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 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

The best way to access oxidative stress in humans is to detect specific and stable markers of oxidative dam- Introduction age using reliable methods. There have been many markers of oxidative damage and various methods for Increased production of reactive species, especially reac- each marker with different advantages and disadvantages tive oxygen species and reactive nitrogen species, or [1]. However, since the discovery of F2-isoprostanes decreased antioxidant capacity can result in the status of (F2-IsoPs) as the most specific marker of lipid peroxda- oxidative stress, which has the tendency to cause oxida- tion by Jason Morrow and Jackson Roberts at Vanderbilt tive damage. Oxidative damage to important macro- University in 1990 in humans [2], F2-IsoPs analyzed by molecules, DNA, lipid, or protein, may lead to distur- mass spectrometry has been well recognized as the most bances of normal physiological functions, which plays reliable marker of oxidative damag and widely applied an important role in pathogenesis of various human dis- in various clinical studies [3,4]. Moreover, in addition to eases or mechanisms of toxicity induced by xenobiotics. F4-neuroprostanes (F4-NPs), various products of lipid

Many of these basic concepts can be found in the book peroxidation related to or similar to formation of F2-IsoPs, of Halliwell and Gutteridge [1]. There is increasing de- including F3-IsoPs, D/E form of IsoPs and NPs, A/J form mand for accessing oxidative stress in humans, which of IsoPs and NPs, isothromboxanes, isofurans and neu- has become an important issue in the field of clinical rofurans, isoketals and neuroketals, F2-dihomo-IsoPs, laboratory science. However, inappropriate markers and and urinary metabolites of 15-F2t-IsoP, are also generated nonspecific assays were often employed in the literature. 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] J Biomed Lab Sci 2010 Vol 22 No 1 1

Dectection of F2-isoprostanes and F4-neuroprostanes

mechanisms with different significance [5,6]. This re- of F2-IsoPs [11,14]. On the other hand, enantiomer of view paper will only focus on F2-IsoPs and F4-NPs be- PGF2α, ent-PGF2α, could be generated from IsoP path- cause they are more important in clinical studies. Clini- way and might account for most of PGF2α found in urine, cal utilities of F2-IsoPs and F4-NPs will be discussed by which was thought to be exclusively from the COX path- including our work on the studies of aneurysmal su- way. It should be noted that PGF2α and ent-PGF2α could barachnoid hemorrhage (aSAH) and traumatic brain in- be differentiated under the analysis of special liquid jury (TBI) in humans [7-9]. chromatography/mass spectrometry (LC/MS), but not GC/NICI-MS [15].

The terminology for F2-IsoPs or 15-F2t-IsoP has Formation and Nomenclature of F2-IsoPs been confusing especially in the early years after the and F -NPs 4 discovery of F2-IsoPs. It was not more unified until the nomenclature system of Taber et al. for IsoPs was ap-

F2-IsoPs is a group of (PG)F2-like com- proved by the Nomenclature Committee, pounds that are generated from arachidonic acid (AA, which was also sanctioned by the International Union of C20:4 ω-6), an abundant polyunsaturated fatty acid pre- Pure and Applied Chemistry (IUPAC) [3,16]. This no- sent in all kinds of cell membranes, via free radical- menclature system named four major regioisomers as 5-, catalyzed lipid peroxidation. It was proved to be inde- 12-, 8, and 15-series regioisomers, which corresponded pendent of action of (COX) because to I-IV regioisomers initially categorized by Morrow et human subjects receiving COX inhibitors did not have al. [2] and provided principles to name different isomers. lower levels of these compounds in body fluids. F2-IsoPs However, many publications or companies still used was named because it has F-type prostane rings similar different old terms of 15-F2t-IsoP, such as 8-iso-PGF2α to PGF2α [2,10,11]. At beginning, it was in fact an acci- and 8-epi-PGF2α, without updates in this aspect. The dental discovery of Morrow et al. when plasma samples chemical structure of 15-F2t-IsoP is almost identical to stored at -20℃ for several months were subjected to that of PGF2α except the stereochemistry as shown by routine analysis by gas chromatography/negative-ion Figure 1. Based on the system of Taber et al., 15-F2t-IsoP chemical-ionization mass spectrometry (GC/NICI-MS) should be pronounced as “15-F-2-transe-isoprostane” for 9α,11β-PGF2, a metabolite of PGD2. Unknown and it was designated in this way because two side PGF2-like compounds, identified as peaks adjacent to chains are oriented trans in respect to hydroxyl groups that of 9α,11β-PGF2 of GC chromatograms, were found on the cyclopentane ring [16]. On the other hand, the in those plasma samples at the levels that were approxi- group of FitzGerald has developed a different nomen- mately 50-fold higher than that in fresh plasma. Freeze- clature system and kept using this system is their publi- thaw cycles of plasma also increased the levels of those cations. That system was developed by Rokach et al and compounds [10]. named four regioisomers as types VI, V, IV, and III re-

F2-IsoPs is initially generated on phospholipids on gioisomers [17], which corresponded to 5-, 12-, 8, and tissues or lipoproteins in plasma as esterified form from 15-series regioisomers of Taber et al. [16]. Moreover, esterified fatty acids and can be released into surround- iPF2α-III denotes 15-F2t-IsoP in the system of Rokach et ing body fluids or circulation as free form mediated by al. [17]. phospholipase A2-like activities. Detection of F2-IsoPs in In 1998, Roberts et al. further proved the presence body fluids therefore can reflect the levels of lipid per- of F4-NPs, which was derived from free radical-mediated oxidation in tissues [11,12]. Stafforini et al. identified lipid peroxidation of docosahexaenoic acid (DHA, C22:6 that intracellular and plasma platelet-activating factor ω-3), both in vitro and in vivo with the same principle as acetylhydrolases (PAH-AH) could be responsible for the the formation of F2-IsoPs from AA. Eight different re- hydrolysis of F2-IsoPs from phospholipids [13]. More- gioisomers for F4-NPs, 4-, 7-, 10-, 11-, 13-, 14-, 17- and over, there are four regioisomers of F2-IsoPs, which are 20-series, were predicted [18]. In this paper, the authors denoted as 5-, 12-, 8, and 15-series regioisomers based showed that F4-NPs was generated in a greater amount on the number of carbon atom of side chains on which than F2-IsoPs from equal amounts of AA and DHA, re- the hydroxyl group is localized. Quantity of 5- and spectively. In addition, esterified F4-NPs was barely de-

15-series regioisomers is much higher than that of other tectable in 1 ml of human plasma, while free F4-NPs in two regioisomers both in vitro and in vivo. Although normal human cerebrospinal fluid (CSF) could be de- each regioisomer theoretically consists of eight racemic tected and the levels were elevated in that of patients diastereomers, 15-F2t-IsoP is the most abundant isomer with Alzheimer’s disease (AD). The nomenclature sys-

2 J Biomed Lab Sci 2010 Vol 22 No 1

2 Fig. 1. Chemical structure of PGF2α, 15-F2t-IsoP, and the deuterium (D)-labeled 15-F2t-IsoP, [ H4]-15-F2t-IsoP.

tem for NPs has also been proposed by Taber and Rob- specimen with limited amount and low concentrations. erts [19]. Furthermore, by using a unique LC/MS ap- GC/NICI-MS is also the reference method for analyzing proach, Yin et al. indicated that 4- and 20-series NPs F2-IsoPs that has been validated for human plasma and were the major regioisomers among eight possible re- urine by the groups of Morrow and Roberts [22,25,26]. gioisomers of NPs both in vitro and in rat brain [20]. NICI mode is a very uncommon technique that is hard to Because DHA is present in all kinds of neural cells manage. It is different from the commonly known elec- in the brain but most enriched in neurons, F4-NPs is tron impact ionization (EI) mode. In NICI mode, the therefore considered as a more selective indicator for analyte derivatized with electron withdrawing groups oxidative damage to neurons or gray matter and is more captures low-energy electron, which is produced by the useful in studying neurodegenerative diseases [21,22]. reaction of high-energy electrons and methane gas, and As proposed by Roberts et al., phosphatidylserine with becomes an unfragmented negative ion, which therefore esterified F4-NPs would be a very distorted molecules has higher abundance and sensitivity compared with the and could significantly affect neuronal dysfunction [18]. fragmented target ion under EI mode. Although there are As shown by many conditions of brain injury in animal commercial EIA kits available using polyclonal antibod- experiments, the extent in the elevation of F4-NPs was ies against 15-F2t-IsoP, EIA is an unreliable method with greater than that of F2-IsoPs [23,24]. 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 Analysis of F -IsoPs and F -NPs and Speci- 2 4 and IsoP pathways with similar structure to 15-F2t-IsoP. men Considerations The specificity tested by the manufacturers of the kits was impossible to be sufficient. If SPE procedures are

F2-IsoPs has been quantified by enzyme immunoassay performed to remove some of other compounds, the re- (EIA), gas chromatography/negative-ion chemical-ioni- covery cannot be normalized unless radioactive [3H]- zation mass spectrometry (GC/NICI-MS), and LC/MS or 15-F2t-IsoP is used as the internal standard, which would liquid chromatography/tandem mass spectrometry not be practical for clinical studies or regular laborato-

(LC/MS/MS), while F4-NPs has only be quantified by ries [27]. Based on our previous experiences, EIA was GC/MS [4,18]. Among them, GC/NICI-MS is the most also prone to be interfered by impurities in reagents used sensitive method that is most suitable for routine quanti- [29]. On the other hand, LC/MS method is superior to fication of F2-IsoPs and F4-NPs in human body fluids, GC/NICI-MS method in identifying different regioisom- which is especially important for clinical studies using ers and diastereomers of F2-IsoPs and F4-NPs [20,30].

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Dectection of F2-isoprostanes and F4-neuroprostanes

However, because of its lower sensitivity compared with greatly affected by the kidney. Instead, major urinary

GC/NICI-MS, LC/MS or LC/MS/MS can only be used metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t- to reliably detect free form of F2-IsoPs levels in urine, in IsoP formed by the metabolism in the lung, should be which levels of F2-IsoPs are about 10 fold or higher than measured to represent systemic levels of oxidative damage that in plasma or CSF. Although few papers have re- [13,11]. However, the internal standard for GC/NICI-MS ported low detection limit for plasma levels of F2-IsoPs analysis of this metabolite is not commercially available detected by LC/MS/MS, these studies in fact measured and therefore cannot be widely applied. total F2-IsoPs, the sum of free and esterified F2-IsoPs, in Detailed procedures for analyzing free and esterified plasma, not free form alone [31,32]. form of F2-IsoPs in body fluids or tissues by GC/NICI- F2-IsoPs and F4-NPs can be analyzed as free form in MS method have been described in details [25,26,36]. It body fluids or esterified form on tissues or in plasma requires sophisticated sample processing and mainte- depending on the specimen available or the questions to nance of GC/NICI-MS instrument. The working flow for be addressed. As stated before for the discovery of free form of F2-IsoPs consists of the following steps: the

F2-IsoPs, samples for F2-IsoPs and F4-NPs have to be mixing of a stable isotope-labeled internal standard, 2 stored at -80℃, not -20℃, and cannot be subjected to [ H4]-15-F2t-IsoP, and samples; adjustment of pH to 3.0 freeze-thaw cycles [10]. The fact that esterified form is to enrich nonionized form of F2-IsoPs; two steps of solid more susceptible to artifact caused by ex vivo oxidation phase extract (SPE) using C18 and silica SPE columns; should be carefully deliberated. Tissues samples or first derivatization to form pentafluorobenzyl esters for plasma samples for analyzing esterified form have to be later formation of negative ion in GC/MS; thin-layer immediately frozen by dry ice or liquid nitrogen and chromatography (TLC) purification; second derivatiza- stored at -80℃ upon collection. Plasma for measuring tion to form trimethylsilyl (TMS) derivative to increase esterified form of F2-IsoPs requires the addition of anti- volatility and prevent hydroxyl groups on F2-IsoPs from oxidants into plasma before freezing, which was not reacting with glass components and columns; and injec- required for detection of free form. Levels of free form tion of final analytes dissolved in undecane into GC/MS was not altered even plasma was left at room tempera- to be analyzed under NICI mode. The structure of inter- 2 ture for 2 hours. It was also unchanged in urine when nal standard, [ H4]-15-F2t-IsoP, is shown in Figure 1. incubated at 37℃ for one week [2,11]. The detection of Four hydrogen atoms on two carbons are replaced by esterified form is therefore more problematic than free four deuterium atoms. The use of C18 and silica SPE form in plasma for clinical studies. Another point should columns employed the principles of reverse phase and be considered is that esterified F2-IsoPs in plasma should normal phase chromatography to remove the majority of only stand for lipid peroxidation in the blood system, not unwanted compounds with the polarity very different whole body, while free form stands for whole-body lev- from F2-IsoPs. Moreover, TLC purification that recovers els or systemic levels of lipid peroxidation [3]. Further- a portion of compounds from TLC plates not only to more, serum should be avoided because small amount of removes excessive reagents after first derivatization, but

F2-IsoPs could be released from platelet via COX path- also further narrows down the number of compounds, way during platelet activation in vitro and levels of among those compounds with similar chemical proper-

F2-IsoPs in serum could be suppressed by administration ties from metabolism, entering GC columns of aspirin in human subjects [33] even though it was not with F2-IsoPs after SPE steps. Examples of GC chroma- a concern for the levels in plasma and urine in vivo tograms for analysis of F2-IsoPs in normal CSF, plasma, [4,11,34]. On the other hand, F2-IsoPs is detectable in all and urine samples are illustrated by Figure 2. It should kinds of body fluids so far investigated in the literature be noted that the peak at m/z 569.4 (peak a) with the 2 by GC/NICI-MS method [11,35]. If possible, the body same retention time of [ H4]-15-F2t-IsoP peak (peak c) is fluids surrounds the tissues to be investigated should be used for quantification, but it includes other isomers of obtained to closely correlate status of oxidative damage F2-IsoPs, so total F2-IsoPs are measured by GC/NICI- and tissue abnormalities, such as cerebrospinal fluid MS method [26]. Moreover, there are much more un- (CSF) for brain dysfunction, bronchoalveolar lavage wanted compounds in urine than CSF or plasma samples, fluid for pulmonary diseases, and synovial fluid for joint which often cause more problems to the maintenance of problems. Although urine samples are often used be- GC/MS. On the other hand, as mentioned above, the cause they are easy to be collected and have very high peak of PGF2α (peak b) might contain ent-PGF2α espe- concentrations of F2-IsoPs, urinary levels of F2-IsoPs cially in urine [15]. may not represent systemic levels because it can be Procedures for analyzing free form of F4-NPs were

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Fig. 2. GC chromatograms for F2-IsoPs analysis of a CSF (A), plasma (B), and urine sample from normal sub- 2 jects. Peaks a, b, and c represents peaks of F2-IsoPs, PGF2α, and [ H4]-15-F2t-IsoP, respectively. For this figure, 2 approximately 100 pg of [ H4]-15-F2t-IsoP was mixed with 0.5 ml of CSF (A) or plasma (B), while 500 pg of 2 [ H4]-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- 2 plying the exact amount of added [ H4]-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 for brain tissues. To analyze esterified form of F2-IsoPs al. [18] and further described in details by Arneson and and F4-NPs, they need to be first converted to free form Roberts [22]. The principles are almost the same except after total lipids are extracted [22,25,26]. First, tissues that the scraping range on TLC plates for F4-NPs is are homogenized in Folch solution containing antioxi- longer than that for F2-IsoPs and the wash solution for dant and reducing agent and extracted to obtain total silica SPE column is slightly different. However, as dis- lipids. Second, after adding NaCl solution, the analyte in cussed in our recent paper, it was confusing that the cut- lower organic layer is recovered. Third, KOH solution is ting range for TLC was wider in the paper of Arneson added to release esterified IsoPs or NPs on phospholipids and Roberts [22] than that in Roberts et al. [18], but the into free form by alkaline hydrolysis reaction after resus- TLC range indicated by Roberts et al. should be fol- pension by methanol. Finally, the solution is neutralized lowed. Moreover, unlike F2-IsoPs, F4-NPs are present as and diluted with water (pH 3.0). The same procedures multiple peaks over a range of retention time, the quanti- for free form described above are then proceeded. fication requires the integration of peak area for multiple peaks and is much more complicated. We have refined Advantages of F -IsoPs and F -NPs as Mark- the analysis of F4-NPs by analyzing oxidized DHA with 2 4 unknown samples for each run of analysis in order to ers of Oxidative Damage in Clinical Studies define the range of peaks to be integrated in samples [8].

As to internal standard used for F4-NPs analysis, al- As reviewed by Roberts and Morrow, there are six major 2 though [ H4]-15-F2t-IsoP has been used, previously there advantages of using F2-IsoPs as a marker to access oxi- 18 was a trend to use [ O2]-17-F4c-NP [37], which could dative damage in vivo [3]. First, F2-IsoPs is a very stable only be obtained from Jason Morrow, as an internal compound. Second, F2-IsoPs is the most specific marker standard. However, we addressed several problems with of lipid peroxidation in vivo compared with other mark- this internal standard, especially the interference by the ers of lipid peroxidation, such as malondialdehyde (MDA) presence of F2-dihomo-IsoPs in human CSF, and indi- or lipid hydroperoxide. Third, F2-IsoPs is readily detect- 2 cated that [ H4]-15-F2t-IsoP should be used [8]. GC able in all kinds of biological tissues and body fluids by chromatograms for analysis of F4-NPs in a normal hu- the GC/NICI-MS methods, which allows the establish- man CSF sample are demonstrated by Figure 3. ment of normal ranges and is valuable for clinical stud-

Esterified F2-IsoPs is abundant in all kinds of tis- ies under various conditions. Fourth, it has been well sues, while esterified F4-NPs is usually conducted only demonstrated that F2-IsoPs levels were elevated in vari-

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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 2 DHA oxidized in vitro. The “*” symbol denotes the peak of [ H4]-15-F2t-IsoP at m/z 573.4. About 200 pg of 2 [ H4]-15-F2t-IsoP was added to 1 ml of CSF before SPE steps and the concentration of F4-NPs was 33 pg/ml rela- 2 2 tive to [ H4]-15-F2t-IsoP in this figure based on the ratio of peak area of F4-NPs to that of [ H4]-15-F2t-IsoP.

ous animal models of oxidative stress. Fifth, F2-IsoPs tion of any cells in the brain, including different neural levels are modulated by status of the antioxidant system. cells, inflammatory cells, and vascular cells, but eleva-

Finally, levels of F2-IsoPs are not influenced by diet. In tion of F4-NPs levels should better reflect neuronal contrast to that, levels of MDA in body fluids can be damage or at least undoubtedly indicate oxidative dam- greatly affected by the lipid content of diet [1]. age to DHA in the brain tissue, but not in the vascular

Because free F2-IsoPs in body fluids can represent compartments of the brain. 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- Biological Activities of F2-IsoPs age and pathological changes or outcome of diseases in clinical studies, providing mechanistic explanations for Different isomers of F2-IsoPs and E2-IsoPs have been the roles of oxidative stress in diseases. It is an addi- shown to exert various biological activities in vitro and tional favorable feature that cannot be compared by in vivo, which may contribute to pathogenesis of diseases measuring protein oxidation products in plasma or DNA [38-40]. Because 15-F2t-IsoP is the major isomer of oxidation products in white blood cells in the assessment F2-IsoPs, most studies have focused on studying the of oxidative stress if the source of oxidative stress is not biological activities of this isomer, but other isomers of within the blood system. On the other hand, none of F2-IsoPs have also been shown to exert biological activi- other markers of oxidative damage measured in CSF or ties. The most well known activities of 15-F2t-IsoP are brain tissue can differentiate major sources of oxidative vasoconstriction on various vascular beds and platelet damage in the brain. When measuring both F2-IsoPs and aggregation [40]. Many studies indicate that these activi-

F4-NPs in CSF, like what has been done in our studies ties are mediated through the interaction of 15-F2t-IsoP for aSAH and TBI in humans [7-9], the increase in with thromboxane A2 (TXA2) receptors, but other studies F2-IsoPs levels is supposed to represent lipid peroxida- have argued for the existence of distinct “IsoP recep-

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tors”, which remains to be identified [38,41]. Hou et al. inflammatory diseases, hypercholesterolemia, and he- tested the biological activities of isomers of F2-IsoPs patic diseases. Moreover, augmentation of F2-IsoPs was other than 15-F2t-IsoP using synthetic compounds. They also often found in human subjects exposed to cigarette demonstrated that several isomers of 15-series and smoke and consuming alcohol. This paper will not again

12-series F2-IsoPs were also strong vasoconstrictors on elaborate on those overwhelming examples about the retinal and cerebral microvasculature by inducing endo- association between F2-IsoPs and human diseases, but thelium-dependent synthesis of TXA2 [42], which was will only focus on specific issues and certain special also found for 15-F2t-IsoP and 2,3-dinor-5,6-dihydro-15 cases published in recent years or not included in the

-F2t-IsoP [38]. The vasoconstrictive effect of 15-F2t-IsoP above mentioned review papers. Few studies using could cause reduced glomerular filtration rate and renal F4-NPs in humans will also be summarized. On the other blood flow in the kidney. It also increased pulmonary hand, because EIA is generally not considered as a reli- arterial pressure in the lung and induced bronchocon- able method for detecting F2-IsoPs, only studies using striction of airway smooth muscle [38]. In addition, the mass spectrometric techniques will be included for the cardiovascular effects of 15-F2t-IsoP have also been discussion. shown [43,44]. Belik et al. also discussed the possible Because F2-IsoPs has been proven to be a specific roles of F2-IsoPs on the control of umbilical vasculature marker of oxidative damage, detection of F2-IsoPs can and adverse effects to the fetus [40]. provide solid evidences and new significance that oxida-

F2-IsoPs also exhibits other important biological ac- tive damage is indeed enhanced in the diseased condi- tivities. 15-F2t-IsoP could stimulate production of 1,4,5- tions investigated, which either have never been proved triphosphate and DNA synthesis in rat aortic smooth before or are not well accepted due to the use of unreli- muscle cells [41] and induced cell proliferation, DNA able markers or methods in the literature. Moreover, synthesis, and expression of endothelin-1 in aortic en- F2-IsoPs or F4-NPs might be used as a novel biochemical dothelial cells [45]. These mitogenic effects might play a marker to monitor severity or outcome of diseases based role in pathological processes of vascular systems, such on the correlation between levels of these markers with as the development of atherosclerosis. On the other clinical parameters. The study of Canter et al. first dem- hand, Comporti et al. showed that 15-F2t-IsoP stimulated onstrated that plasma levels of free F2-IsoPs correlated collagen synthesis, DNA synthesis, and cell proliferation with degree of heteroplasmy of the pathogenic mito- in hepatic stellate cells from normal liver; and increased chondrial DNA mutation, mtA8344G mutation causing production of transforming growth factor-β1 in a inherited myoclonic epilepsy and ragged red fibers, in a promonocyte cell line. These effects might be related to large family [55]. Matayatsuk et al. first used F2-IsoPs to the pathogenesis of liver fibrosis [46]. Furthermore, prove that oxidative stress was increased in patients with Benndorf et al. demonstrated that several isoprostanes β-thalassemia by showing that plasma levels of total could inhibit vascular endothelial growth factor-induced F2-IsoPs and urinary levels of free F2-IsoPs, but not lev- angiogensis in vivo, which might be linked to coronary els of erythrocyte MDA measured as thiobarbituric heart disease and capillary rarefaction in diseases with acid-reactive substances, were increased in thalassemic increased oxidative stress [47]. 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 F2-IsoPs and F4-NPs in Clinical Studies improved the diagnostic and predictive outcomes of clinical measures, including quantitative magnetic reso-

The use of F2-IsoPs as a marker of oxidative damage has nance imaging [57]. Kelly et al. first provided the evi- been applied in various clinical studies and elevation of dence for the increase of free F2-IsoPs levels in plasma F2-IsoPs has also been found in different human condi- during acute ischemic stroke [58]. Moreover, Seet et al. tions. Effect of antioxidant intervention on levels of first found that total F2-IsoPs levels in plasma, normal-

F2-IsoPs has also been investigated in many reports. ized by AA concentrations, were higher during febrile Those findings have been discussed in several excellent stage than that convalescent stage in patients with den- review papers [3,4,21,35,40,48-54]. F2-IsoPs was most gue fever [59]. The report of De Felice et al. first dem- well accepted to be increased or play an important role onstrated that plasma levels of F2-IsoPs were increased in the pathogenesis of diseases in the following categories in patients with Rett syndrome, a neurodevelopmental of human diseases: pulmonary diseases, neurodegenera- disorder caused by mutations in X chromosome, and tive diseases, cardiovascular diseases, type 2 diabetes, correlated with greater phenotype severity [60]. Mon-

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Dectection of F2-isoprostanes and F4-neuroprostanes

neret et al. also found that urinary levels of F2-IsoPs damage has indeed suppressed by antioxidant supple- were increased in patients with severe obstructive sleep mentation. However, the effect of antioxidants on apnea and correlated with carotid intima-media thickness F4-NPs remains to be investigated and more studies are and intermittent hypoxia in nonobese patients [61]. needed to address whether suppression of oxidative Results from our laboratory first showed that damage correlated with the improvement of clinical in-

F2-IsoPs and F4-NPs in CSF and plasma levels of F2-IsoPs dices or outcome. 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. Conclusion However, in the study of aSAH, only CSF levels of

F2-IsoPs and F4-NPs, but not plasma F2-IsoPs levels, Although F2-IsoPs is difficult to be measured, the use- correlated with degree of hemorrhage before surgery and fulness of this marker for clinical studies has been poor outcome three months after surgery. Moreover, documented by numerous publications worldwide. levels of F4-NPs, but not F2-IsoPs, in CSF at early time GC/NICI-MS is still the most sensitive and reliable points could predict 3-month outcome. The results not method to routinely quantify large amount of clinical only demonstrated the increase of oxidative damage in samples in all kinds of body fluids, allowing more patients, but also provided mechanistic explanations that meaningful and closer investigation on oxidative stress hemoglobin could be the source of oxidative stress and in affected organs. Detection F2-IsoPs and F4-NPs has neuronal oxidative damage might be the cause of poor offered a cutting-edge tool to reliably assess oxidative outcome. These findings also addressed the issue that stress in vivo with several distinct advantages for clinical body fluids adjacent to the affected organs should be studies, while F4-NPs is a powerful marker to investigate used to get better correlation with clinical parameters neurological disorders. It can be expected that important since any alterations in plasma would be diluted by sys- roles of oxidative stress in human diseases will be fur- temic levels. It might be especially important for neuro- ther explored in more human conditions by using these logical disorders. As shown by studies for AD, only two markers in the future.

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 Acknowledgements hand, our study on aSAH in fact was the only publica- tion detecting F4-NPs in human CSF [8] in the literature This work was supported by grants NSC91-2314-B-182- besides that for AD in the original paper discovering 072, NSC96-2320-B-182-018, and NSC97-2320-B-182-

F4-NPs in humans [18], while our another study for TBI 012-MY3 from National Science Council, Taiwan. The is still ongoing [9]. As to detection of F4-NPs in human establishment of techniques for analyzing F2-IsoPs and brain tissues, it was also only conducted for AD and was F4-NPs by GC/NICI-MS in the author’s laboratory was found to be elevated in AD patients in certain regions of assisted by Dr. Jason Morrow at Vanderbilt University. brain [37,62]. This paper was also written in memory of Dr. Morrow. 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 References many studies [4]. Antioxidant supplementation generally did not affect status of F2-IsoPs in healthy subjects, but 1. Halliwell B, Gutteridge MC. Free Radicals in Biology and Medicine. 4th ed. New York: Oxford University Press, was found to be more effective in lowering F2-IsoPs lev- els in patients with type 2 diabetes and hypercholes- 2007. 2. Morrow JD, Hill KE, Burk RF, et al. A series of pros- terolemia. It also could decrease F2-IsoPs levels in taglandin F2-like compounds are produced in vivo in smokers and ultramarathon runners. From these studies, humans by a non-cyclooxygenase, free radical-catalyzed it was interesting to note that some antioxidants were mechanism. Proc Natl Acad Sci U S A 1990; 87: 9383-7. more effective than the other for different stress condi- 3. Roberts LJ, Morrow JD. Measurement of F2-isoprostanes tions. For examples, different tocopherols, but not vita- as an index of oxidative stress in vivo. Free Radic Biol min C or coenzyme Q10, could decrease F2-IsoPs levels Med 2000; 28: 505-13. 4. Basu S. F2-isoprostanes in human health and diseases: in patients with type 2 diabetes. Therefore, F2-IsoPs can serve as a useful marker to monitor whether oxidative from molecular mechanisms to clinical implications. An-

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綜 論

F2-isoprostanes 和 F4-neuropstanes 之偵測和臨床研究

顏秀娟

長庚大學醫學生物技術研究所/醫學生物技術暨檢驗學系

偵測安定之氧化傷害產物是活體中評估氧化壓力之最可靠做法。F2-isoprostanes (F2-IsoPs)和F4-

neuropstanes (F4-NPs)是最具專一性之脂質過氧化指標，且在臨床研究上，在很多方面較其他氧化

傷害指標具優勢。 F2-IsoPs是各種細胞中富含的花生四烯酸經過氧化作用而產生；而 F4-NPs則自神

經元細胞較多之二十二碳六烯酸生成。而且， F2-IsoPs已知有血管收縮、血小板凝集等生物活性。

氣相層析/負離子化學游離質譜分析法是定量 F2-IsoPs和F4-NPs之參考方法，也是具最高靈敏度之方

法。F2-IsoPs不只是一個在各種體液皆能被偵測到之脂質過氧化黃金指標，而且是一個因其生物活 性而可造成疾病之病因、一個可評估疾病嚴重程度或預測癒後之指標、或是一個監控抗氧化物治

療效果之利器。因需取得腦脊髓液或腦組織，偵測F4-NPs之臨床研究很少，但其在選擇性地評估

神經元之氧化傷害方面較 F2-IsoPs有用。本文將討論以上各議題，並偏重評論臨床研究時之優勢及 考量。

關鍵詞：脂質過氧化、氣相層析 /負離子化學游離質譜分析法、體液、神經元

收稿日期：99 年 3 月 9 日 通訊作者：顏秀娟副教授，長庚大學醫學生物技術暨檢驗學系，桃園縣 333 龜山鄉文化一路 259 號 電話：(03) 2118800 轉 5207 傳真：(03)2118692 電子郵件：[email protected]

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