs levels were elevated in vari5

Dectection of F2-isoprostanes and F4-neuroprostanes

Fig. 3. GC chromatograms for F4-NPs analysis of a normal human CSF sample. The area above the dashed line
and under the curve at m/z 593.5 was integrated for F4-NPs. The range of integration was defined by peaks of
DHA oxidized in vitro. The * symbol denotes the peak of [2H4]-15-F2t-IsoP at m/z 573.4. About 200 pg of
[2H4]-15-F2t-IsoP was added to 1 ml of CSF before SPE steps and the concentration of F4-NPs was 33 pg/ml relative to [2H4]-15-F2t-IsoP in this figure based on the ratio of peak area of F4-NPs to that of [2H4]-15-F2t-IsoP.

ous animal models of oxidative stress. Fifth, F2-IsoPs

levels are modulated by status of the antioxidant system.
Finally, levels of F2-IsoPs are not influenced by diet. In
contrast to that, levels of MDA in body fluids can be
greatly affected by the lipid content of diet [1].
Because free F2-IsoPs in body fluids can represent
steady-state levels of oxidative damage in whole body or
specific organs, the detection of free F2-IsoPs in different
body fluids is useful in relating levels of oxidative damage and pathological changes or outcome of diseases in
clinical studies, providing mechanistic explanations for
the roles of oxidative stress in diseases. It is an additional favorable feature that cannot be compared by
measuring protein oxidation products in plasma or DNA
oxidation products in white blood cells in the assessment
of oxidative stress if the source of oxidative stress is not
within the blood system. On the other hand, none of
other markers of oxidative damage measured in CSF or
brain tissue can differentiate major sources of oxidative
damage in the brain. When measuring both F2-IsoPs and
F4-NPs in CSF, like what has been done in our studies
for aSAH and TBI in humans [7-9], the increase in
F2-IsoPs levels is supposed to represent lipid peroxida6

tion of any cells in the brain, including different neural

cells, inflammatory cells, and vascular cells, but elevation of F4-NPs levels should better reflect neuronal
damage or at least undoubtedly indicate oxidative damage to DHA in the brain tissue, but not in the vascular
compartments of the brain.

Biological Activities of F2-IsoPs

Different isomers of F2-IsoPs and E2-IsoPs have been
shown to exert various biological activities in vitro and
in vivo, which may contribute to pathogenesis of diseases
[38-40]. Because 15-F2t-IsoP is the major isomer of
F2-IsoPs, most studies have focused on studying the
biological activities of this isomer, but other isomers of
F2-IsoPs have also been shown to exert biological activities. The most well known activities of 15-F2t-IsoP are
vasoconstriction on various vascular beds and platelet
aggregation [40]. Many studies indicate that these activities are mediated through the interaction of 15-F2t-IsoP
with thromboxane A2 (TXA2) receptors, but other studies
have argued for the existence of distinct IsoP recepJ Biomed Lab Sci 2010 Vol 22 No 1

tors, which remains to be identified [38,41]. Hou et al.

tested the biological activities of isomers of F2-IsoPs
other than 15-F2t-IsoP using synthetic compounds. They
demonstrated that several isomers of 15-series and
12-series F2-IsoPs were also strong vasoconstrictors on
retinal and cerebral microvasculature by inducing endothelium-dependent synthesis of TXA2 [42], which was
also found for 15-F2t-IsoP and 2,3-dinor-5,6-dihydro-15
-F2t-IsoP [38]. The vasoconstrictive effect of 15-F2t-IsoP
could cause reduced glomerular filtration rate and renal
blood flow in the kidney. It also increased pulmonary
arterial pressure in the lung and induced bronchoconstriction of airway smooth muscle [38]. In addition, the
cardiovascular effects of 15-F2t-IsoP have also been
shown [43,44]. Belik et al. also discussed the possible
roles of F2-IsoPs on the control of umbilical vasculature
and adverse effects to the fetus [40].
F2-IsoPs also exhibits other important biological activities. 15-F2t-IsoP could stimulate production of 1,4,5triphosphate and DNA synthesis in rat aortic smooth
muscle cells [41] and induced cell proliferation, DNA
synthesis, and expression of endothelin-1 in aortic endothelial cells [45]. These mitogenic effects might play a
role in pathological processes of vascular systems, such
as the development of atherosclerosis. On the other
hand, Comporti et al. showed that 15-F2t-IsoP stimulated
collagen synthesis, DNA synthesis, and cell proliferation
in hepatic stellate cells from normal liver; and increased
production of transforming growth factor-1 in a
promonocyte cell line. These effects might be related to
the pathogenesis of liver fibrosis [46]. Furthermore,
Benndorf et al. demonstrated that several isoprostanes
could inhibit vascular endothelial growth factor-induced
angiogensis in vivo, which might be linked to coronary
heart disease and capillary rarefaction in diseases with
increased oxidative stress [47].

F2-IsoPs and F4-NPs in Clinical Studies

The use of F2-IsoPs as a marker of oxidative damage has
been applied in various clinical studies and elevation of
F2-IsoPs has also been found in different human conditions. Effect of antioxidant intervention on levels of
F2-IsoPs has also been investigated in many reports.
Those findings have been discussed in several excellent
review papers [3,4,21,35,40,48-54]. F2-IsoPs was most
well accepted to be increased or play an important role
in the pathogenesis of diseases in the following categories
of human diseases: pulmonary diseases, neurodegenerative diseases, cardiovascular diseases, type 2 diabetes,
J Biomed Lab Sci 2010 Vol 22 No 1

inflammatory diseases, hypercholesterolemia, and hepatic diseases. Moreover, augmentation of F2-IsoPs was
also often found in human subjects exposed to cigarette
smoke and consuming alcohol. This paper will not again
elaborate on those overwhelming examples about the
association between F2-IsoPs and human diseases, but
will only focus on specific issues and certain special
cases published in recent years or not included in the
above mentioned review papers. Few studies using
F4-NPs in humans will also be summarized. On the other
hand, because EIA is generally not considered as a reliable method for detecting F2-IsoPs, only studies using
mass spectrometric techniques will be included for the
discussion.
Because F2-IsoPs has been proven to be a specific
marker of oxidative damage, detection of F2-IsoPs can
provide solid evidences and new significance that oxidative damage is indeed enhanced in the diseased conditions investigated, which either have never been proved
before or are not well accepted due to the use of unreliable markers or methods in the literature. Moreover,
F2-IsoPs or F4-NPs might be used as a novel biochemical
marker to monitor severity or outcome of diseases based
on the correlation between levels of these markers with
clinical parameters. The study of Canter et al. first demonstrated that plasma levels of free F2-IsoPs correlated
with degree of heteroplasmy of the pathogenic mitochondrial DNA mutation, mtA8344G mutation causing
inherited myoclonic epilepsy and ragged red fibers, in a
large family [55]. Matayatsuk et al. first used F2-IsoPs to
prove that oxidative stress was increased in patients with
-thalassemia by showing that plasma levels of total
F2-IsoPs and urinary levels of free F2-IsoPs, but not levels of erythrocyte MDA measured as thiobarbituric
acid-reactive substances, were increased in thalassemic
patients [56]. Results of de Leon et al. indicated that
CSF levels of F2-IsoPs might be useful in monitoring the
course of AD and its early detection because F2-IsoPs
improved the diagnostic and predictive outcomes of
clinical measures, including quantitative magnetic resonance imaging [57]. Kelly et al. first provided the evidence for the increase of free F2-IsoPs levels in plasma
during acute ischemic stroke [58]. Moreover, Seet et al.
first found that total F2-IsoPs levels in plasma, normalized by AA concentrations, were higher during febrile
stage than that convalescent stage in patients with dengue fever [59]. The report of De Felice et al. first demonstrated that plasma levels of F2-IsoPs were increased
in patients with Rett syndrome, a neurodevelopmental
disorder caused by mutations in X chromosome, and
correlated with greater phenotype severity [60]. Mon7

Dectection of F2-isoprostanes and F4-neuroprostanes

neret et al. also found that urinary levels of F2-IsoPs

were increased in patients with severe obstructive sleep
apnea and correlated with carotid intima-media thickness
and intermittent hypoxia in nonobese patients [61].
Results from our laboratory first showed that
F2-IsoPs and F4-NPs in CSF and plasma levels of F2-IsoPs
were increased in patients following the onset of aSAH,
a common nontraumatic hemorrhagic stroke [7,8], and in
patients with TBI [9] by using GC/NICI-MS method.
However, in the study of aSAH, only CSF levels of
F2-IsoPs and F4-NPs, but not plasma F2-IsoPs levels,
correlated with degree of hemorrhage before surgery and
poor outcome three months after surgery. Moreover,
levels of F4-NPs, but not F2-IsoPs, in CSF at early time
points could predict 3-month outcome. The results not
only demonstrated the increase of oxidative damage in
patients, but also provided mechanistic explanations that
hemoglobin could be the source of oxidative stress and
neuronal oxidative damage might be the cause of poor
outcome. These findings also addressed the issue that
body fluids adjacent to the affected organs should be
used to get better correlation with clinical parameters
since any alterations in plasma would be diluted by systemic levels. It might be especially important for neurological disorders. As shown by studies for AD, only
F2-IsoPs levels in CSF, but not plasma or urine, were
increased in different stages of AD patients and correlated with clinical parameters [18,52,57]. On the other
hand, our study on aSAH in fact was the only publication detecting F4-NPs in human CSF [8] in the literature
besides that for AD in the original paper discovering
F4-NPs in humans [18], while our another study for TBI
is still ongoing [9]. As to detection of F4-NPs in human
brain tissues, it was also only conducted for AD and was
found to be elevated in AD patients in certain regions of
brain [37,62].
As listed in the review paper of Basu, effects of different antioxidant interventions on levels of F2-IsoPs
have been investigated in healthy or stressed subjects in
many studies [4]. Antioxidant supplementation generally
did not affect status of F2-IsoPs in healthy subjects, but
was found to be more effective in lowering F2-IsoPs levels in patients with type 2 diabetes and hypercholesterolemia. It also could decrease F2-IsoPs levels in
smokers and ultramarathon runners. From these studies,
it was interesting to note that some antioxidants were
more effective than the other for different stress conditions. For examples, different tocopherols, but not vitamin C or coenzyme Q10, could decrease F2-IsoPs levels
in patients with type 2 diabetes. Therefore, F2-IsoPs can
serve as a useful marker to monitor whether oxidative
8

damage has indeed suppressed by antioxidant supplementation. However, the effect of antioxidants on
F4-NPs remains to be investigated and more studies are
needed to address whether suppression of oxidative
damage correlated with the improvement of clinical indices or outcome.

Conclusion
Although F2-IsoPs is difficult to be measured, the usefulness of this marker for clinical studies has been
documented by numerous publications worldwide.
GC/NICI-MS is still the most sensitive and reliable
method to routinely quantify large amount of clinical
samples in all kinds of body fluids, allowing more
meaningful and closer investigation on oxidative stress
in affected organs. Detection F2-IsoPs and F4-NPs has
offered a cutting-edge tool to reliably assess oxidative
stress in vivo with several distinct advantages for clinical
studies, while F4-NPs is a powerful marker to investigate
neurological disorders. It can be expected that important
roles of oxidative stress in human diseases will be further explored in more human conditions by using these
two markers in the future.

Acknowledgements
This work was supported by grants NSC91-2314-B-182072, NSC96-2320-B-182-018, and NSC97-2320-B-182012-MY3 from National Science Council, Taiwan. The
establishment of techniques for analyzing F2-IsoPs and
F4-NPs by GC/NICI-MS in the authors laboratory was
assisted by Dr. Jason Morrow at Vanderbilt University.
This paper was also written in memory of Dr. Morrow.

References
1.

2.

3.

4.

Halliwell B, Gutteridge MC. Free Radicals in Biology and

Medicine. 4th ed. New York: Oxford University Press,
2007.
Morrow JD, Hill KE, Burk RF, et al. A series of prostaglandin F2-like compounds are produced in vivo in
humans by a non-cyclooxygenase, free radical-catalyzed
mechanism. Proc Natl Acad Sci U S A 1990; 87: 9383-7.
Roberts LJ, Morrow JD. Measurement of F2-isoprostanes
as an index of oxidative stress in vivo. Free Radic Biol
Med 2000; 28: 505-13.
Basu S. F2-isoprostanes in human health and diseases:
from molecular mechanisms to clinical implications. An-

J Biomed Lab Sci 2010 Vol 22 No 1

5.

6.
7.

8.

9.

10.

11.
12.

13.

14.
15.

16.
17.

18.

19.

20.

21.

tioxid Redox Signal 2008; 10: 1405-34.

Roberts LJ, Fessel JP. The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid
peroxidation. Chem Phys Lipids 2004; 128: 173-86.
Roberts LJ, Milne GL. Isoprostanes. J Lipid Res 2009;50
Suppl: S219-S223.
Lin CL, Hsu YT, Lin TK, et al. Increased levels of
F2-isoprostanes following aneurysmal subarachnoid
hemorrhage in humans. Free Radic Biol Med 2006; 40:
1466-73.
Hsieh YP, Lin CL, Shiue AL, et al. Correlation of
F4-neuroprostanes levels in cerebrospinal fluid with
outcome of aneurysmal subarachnoid hemorrhage in
humans. Free Radic Biol Med 2009; 47: 814-24.
Chen TW, Lin CL, Yen HC. Elevation of F2-isoprostanes
and F4-neuroprostanes levels in cerebrospinal fluid of
patients with traumatic brain injury [Abstract]. Free Radic
Biol Med 2009; 47: s108.
Morrow JD, Harris TM, Roberts LJ. Noncyclooxygenase
oxidative formation of a series of novel prostaglandins:
analytical ramifications for measurement of eicosanoids.
Anal Biochem 1990; 184: 1-10.
Roberts LJ, Morrow JD. The generation and actions of
isoprostanes. Biochim Biophys Acta 1997; 1345: 121-35.
Morrow JD, Awad JA, Boss HJ, et al. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed
in situ on phospholipids. Proc Natl Acad Sci U S A 1992;
89: 10721-5.
Stafforini DM, Sheller JR, Blackwell TS, et al. Release of
free F2-isoprostanes from esterified phospholipids is
catalyzed by intracellular and plasma platelet-activating
factor acetylhydrolases. J Biol Chem 2006; 281: 461623.
Milne GL, Yin H, Morrow JD. Human biochemistry of the
isoprostane pathway. J Biol Chem 2008; 283: 15533-7.
Yin H, Gao L, Tai HH, et al. Urinary prostaglandin F2 is
generated from the isoprostane pathway and not the
cyclooxygenase in humans. J Biol Chem 2007; 282:
329-36.
Taber DF, Morrow JD, Roberts LJ. A nomenclature system for the isoprostanes. Prostaglandins 1997; 53: 63-7.
Rokach J, Khanapure SP, Hwang SW, et al. Nomenclature of isoprostanes: a proposal. Prostaglandins 1997;
54: 853-73.
Roberts LJ, Montine TJ, Markesbery WR, et al. Formation of isoprostane-like compounds (neuroprostanes) in
vivo from docosahexaenoic acid. J Biol Chem 1998;
273: 13605-12.
Taber DF, Roberts LJ. Nomenclature systems for the
neuroprostanes and for the neurofurans. Prostaglandins
Other Lipid Mediat 2005; 78: 14-8.
Yin H, Musiek ES, Gao L, et al. Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo. J Biol Chem 2005;
280: 26600-11.
Montine KS, Quinn JF, Zhang J, et al. Isoprostanes and
related products of lipid peroxidation in neurodegenera-

J Biomed Lab Sci 2010 Vol 22 No 1

tive diseases. Chem Phys Lipids 2004; 128: 117-24.

22. Arneson KO, Roberts LJ. Measurement of products of
docosahexaenoic acid peroxidation, neuroprostanes,
and neurofurans. Methods Enzymol 2007; 433: 127-43.
23. Montine TJ, Milatovic D, Gupta RC, et al. Neuronal oxidative damage from activated innate immunity is EP2
receptor-dependent. J Neurochem 2002; 83: 463-70.
24. Milne GL, Morrow JD, Picklo MJ, Sr. Elevated oxidation
of docosahexaenoic acid, 22:6 (n-3), in brain regions of
rats undergoing ethanol withdrawal. Neurosci Lett 2006;
405: 172-4.
25. Milne GL, Yin H, Brooks JD, et al. Quantification of
F2-isoprostanes in biological fluids and tissues as a
measure of oxidant stress. Methods Enzymol 2007; 433:
113-26.
26. Liu W, Morrow JD, Yin H. Quantification of
F2-isoprostanes as a reliable index of oxidative stress in
vivo using gas chromatography-mass spectrometry
(GC-MS) method. Free Radic Biol Med 2009; 47: 1101-7.
27. Proudfoot J, Barden A, Mori TA, et al. Measurement of
urinary F2-isoprostanes as markers of in vivo lipid peroxidation-A comparison of enzyme immunoassay with
gas chromatography/mass spectrometry. Anal Biochem
1999; 272: 209-15.
28. :Isoprostanes
Neuroprostanes 2009; 2: 53-67
29. Yen HC, Cheng HS, Hsu YT, et al. Effects of age and
health status on levels of urinary 15-F2t-isoprostane. J
Biomed Lab Sci 2001; 13: 24-8.
30. Yin H, Porter NA, Morrow JD. Separation and identification of F2-isoprostane regioisomers and diastereomers
by novel liquid chromatographic/mass spectrometric
methods. J Chromatogr B Analyt Technol Biomed Life
Sci 2005; 827: 157-64.
31. Taylor AW, Bruno RS, Frei B, et al. Benefits of prolonged
gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of
plasma total 15-series F-isoprostanes. Anal Biochem
2006; 350: 41-51.
32. Sircar D, Subbaiah PV. Isoprostane measurement in
plasma and urine by liquid chromatography-mass spectrometry with one-step sample preparation. Clin Chem
2007; 53: 251-8.
33. Pratico D, Lawson JA, FitzGerald GA. Cyclooxygenasedependent formation of the isoprostane, 8-epi prostaglandin F2. J Biol Chem 1995; 270: 9800-8.
34. Reilly M, Delanty N, Lawson JA, et al. Modulation of
oxidant stress in vivo in chronic cigarette smokers. Circulation 1996; 94: 19-25.
35. Basu S. Isoprostanes: novel bioactive products of lipid
peroxidation. Free Radic Res 2004; 38: 105-22.
36. Milne GL, Sanchez SC, Musiek ES, et al. Quantification
of F2-isoprostanes as a biomarker of oxidative stress.
Nat Protoc 2007; 2: 221-6.
37. Musiek ES, Cha JK, Yin H, et al. Quantification of F-ring
isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a

Dectection of F2-isoprostanes and F4-neuroprostanes

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.
49.

10

stable isotope dilution mass spectrometric assay. J

Chromatogr B Analyt Technol Biomed Life Sci 2004;
799: 95-102.
Morrow JD. The isoprostanes - unique products of arachidonate peroxidation: their role as mediators of oxidant
stress. Curr Pharm Des 2006; 12: 895-902.
Comporti M, Signorini C, Arezzini B, et al. F2-isoprostanes
are not just markers of oxidative stress. Free Radic Biol
Med 2008; 44: 247-56.
Belik J, Gonzalez-Luis GE, Perez-Vizcaino F, et al. Isoprostanes in fetal and neonatal health and disease. Free
Radic Biol Med 2010; 48: 177-88.
Fukunaga M, Makita N, Roberts LJ, et al. Evidence for
the existence of F2-isoprostane receptors on rat vascular
smooth muscle cells. Am J Physiol 1993; 264: C1619C1624.
Hou X, Roberts LJ, Gobeil F, Jr., et al. Isomer-specific
contractile effects of a series of synthetic F2-isoprostanes
on retinal and cerebral microvasculature. Free Radic
Biol Med 2004; 36: 163-72.
Mobert J, Becker BF, Zahler S, et al. Hemodynamic
effects of isoprostanes (8-iso-prostaglandin F2 and E2)
in isolated guinea pig hearts. J Cardiovasc Pharmacol
1997; 29: 789-94.
Audoly LP, Rocca B, Fabre JE, et al. Cardiovascular
responses to the isoprostanes iPF2-III and iPE2-III are
mediated via the thromboxane A2 receptor in vivo. Circulation 2000; 101: 2833-40.
Yura T, Fukunaga M, Khan R, et al. Free-radical-generated
F2-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells. Kidney Int 1999;
56: 471-8.
Comporti M, Arezzini B, Signorini C, et al. F2-isoprostanes
stimulate collagen synthesis in activated hepatic stellate
cells: a link with liver fibrosis? Lab Invest 2005; 85:
1381-91.
Benndorf RA, Schwedhelm E, Gnann A, et al. Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation,
and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A2 receptor: a potential link between oxidative stress and
impaired angiogenesis. Circ Res 2008; 103: 1037-46.
Basu S, Helmersson J. Factors regulating isoprostane
formation in vivo. Antioxid Redox Signal 2005; 7: 221-35.
Kaviarasan S, Muniandy S, Qvist R, et al. F2-isoprostanes

50.

51.

52.

53.
54.

55.

56.

57.

58.

59.
60.

61.

62.

as novel biomarkers for type 2 diabetes: a review. J Clin

Biochem Nutr 2009; 45: 1-8.
Morrow JD, Roberts LJ. The isoprostanes: their role as
an index of oxidant stress status in human pulmonary
disease. Am J Respir Crit Care Med 2002; 166: S25-30.
Montine TJ, Montine KS, McMahan W, et al.
F2-isoprostanes in Alzheimer and other neurodegenerative diseases. Antioxid Redox Signal 2005; 7: 269-75.
Montine TJ, Quinn J, Kaye J, et al. F2-isoprostanes as
biomarkers of late-onset Alzheimer's disease. J Mol
Neurosci 2007; 33: 114-9.
Moore K. Isoprostanes and the liver. Chem Phys Lipids
2004; 128: 125-33.
Morrow JD. Quantification of isoprostanes as indices of
oxidant stress and the risk of atherosclerosis in humans.
Arterioscler Thromb Vasc Biol 2005; 25: 279-86.
Canter JA, Eshaghian A, Fessel J, et al. Degree of heteroplasmy reflects oxidant damage in a large family with
the mitochondrial DNA A8344G mutation. Free Radic
Biol Med 2005; 38: 678-83.
Matayatsuk C, Lee CY, Kalpravidh RW, et al. Elevated
F2-isoprostanes in thalassemic patients. Free Radic Biol
Med 2007; 43: 1649-55.
de Leon MJ, Mosconi L, Li J, et al. Longitudinal CSF
isoprostane and MRI atrophy in the progression to AD. J
Neurol 2007; 254: 1666-75.
Kelly PJ, Morrow JD, Ning M, et al. Oxidative stress and
matrix metalloproteinase-9 in acute ischemic stroke: the
Biomarker Evaluation for Antioxidant Therapies in Stroke
(BEAT-Stroke) study. Stroke 2008; 39: 100-4.
Seet RC, Lee CY, Lim EC, et al. Oxidative damage in
dengue fever. Free Radic Biol Med 2009;47:375-80.
De Felice C, Ciccoli L, Leoncini S, et al. Systemic oxidative stress in classic Rett syndrome. Free Radic Biol
Med 2009; 47: 440-8.
Monneret D, Pepin JL, Godin-Ribuot D, et al. Association of urinary 15-F2t-isoprostane level with oxygen desaturation and carotid intima-media thickness in nonobese sleep apnea patients. Free Radic Biol Med 2010;
48: 619-25.
Reich EE, Markesbery WR, Roberts LJ, et al. Brain
regional quantification of F-ring and D-/E-ring isoprostanes and neuroprostanes in Alzheimer's disease.
Am J Pathol 2001; 158: 293-7.

J Biomed Lab Sci 2010 Vol 22 No 1

F2-isoprostanes F4-neuropstanes

F2-isoprostanes (F2-IsoPs)F4neuropstanes (F4-NPs)

F2-IsoPsF4-NPs
F2-IsoPs
/F2-IsoPsF4-NPs
F2-IsoPs

F4-NPs
F2-IsoPs

99 3 9
333 259
(03) 2118800 5207 (03)2118692 yen@mail.cgu.edu.tw

J Biomed Lab Sci 2010 Vol 22 No 1

11