01 Detection of F2 Isoprostanes an (1)

J Biomed Lab Sci 2010 Vol 22 No 1

1

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 out-come of diseases, or a tool to monitor the effectiveness of antioxidant therapy. Clinical studies de-tecting 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 reac-tive oxygen species and reactive nitrogen species, or decreased antioxidant capacity can result in the status of oxidative stress, which has the tendency to cause oxida-tive damage. Oxidative damage to important macro-molecules, DNA, lipid, or protein, may lead to distur-bances of normal physiological functions, which plays an important role in pathogenesis of various human dis-eases 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 de-mand 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 dam-age 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 peroxda-tion 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 neu-rofurans, 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: [email protected]

Mini Review

Dectection of F2-isoprostanes and F4-neuroprostanes

2


mechanisms with different significance [5,6]. This re-view paper will only focus on F2-IsoPs and F4-NPs be-cause they are more important in clinical studies. Clini-cal utilities of F2-IsoPs and F4-NPs will be discussed by including our work on the studies of aneurysmal su-barachnoid hemorrhage (aSAH) and traumatic brain in-jury (TBI) in humans [7-9].

Formation and Nomenclature of F2-IsoPs and F4-NPs

F2-IsoPs is a group of prostaglandin (PG)F2-like com-pounds that are generated from arachidonic acid (AA, C20:4 -6), an abundant polyunsaturated fatty acid pre-sent in all kinds of cell membranes, via free radical- catalyzed lipid peroxidation. It was proved to be inde-pendent 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 acci-dental 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 approxi-mately 50-fold higher than that in fresh plasma. Freeze- thaw 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 surround-ing 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 per-oxidation 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]. More-over, 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

of F2-IsoPs [11,14]. On the other hand, enantiomer of PGF2, ent-PGF2, could be generated from IsoP path-way and might account for most of PGF2 found in urine, which was thought to be exclusively from the COX path-way. 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 ap-proved by the Eicosanoid Nomenclature Committee, which was also sanctioned by the International Union of Pure and Applied Chemistry (IUPAC) [3,16]. This no-menclature 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 nomen-clature system and kept using this system is their publi-cations. That system was developed by Rokach et al and named four regioisomers as types VI, V, IV, and III re-gioisomers [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 re-gioisomers 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, re-spectively. In addition, esterified F4-NPs was barely de-tectable in 1 ml of human plasma, while free F4-NPs in normal human cerebrospinal fluid (CSF) could be de-tected and the levels were elevated in that of patients with Alzheimers disease (AD). The nomenclature sys-


3

tem for NPs has also been proposed by Taber and Rob-erts [19]. Furthermore, by using a unique LC/MS ap-proach, Yin et al. indicated that 4- and 20-series NPs were the major regioisomers among eight possible re-gioisomers 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 Speci-men 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 quanti-fication of F2-IsoPs and F4-NPs in human body fluids, which is especially important for clinical studies using

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 elec-tron 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 antibod-ies 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 re-covery 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 laborato-ries [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 regioisom-ers and diastereomers of F2-IsoPs and F4-NPs [20,30].

Fig. 1. Chemical structure of PGF2, 15-F2t-IsoP, and the deuterium (D)-labeled 15-F2t-IsoP, [2H4]-15-F2t-IsoP.


4


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 re-ported 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 anti-oxidants 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 tempera-ture 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 lev-els or systemic levels of lipid peroxidation [3]. Further-more, serum should be avoided because small amount of F2-IsoPs could be released from platelet via COX path-way 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 be-cause 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

greatly affected by the kidney. Instead, major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t- IsoP 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/NICI- MS method have been described in details [25,26,36]. It requires sophisticated sample processing and mainte-nance 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 derivatiza-tion to form trimethylsilyl (TMS) derivative to increase volatility and prevent hydroxyl groups on F2-IsoPs from reacting with glass components and columns; and injec-tion of final analytes dissolved in undecane into GC/MS to be analyzed under NICI mode. The structure of inter-nal 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 proper-ties from eicosanoids metabolism, entering GC columns with F2-IsoPs after SPE steps. Examples of GC chroma-tograms 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/NICI- MS method [26]. Moreover, there are much more un-wanted 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 espe-cially in urine [15].

Procedures for analyzing free form of F4-NPs were


5

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 dis-cussed in our recent paper, it was confusing that the cut-ting 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 fol-lowed. Moreover, unlike F2-IsoPs, F4-NPs are present as multiple peaks over a range of retention time, the quanti-fication 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, al-though [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 indi-cated that [2H4]-15-F2t-IsoP should be used [8]. GC chromatograms for analysis of F4-NPs in a normal hu-man CSF sample are demonstrated by Figure 3.

Esterified F2-IsoPs is abundant in all kinds of tis-sues, while esterified F4-NPs is usually conducted only

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 antioxi-dant 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 resus-pension 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 Mark-ers 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 oxi-dative 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 mark-ers of lipid peroxidation, such as malondialdehyde (MDA) or lipid hydroperoxide. Third, F2-IsoPs is readily detect-able in all kinds of biological tissues and body fluids by the GC/NICI-MS methods, which allows the establish-ment of normal ranges and is valuable for clinical stud-ies under various conditions. Fourth, it has been well demonstrated that F2-IsoPs levels were elevated in vari-

Fig. 2. GC chromatograms for F2-IsoPs analysis of a CSF (A), plasma (B), and urine sample from normal sub-jects. 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 multi-plying 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.


6


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 dam-age and pathological changes or outcome of diseases in clinical studies, providing mechanistic explanations for the roles of oxidative stress in diseases. It is an addi-tional 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 peroxida-

tion of any cells in the brain, including different neural cells, inflammatory cells, and vascular cells, but eleva-tion of F4-NPs levels should better reflect neuronal damage or at least undoubtedly indicate oxidative dam-age 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 activi-ties. The most well known activities of 15-F2t-IsoP are vasoconstriction on various vascular beds and platelet aggregation [40]. Many studies indicate that these activi-ties 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 recep-

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 rela-tive 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.


7

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 endo-thelium-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 bronchocon-striction 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 ac-tivities. 15-F2t-IsoP could stimulate production of 1,4,5- triphosphate and DNA synthesis in rat aortic smooth muscle cells [41] and induced cell proliferation, DNA synthesis, and expression of endothelin-1 in aortic en-dothelial 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 condi-tions. 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, neurodegenera-tive diseases, cardiovascular diseases, type 2 diabetes,

inflammatory diseases, hypercholesterolemia, and he-patic 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 reli-able 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 oxida-tive damage is indeed enhanced in the diseased condi-tions investigated, which either have never been proved before or are not well accepted due to the use of unreli-able 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 dem-onstrated that plasma levels of free F2-IsoPs correlated with degree of heteroplasmy of the pathogenic mito-chondrial 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 lev-els 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 reso-nance imaging [57]. Kelly et al. first provided the evi-dence 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, normal-ized by AA concentrations, were higher during febrile stage than that convalescent stage in patients with den-gue fever [59]. The report of De Felice et al. first dem-onstrated 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]. Mon-


8


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 sys-temic levels. It might be especially important for neuro-logical 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 corre-lated with clinical parameters [18,52,57]. On the other hand, our study on aSAH in fact was the only publica-tion 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 dif-ferent 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 lev-els in patients with type 2 diabetes and hypercholes-terolemia. 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 condi-tions. For examples, different tocopherols, but not vita-min 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

damage has indeed suppressed by antioxidant supple-mentation. 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 in-dices or outcome.

Conclusion

Although F2-IsoPs is difficult to be measured, the use-fulness 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 fur-ther explored in more human conditions by using these two markers in the future.

Acknowledgements

This work was supported by grants NSC91-2314-B-182- 072, NSC96-2320-B-182-018, and NSC97-2320-B-182- 012-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. Halliwell B, Gutteridge MC. Free Radicals in Biology and Medicine. 4th ed. New York: Oxford University Press, 2007.

2. Morrow JD, Hill KE, Burk RF, et al. A series of pros-taglandin 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.

3. Roberts LJ, Morrow JD. Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Radic Biol Med 2000; 28: 505-13.

4. Basu S. F2-isoprostanes in human health and diseases: from molecular mechanisms to clinical implications. An-


9

tioxid Redox Signal 2008; 10: 1405-34. 5. Roberts LJ, Fessel JP. The biochemistry of the iso-

prostane, neuroprostane, and isofuran pathways of lipid peroxidation. Chem Phys Lipids 2004; 128: 173-86.

6. Roberts LJ, Milne GL. Isoprostanes. J Lipid Res 2009;50 Suppl: S219-S223.

7. 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.

8. 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.

9. 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.

10. 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.

11. Roberts LJ, Morrow JD. The generation and actions of isoprostanes. Biochim Biophys Acta 1997; 1345: 121-35.

12. 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.

13. 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: 4616- 23.

14. Milne GL, Yin H, Morrow JD. Human biochemistry of the isoprostane pathway. J Biol Chem 2008; 283: 15533-7.

15. 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.

16. Taber DF, Morrow JD, Roberts LJ. A nomenclature sys-tem for the isoprostanes. Prostaglandins 1997; 53: 63-7.

17. Rokach J, Khanapure SP, Hwang SW, et al. Nomencla-ture of isoprostanes: a proposal. Prostaglandins 1997; 54: 853-73.

18. Roberts LJ, Montine TJ, Markesbery WR, et al. Forma-tion of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J Biol Chem 1998; 273: 13605-12.

19. Taber DF, Roberts LJ. Nomenclature systems for the neuroprostanes and for the neurofurans. Prostaglandins Other Lipid Mediat 2005; 78: 14-8.

20. Yin H, Musiek ES, Gao L, et al. Regiochemistry of neu-roprostanes generated from the peroxidation of doco-sahexaenoic acid in vitro and in vivo. J Biol Chem 2005; 280: 26600-11.

21. Montine KS, Quinn JF, Zhang J, et al. Isoprostanes and related products of lipid peroxidation in neurodegenera-

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 oxi-dative 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 per-oxidation-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 identifica-tion 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 chro-matography-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 spec-trometry with one-step sample preparation. Clin Chem 2007; 53: 251-8.

33. Pratico D, Lawson JA, FitzGerald GA. Cyclooxygenase- dependent formation of the isoprostane, 8-epi pros-taglandin 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. Cir-culation 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) de-rived from docosahexaenoic acid in vivo in humans by a


10


stable isotope dilution mass spectrometric assay. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 799: 95-102.

38. Morrow JD. The isoprostanes - unique products of ara-chidonate peroxidation: their role as mediators of oxidant stress. Curr Pharm Des 2006; 12: 895-902.

39. 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.

40. Belik J, Gonzalez-Luis GE, Perez-Vizcaino F, et al. Iso-prostanes in fetal and neonatal health and disease. Free Radic Biol Med 2010; 48: 177-88.

41. 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: C1619- C1624.

42. 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.

43. 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.

44. 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. Cir-culation 2000; 101: 2833-40.

45. Yura T, Fukunaga M, Khan R, et al. Free-radical-generated F2-isoprostane stimulates cell proliferation and endo-thelin-1 expression on endothelial cells. Kidney Int 1999; 56: 471-8.

46. 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.

47. Benndorf RA, Schwedhelm E, Gnann A, et al. Iso-prostanes inhibit vascular endothelial growth fac-tor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angio-genesis in vivo via activation of the thromboxane A2 re-ceptor: a potential link between oxidative stress and impaired angiogenesis. Circ Res 2008; 103: 1037-46.

48. Basu S, Helmersson J. Factors regulating isoprostane formation in vivo. Antioxid Redox Signal 2005; 7: 221-35.

49. Kaviarasan S, Muniandy S, Qvist R, et al. F2-isoprostanes

as novel biomarkers for type 2 diabetes: a review. J Clin Biochem Nutr 2009; 45: 1-8.

50. 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.

51. Montine TJ, Montine KS, McMahan W, et al. F2-isoprostanes in Alzheimer and other neurodegenera-tive diseases. Antioxid Redox Signal 2005; 7: 269-75.

52. 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.

53. Moore K. Isoprostanes and the liver. Chem Phys Lipids 2004; 128: 125-33.

54. 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.

55. Canter JA, Eshaghian A, Fessel J, et al. Degree of het-eroplasmy reflects oxidant damage in a large family with the mitochondrial DNA A8344G mutation. Free Radic Biol Med 2005; 38: 678-83.

56. Matayatsuk C, Lee CY, Kalpravidh RW, et al. Elevated F2-isoprostanes in thalassemic patients. Free Radic Biol Med 2007; 43: 1649-55.

57. 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.

58. 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.

59. Seet RC, Lee CY, Lim EC, et al. Oxidative damage in dengue fever. Free Radic Biol Med 2009;47:375-80.

60. De Felice C, Ciccoli L, Leoncini S, et al. Systemic oxida-tive stress in classic Rett syndrome. Free Radic Biol Med 2009; 47: 440-8.

61. Monneret D, Pepin JL, Godin-Ribuot D, et al. Associa-tion of urinary 15-F2t-isoprostane level with oxygen de-saturation and carotid intima-media thickness in nono-bese sleep apnea patients. Free Radic Biol Med 2010; 48: 619-25.

62. Reich EE, Markesbery WR, Roberts LJ, et al. Brain regional quantification of F-ring and D-/E-ring iso-prostanes and neuroprostanes in Alzheimer's disease. Am J Pathol 2001; 158: 293-7.


11

F2-isoprostanes F4-neuropstanes

/

F2-isoprostanes (F2-IsoPs)F4- neuropstanes (F4-NPs)F2-IsoPsF4-NPs

F2-IsoPs/F2-IsoPsF4-NPs

F2-IsoPsF4-NPs

F2-IsoPs

/

99 3 9 333 259 (03) 2118800 5207 (03)2118692 [email protected]

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown

/Description >>> setdistillerparams> setpagedevice

01 Detection of F2 Isoprostanes an (1)

Documents

Transcript of 01 Detection of F2 Isoprostanes an (1)