F2-isoprostane formation, measurement and interpretation: The role of exercise

Progress in Lipid Research 50 (2011) 89–103

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Progress in Lipid Research journal homepage: www.elsevier.com/locate/plipres

Review

F2-isoprostane formation, measurement and interpretation: The role of exercise Michalis G. Nikolaidis a,⇑, Antonios Kyparos b, Ioannis S. Vrabas b a b

Institute of Human Performance and Rehabilitation, Center for Research and Technology – Thessaly, Trikala, Greece Exercise Physiology and Biochemistry Laboratory, Department of Physical Education and Sport Sciences at Serres, Aristotle University of Thessaloniki, Serres, Greece

a r t i c l e

i n f o

Article history: Received 26 August 2010 Accepted 2 October 2010

Keywords: Antioxidant Biomarker Free radical Lipid peroxidation Reactive species Training

a b s t r a c t The level of F2-isoprostanes (F2-IsoP) in blood or urine is widely regarded as the reference marker for the assessment of oxidative stress. As a result, nowadays, F2-IsoP is the most frequently measured oxidative stress marker. Nevertheless, determining F2-IsoP is a challenging task and the measurement is neither free of mishaps nor straightforward. This review presents for the first time the effect of acute and chronic exercise on F2-IsoP levels in plasma, urine and skeletal muscle, placing emphasis on the origin, the methodological caveats and the interpretation of F2-IsoP alterations. From data analysis, the following effects of exercise have emerged: (i) acute exercise clearly increases F2-IsoP levels in plasma and this effect is generally short-lived, (ii) acute exercise and increased contractile activity markedly increase F2-IsoP levels in skeletal muscle, (iii) chronic exercise exhibits trend for decreased F2-IsoP levels in urine but further research is needed. Theoretically, it seems that significant amounts of F2-IsoP can be produced not only from phospholipids but from neutral lipids as well. The origin of F2-IsoP detected in plasma and urine (as done by almost all studies in humans) remains controversial, as a multitude of tissues (including skeletal muscle and plasma) can independently produce F2-IsoP. Ó 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6.

7. 8. 9. 10. 11. 12. 13. 14.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation and nomenclature of F2-IsoP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodologies for measurement of F2-IsoP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Criteria for study inclusion and methodological issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of acute exercise on F2-IsoP levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Skeletal muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of chronic exercise on F2-IsoP levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of F2-IsoP in skeletal muscle and plasma lipids: influence of arachidonic acid content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of F2-IsoP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The origin of F2-IsoP detected in plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The origin of F2-IsoP detected in urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do F2-IsoP measured in plasma or urine after exercise reflect changes in skeletal muscle? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do changes in F2-IsoP after exercise correlate with changes in other markers of lipid peroxidation?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do EIA and MS assays provide comparable results? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F2-IsoP are not simply markers of oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90 90 91 91 92 92 93 93 94 94 94 95 95 96 97 97 98 98 98

Abbreviations: EDL, extensor digitorum longus; EIA, enzyme immunoassay; F2-IsoP, F2-isoprostanes; GC, gas chromatography; HO2 , hydroperoxyl radical; H2O2, hydrogen peroxide; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NO, nitric oxide; O2 , superoxide radical; OH, hydroxyl radical; PLA2, phospholipase A2; RO, alkoxyl radical; ROO, peroxyl radical; TBARS, thiobarbituric acid reactive substances; VO2max, maximal oxygen consumption. ⇑ Corresponding author. Address: Institute of Human Performance and Rehabilitation, Center for Research and Technology, Thessaly, Syggrou 32, 42100 Trikala, Greece. Tel.: +30 24310 63169; fax: +30 24310 63191. E-mail address: [email protected] (M.G. Nikolaidis). 0163-7827/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2010.10.002

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Methodological considerations for measuring F2-IsoP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 15.1. Controlling ex vivo oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 15.2. The choice of matrix: plasma or urine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 15.3. The choice of F2-IsoP isomer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 15.4. The choice of lipid class: free, esterified or total?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 15.5. Expressing F2-IsoP concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

1. Introduction One of the greatest challenges in the field of redox biology is the identification of a reliable non-invasive marker to assess oxidative stress in vivo [1,2]. Most methods available to assess oxidative stress are adequate for in vitro experiments, yet they suffer from a lack of reliability and/or validity in terms of specificity to the substrate oxidized when applied to complex biological fluids and tissues [3]. However, a considerable body of evidence indicates that measurement of isoprostanes (IsoP; prostaglandin-like compounds produced primarily from arachidonic acid catalysed by reactive oxygen and nitrogen species, hereinafter called reactive species) in body fluids such as plasma and urine provides a reliable approach to assess oxidative stress in vivo [4,5]. In fact, the level of one abundant IsoP stereoisomer, 15-F2t-IsoP, in blood or urine is widely regarded as the ‘‘gold standard” marker for the assessment of oxidative stress [6–8]. As a result, the number of studies measuring IsoP levels in the biomedicine field has been increasing exponentially every year since the 1990, when the IsoP were discovered [9]. The trend for increased IsoP measure as a marker of oxidative damage is also noticeable in the redox biology of exercise field, in which IsoP has been currently assessed in more than 60 exercise studies (updated on August 25th, 2010 in PubMed). Nevertheless, determining IsoP is a challenging task and the measurement is neither free of mishaps nor straightforward. In addition, several crucial decisions have to be made before embarking on IsoP measurements. For example, it has to be decided whether to measure free, esterified or total IsoP and whether the assessment will be performed in skeletal muscle, plasma or urine. It is common practice that the type of IsoP and the matrix used to assess them are frequently decided on ‘‘common sense” of what is considered ‘‘the right thing to do” rather on solid principles. The same also holds true for the interpretation of the IsoP changes, particularly considering the dual role of IsoP, that is, as a marker of

oxidative stress and as mediators of vital biological effects [10,11]. In addition, none of the reviews devoted to this topic have examined the role of skeletal muscle or exercise on IsoP production. Exercise is a physiological stimulus that may exert distinctive effects on IsoP metabolism and IsoP levels; in that case a special interpretational framework for the description and comprehension of these effects may be required. As an illustration to this, the typical increase of total antioxidant capacity frequently reported after exercise (e.g. [12,13]) is not related, at least directly, to an orchestrated change in the antioxidant components of plasma, rather it is largely a result of increased uric acid production [13], which is in turn the product of increased ATP degradation during exercise [14]. Therefore, this review aims at presenting for the first time the effect of acute and chronic exercise on IsoP levels in plasma, urine and skeletal muscle, placing emphasis on the origin, the methodological caveats and the interpretation of IsoP alterations. We hope the information presented herein will be appealing to both exercise scientists and biological scientists interested in IsoP biochemistry and physiology.

2. Formation and nomenclature of F2-IsoP Since all the relevant exercise studies have focused on the socalled F2-IsoP of the 15 series (nomenclature explained below), only the production of these IsoP will be presented in this review based on the mechanism proposed by Milne et al. [15], which resides in the earlier work of Pryor et al. [16]. Nonetheless, it is worth mentioning that controversy exists regarding the exact mechanism of F2-IsoP generation [17,18]. Briefly, reactive species can attack arachidonic acid carboxyl chain at three different sites and abstract a bis-allylic hydrogen [15]. Several reactive species can abstract the first hydrogen atom and include the radicals: hydroxyl (OH), alkoxyl (RO), peroxyl (ROO) and possibly hydroperoxyl (HO2 ) but

Fig. 1. Chemical structure of arachidonic acid and of the four F2-IsoP regioisomers. Each of the four regioisomers are comprised of 16 stereoisomers.

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3. Methodologies for measurement of F2-IsoP

Fig. 2. Chemical structure (including relative stereochemistry for the stereogenic centers at C8, C9, C11, C12 and C15) of 15-F2t-IsoP, the most frequently studied IsoP isomer in biology and medicine. For those interested, the structures of all 16 isomers of 15-F2-IsoP are depicted in Rokach et al. [17].

not hydrogen peroxide (H2O2) or superoxide (O2 ) [19]. This process results in a delocalized pentadienyl carbon-centered radical. Subsequently, an oxygen molecule is inserted and a peroxyl radical is formed. These peroxyl radicals undergo further cyclizations, followed by the addition of another oxygen molecule to yield bicyclic endoperoxide molecules. These intermediates are then reduced to F2-IsoP or rearranged to form prostaglandin E2- and D2-regioisomers [15]. Four different F2-IsoP regioisomers (5-, 8-, 12- and 15-series regioisomers – depending on the carbon atom to which the allylic hydroxyl is attached) can be generated, each of the regioisomers theoretically comprised of 8 racemic diastereomers (i.e., 16 stereoisomers; Fig. 1). The most studied of the IsoP is a 15-series IsoP called ‘‘15-F2t-IsoP” (due essentially to its widespread commercial availability). Considering that there are theoretically 3 IsoP classes (i.e., F2-IsoP, D2-IsoP and E2-IsoP; each class comprising 4 series and each series comprising 16 stereoisomers, which sum in total 3  4  16 = 192 isomers – for details, see next paragraph), it is evident that 15-F2t-IsoP represents only a small percentage of the total F2-IsoP and an even smaller fraction of the total IsoP. Noteworthy, although IsoP are regularly reported to be formed by a non-enzymatic (i.e., reactive species-induced) mechanism, formation of IsoP through reactive species-independent mechanisms have also been reported by several groups [20–26]. For a comprehensive overview of the chemistry and biochemistry of IsoP (including cyclic polyunsaturated metabolites formed non-enzymatically) the interested reader is referred to Jahn et al. [27]. Undoubtedly, there is much debate as to the appropriate nomenclature of IsoP, a fact that brings about confusion to the general scientific community. For instance, the three systems proposed by Taber et al. [28], Rokach et al. [17] and Mueller [29] lead to completely different names for the same structures. The Taber et al. [28] nomenclature was approved by the International Union of Pure and Applied Chemistry and it is widely used now in the IsoP field, thus will be used throughout this review to avoid confusion. According to Taber et al. [28], isoprostanes are abbreviated as ‘‘IsoP” and the 3 different classes of IsoP are referred to as F2-, E2- and D2-IsoP (according to their ring substitution pattern). Dehydration of E2- and D2-IsoP produces cyclopentenone-A2- and J2-IsoP both in vitro and in vivo [30]. F2-IsoP are further divided into four series (5-F2, 8-F2, 12-F2 and 15-F2) to refer to each of the different regioisomers that are formed based on the location of the side chain hydroxyl group, with C-1 being the carboxyl group. Based on this nomenclature system, the most frequently studied IsoP is designated as 15-F2t-IsoP, the subscript ‘‘t” indicating that the side chains are oriented trans in relation to the cyclopentane ring hydroxyls (Fig. 2). The formal name of 15-F2t-IsoP isomer is 9a,11a,15S-trihydroxy-(8b,12b)-prosta-5Z,13E-dien-1oic acid and it is also known as 8-iso PGF2a, 8-epi PGF2a, 8-isoprostane, iPF2a-III, 15(S)-8-iso-PGF2a or 15(S)-8-epi-PGF2a. It is worth mentioning that 15-F2t-IsoP is not the most abundant isomer; in fact, other isomers belonging to the 5-F2 series, such as 5-F2c-IsoP, are 20-fold greater than 15-F2t-IsoP in urine [31].

A variety of analytical methods have been developed for the determination of F2-IsoP. These methods include mass spectrometry (MS) detection coupled to gas chromatography (GC) or liquid chromatography (LC) separation, and detection using immunological approaches (both radio and enzymatic). Most of the studies in biology and medicine have measured F2-IsoP employing the first method developed, i.e., through GC–MS. Recently, HPLC coupled with tandem MS (MS/MS) have also been developed. GC–MS methods quantify some or all possible F2-IsoP stereoisomers while LC– MS methods permit separation and identification of selected regioisomers and diastereomers of F2-IsoP [32]. Enzyme immunoassay (EIA) kits allow determination of the single isomer 15-F2tIsoP with minimal cross-reactivity with other IsoP isomers or related compounds according to the manufacturers, although controversy still exists regarding the validity of this technique (described later on in ‘‘13. Do EIA and MS assays provide comparable results?”; [33,34]). A promising but not often used technique to selectively isolate and concentrate 15-F2t-IsoP is immunoaffinity chromatography followed by LC–MS spectrometry and especially MS/MS [35,36]. Indeed, in LC–MS, immunoaffinity column extraction of 15-F2t-IsoP is considered indispensable for accurate analysis and superior to solid phase extraction [36]. Immunoaffinity column chromatography can also be used before using an EIA kit. It is noteworthy, that despite commercially available EIA kits employ an antibody raised against a specific F2-IsoP (i.e., 15-F2tIsoP), cross-reactivity with other isomers and metabolites is also exhibited (for more information, see the relevant kit booklets from Cayman Chemical, Enzo Life Sciences and Oxford Biomedical Research). Furthermore, even in the studies that chromatography coupled with mass spectrometry were employed, identification and quantitation was the result of comparison with authentic samples [37,38]. This means that 15-F2t-IsoP and its enantiomer will appear as single peaks in GC and LC [18]. As a result, unless chiral chromatography or diastereomeric derivatization is used, separation of the enantiomers is not feasible [18]. To this end, in the studies presented in this review, unless a method that clearly detects a single isomer only (regardless of the detection method employed) is implemented, data are expressed as F2-IsoP. Nhe same policy has been followed in most of the papers that will be presented below. For a detailed description of the advantages and disadvantages of the available F2-IsoP analysis methods and techniques, the reader is referred to the following authoritative reviews [36,39,40].

4. Criteria for study inclusion and methodological issues The literature reviewed below refers to both rats and humans. There was no restriction to the type of exercise used. Accordingly, studies that employed either acute or chronic exercise protocols were analyzed. Relevant studies that employed non-physiological exercise models, namely in vitro muscle stimulation [41] or in situ muscle stimulation [42] are also included, mainly because they provide access to skeletal muscle and offer mechanistic insights for the data derived from physiologic models. In addition, no restriction to the technique employed for F2-IsoP determination was applied (i.e., GC–MS, HPLC-MS/MS or immunoassays). Finally, the type of specimen collected and analyzed was delimited to urine, blood and skeletal muscle. These criteria were fulfilled by 45 studies that will be presented hereinafter. Differences described throughout this review as a result of exercise are in reference to the resting condition (when dealing with acute exercise) or the untrained condition (when dealing with chronic exercise). In addition, this comparison refers only to the groups that did not subject to any other intervention (e.g., nutri-

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Significant effects

Non-significant effects

55

F2-IsoP change (%)

63

57 70 69

64 66

67

68

56 43

65

52

44 49 50 53 54 59 45 60

61 62

46

58 47 51

48

71 72 Fig. 3. The effect of acute exercise on plasma F2-IsoP levels. The maximum post-exercise change compared with the resting concentration in plasma F2-IsoP is presented. The number on the bars indicates the number of the reference.

tional, environmental) except for the exercise, that could had independently affected lipid peroxidation levels. Whenever the term significant is used, this reflects the outcome of statistical comparisons reported in the original papers. Data were extracted mostly from figures as well as from text and tables of the original papers. When the effects of exercise are presented cumulatively only the maximum post-exercise change compared with the resting concentration in F2-IsoP is presented (in cases where multiple samplings took place). For the sake of simplicity, we use the term ‘‘plasma” even for studies in which serum was actually analyzed. The vast majority of the studies apparently measured free F2-IsoP and only two of them total F2-IsoP in plasma (i.e., free and esterified; [43,44]). It is worth mentioning that most of the studies did not explicitly report the type of F2-IsoP measured (i.e., free, esterified or total). This limitation can be overcome to a degree if an alkaline hydrolysis step resulting in the release of the esterified F2-IsoP was also performed in these studies. In this case the type of F2-IsoP measured could be deduced more accurately. The above mentioned limitation does not apply if F2-IsoP were determined in urine because the lipid content in the urine is minimal [9]. Therefore, F2-IsoP in the urine is free F2-IsoP rather than esterified. On the other hand, it is indispensable to report the type of F2-IsoP measured when the matrix contains substantial amount of lipids, as it is the case in plasma and skeletal muscle. 5. The effect of acute exercise on F2-IsoP levels 5.1. Plasma As Fig. 3 shows, 20 of the total 30 relevant human and animal studies reported significantly increased levels of plasma F2-IsoP after exercise compared to resting levels [43–62], whereas the rest of them reported no significant changes [63–71], except for one study that reported significantly decreased levels after short-term (14 min) intense exercise [72]. The significant increases in F2-IsoP ranged from 13% to 181% (on average 59%). It is worthy of note that 7 of the 9 studies that found non-statistically significant effects of exercise on plasma F2-IsoP levels reported relatively large increases in plasma F2-IsoP levels ranging from 3% to 150% (on average 54%) [65,66,68–70], whereas the other two studies reported either marginal decreases ( 1.9%; [71]) or virtually no changes

[64] after exercise compared to baseline values. It appears that if the statistical power in these studies was higher, increased lipid peroxidation levels post-exercise would have been found in most of them. Collectively, we believe that there is strong evidence – although non-statistically justified in some cases – that acute exercise increases F2-IsoP levels in plasma post-exercise. A worth asking question derived from the findings of the studies mentioned above is how long after exercise the reported changes in plasma F2-IsoP would last. Addressing this question is important, because it will provide useful information regarding the proper sampling time-points after exercise. However, this is a difficult question to answer given that in the majority of the relevant studies that reported significant effects of exercise on plasma F2-IsoP samples were collected just immediately after exercise (1– 15 min after exercise; [45,47,52,56–59,72,73]) or at some other early post-exercise point (up to 90 min post-exercise; [46,48,53– 55,62]). In the rest of the relevant studies plasma samples were collected up to 24 h post-exercise [43,44,49,51,60,61], except for one study in which samples were collected up to 5 days post-exercise [50]. Judging from the studies that measured F2-IsoP levels immediately after exercise and at least one late post-exercise time point (i.e., 1 h – 5 days) it appears that F2-IsoP levels return to baseline values in less than 1 h after exercise cessation [43,44,48,50,51,54,55,61]. Nevertheless, three studies that provided early along with late post-exercise data, found increased levels of F2-IsoP at 1 h [46,49] and at 24 h [60] post-exercise. Based on these rather fragmentary data it seems that the effect of acute exercise on F2-IsoP levels of plasma is generally short-lived. This is supported by the relatively short elimination half-life of 15F2t-IsoP in plasma, which was reported to be about 4 min in rabbits [74] and 16 min in rats [75]. Undoubtedly, similar kinetic data for humans are eagerly needed. Another interesting question related to the issue discussed in this review is whether antioxidant supplementation affects the response of plasma F2-IsoP levels to acute exercise. This research is pivotal taking into account that many athletes consume antioxidants in the belief they will reduce cell damage and attenuate fatigue during daily training. In fact, undertaking such practices may even attenuate muscle adaptations to chronic exercise [76,77], although ergogenic [78] or no effects [79] of antioxidant supplementation on human performance have also been presented.

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Mastaloudis et al. [50] examined the effect of 6 weeks of supplementation with ascorbic acid (1000 mg per day) and a-tocopherol (300 mg per day; the vitamin E form most intensely investigated and the major lipid soluble antioxidant of lipoproteins and membranes; [80]) in trained but non-competitive runners during and after a 50 km ultramarathon. Although plasma F2-IsoP levels were similar in placebo and supplementation groups at baseline, F2-IsoP increased during the run only in the placebo group. Immediately post-race, when oxidative stress was maximal, F2-IsoP concentrations were negatively correlated both with a-tocopherol (r = 0.78) and with ascorbic acid (r = 0.64), providing further evidence that antioxidants were at least partially responsible for preventing lipid peroxidation. Briefly, antioxidant supplementation completely inhibited exercise-induced lipid peroxidation but had no effect on resting levels of lipid peroxidation. Based on this finding, it is probable that the resting levels of many oxidative stress markers (including F2-IsoP) can give much less information compared to the ones modified by an acute exercise session. In other words, it may be easier to find an effect of antioxidant supplementation on blood redox status after exercise than at rest. This renders exercise as a convenient model to study redox status homeostasis. Sacheck et al. [70] investigated the effects of 12 weeks of supplementation with a-tocopherol (1000 mg per day) in young and elderly individuals after a downhill run for 45 min at 75% VO2max (maximal oxygen consumption) and collected blood samples immediately post-exercise as well as at 6 h and 1 and 3 days post-exercise. It is worth noting, that the exercise protocol chosen (i.e., downhill run) induces severe muscle damage that peaks 2– 3 days post-exercise [81,82]. Therefore, to collect blood samples up to 3 days post-exercise proved a prudent choice since the effect of downhill run on F2-IsoP in plasma was delayed and appeared only at 1 and 3 days post-exercise in both young and elderly men before supplementation. After supplementation in young men, atocopherol prevented any significant rise in F2-IsoP following exercise up to 3 days, however, baseline post-supplementation levels were actually higher than that detected in the pre-supplementation visit. On the other hand, a-tocopherol reduced plasma F2-IsoP in the old men at baseline and at day 1 post-exercise. Thus, atocopherol supplementation suppressed some post-exercise elevations in plasma F2-IsoP at different time-points between young and elderly men as well as acted as a pro-oxidant at baseline values in young men and as an antioxidant in elderly men. Watson et al. [83] determined the effects of a 2-week dietary antioxidant restriction on plasma F2-IsoP in trained athletes. The exercise protocol consisted of initial run for 30 min at 60% VO2max, followed by an incremental running test to exhaustion. The dietary intervention made a significant difference to F2-IsoP in response to exercise. This difference was not evident at rest where similar concentrations of F2-IsoP were observed after implementing either the habitual or restricted antioxidant diets, indicating that both diets were equally capable of inhibiting the rate of reactive species generation at rest. Conversely, despite the fact that post-exercise plasma F2-IsoP levels did not alter following the habitual diet (which was rich in antioxidants), they did increase following the restricted antioxidant diet. This is suggesting that antioxidant capacity was reduced in antioxidant restricted individuals and were not as capable to mitigate the increase in reactive species production during exercise. This study corroborates the findings of Mastaloudis et al. [50] pointing out that an exercise stimulus was required for bringing up the effects of modifications in antioxidant consumption. McAnulty et al. [55] examined the effect of 2-month supplementation with a-tocopherol (542 mg per day) on plasma F2-IsoP levels in trained athletes after a triathlon race. Plasma F2-IsoP increased during the race in both the supplemented and placebo groups, even though, the post-exercise increase was higher in the a-tocopherol-supplemented group. Namely, this study indicated

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that prolonged doses of a-tocopherol supplementation provoked a pro-oxidant state. Additionally, based on the fact that baseline F2-IsoP values did not differ between the two groups, this study supports the notion, also substantiated by Mastaloudis et al. [50] and Watson et al. [83], that exercise may serve as stimulus for discriminating whether or not a change in redox status takes place. Nieman et al. [59] studied the influence of 1-week supplementation with ascorbic acid (1500 mg per day) on plasma F2-IsoP during and after an 80 km ultramarathon in trained runners. F2-IsoP values were increased during running in both ascorbic acid and placebo groups, indicating that the ultramarathon run induced oxidative stress. Nevertheless, F2-IsoP tended to be higher in the ascorbic acid group compared with the placebo group both at rest and during exercise (P = 0.051). These data indicate that ascorbic acid supplementation, despite not fully justified statistically, may act as a pro-oxidant at rest as well as during and after a competitive ultramarathon race. In summary, the studies investigated the effect of antioxidant supplementation on F2-IsoP levels during exercise reported divergent results, including increased F2-IsoP levels [55,59], decreased F2-IsoP levels [50] or mixed outcomes [70]. These discrepancies may be explained-among others-by differences in antioxidant type, dosage and duration of supplementation as well as training state and type of exercise. Probably the most interesting finding emanated from this limited research is that supplementation with ascorbic acid [59] or a-tocopherol [55] in some cases may not decrease, as intuitively expected, but rather augment lipid peroxidation. There is an ongoing discussion in the literature on the possible pro-oxidant role of ascorbic acid [84] and a-tocopherol [80]. 5.2. Urine Nieman et al. [69] determined the response of F2-IsoP levels after a triathlon race lasting approximately 12 h, collecting urine samples shortly after exercise and not 12–24 h post-race, which is the usual time window in similar cases. F2-IsoP markedly increased by 89% at 5–10 min post-race and by 107% at 1.5 h postrace. Apparently, there was enough time for IsoP to accumulate in urine during the 12 h of the run. On the contrary, McAnulty et al. [68] did not report any effect of a 160-km ultramarathon lasting approximately 26 h on F2-IsoP levels in urine collected 5– 15 min after the race. Lastly, Rietjens et al. [85] found that the levels of F2-IsoP in urine collected 24 h after a single session of resistance exercise increased by 40% compared to the pre exercise baseline values. No doubt, further research is needed to confirm or not whether acute exercise is able to increase the levels of F2IsoP in urine. 5.3. Skeletal muscle Only one study has used exercise as a physiological model to examine the effects of physical activity on F2-IsoP levels in skeletal muscle (Fig. 4). In detail, Karamouzis et al. [86] evaluated the response of muscle interstitial F2-IsoP levels during intensive cycle exercise. To accomplish this, the researchers employed the in vivo microdialysis technique, which permits access to a specific site of F2-IsoP production, thus allowing the continuous measurement of F2-IsoP at rest and during exercise [87]. They reported that the levels of F2-IsoP in the interstitial fluid (i.e., the extracellular solution that bathes and surrounds the muscle cells) of the vastus lateralis muscle of humans increased by 193% during exercise. The most interesting finding emerged from this study is that large amounts of F2-IsoP rapidly escape from skeletal muscle cells during exercise and diffuse into the interstitial space before entering into blood via blood capillaries.

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500

F2-IsoP (pg/mL)

400 300 200 100 0 At rest

During exercise

Fig. 4. F2-IsoP concentration in the interstitial fluid of skeletal muscle during exercise. The dialysate fluid samples were collected after an equilibration period of 20 min at rest and during 30 min of cycling exercise. *P < 0.05 (modified from Karamouzis et al. [86] with kind permission from Elsevier Ltd.).

The rest of the studies that measured F2-IsoP levels in skeletal muscle have employed exercise animal models that do not adequately mimic human physical activity (according to the classification by Booth and Thomason [88]). Goodman et al. [41] examined the effects of in vitro electrical stimulation of extensor digitorum longus (EDL) muscle in rats. EDL muscles were subjected either to 10-s continuous stimulation at a frequency of 100 Hz or 3-min intermittent stimulation (1-s stimulation at 100 Hz followed by 4-s recovery). F2-IsoP production during fatiguing in vitro contractions increased by 55% only after the 3-min intermittent stimulation. Moreover, two independent studies from the same research group, reported either 56% [89] or 158% increase [42] of F2-IsoP levels in tibialis anterior muscles of rats subjected to 1-min in situ contractions at a frequency of 10 Hz. In summary, based on the rather limited data available, it seems that acute exercise [86] and increased contractile activity [41,42,89] noticeably augments the production of F2-IsoP in skeletal muscle. 6. The effect of chronic exercise on F2-IsoP levels

6.1. Plasma Changes in plasma F2-IsoP levels in response to chronic exercise are less apparent than those detected in response to acute exercise. A probable reason could be the much smaller magnitude of the chronic changes in the concentration of plasma F2-IsoP compared with the acute changes. Moreover, obtaining a clear picture is confounded by the insufficient number of relevant studies on chronic exercise. Galassetti et al. [90] investigated the effects of chronic exercise for 7 days, 3 h/day at 75% VO2max on plasma F2-IsoP levels in humans. Caloric intake was either 110% or 75% of caloric expenditure. Plasma F2-IsoP were similarly reduced after 7 days of exercise in the high-calorie ( 23%) and low-calorie ( 31%) groups. Interestingly, this reduction below baseline levels was still present 1 week after the end of the exercise program ( 20% in the high-calorie group and 23% in the low-calorie group). Based on these data, a reduction in plasma lipid peroxidation may occur relatively early during intense chronic exercise training, independently of caloric intake. Kelly et al. [91] examined the influence of chronic exercise on plasma F2-IsoP levels in overweight children. The exercise intensity and duration was gradually increased throughout the course of the 8-week training program (from 55% at week 1 for 30 min to 75% at week 8 for 50 min per session). Chronic exercise did not affect the resting levels of F2-IsoP, despite a non-significant 14% decrease was noted. Likewise, Moien-Afshari et al. [92] reported a non-signifi-

cant 11% decrease in plasma F2-IsoP levels in mice exercised for 8 weeks. In a cross-sectional study, Watson et al. [93] compared the resting levels of plasma F2-IsoP between trained and untrained individuals. They also reported a non-significant decrease in plasma F2-IsoP levels by 18% in trained individuals compared to sedentary controls. Collectively, despite 3 of the 4 relevant studies [91–93] reported non-significant differences after chronic exercise (employing either longitudinal or cross-sectional designs) and only one reported reduced F2-IsoP levels, a trend for decreased values in plasma F2-IsoP after chronic exercise is evident. An indirect support to these findings is the reduced levels by 11% of arachidonic acid detected in the phospholipid fraction of plasma after chronic exercise [94]. Since F2-IsoP are rapidly cleared from the circulation, measurements of F2-IsoP in plasma may not reliably reflect the chronic stress imposed by long-term exercise programs. 6.2. Urine Campbell et al. [95] examined the effect of a yearlong exercise intervention on spot urine F2-IsoP in 173 sedentary, overweight or obese and postmenopausal women. The exercise intervention progressed to P45 min per day of moderate-intensity aerobic exercise (60–75% of maximal heart rate), 5 days per week, by the 8th week of the trial, where it was maintained to the end of study. The authors observed a modest reduction ( 6%) in F2-IsoP overall that was not statistically significant. However, when participants were stratified by gain in aerobic fitness, exercisers who increased VO2max more than 15% exhibited significantly decreased levels of F2-IsoP by 14%. These data suggest that aerobic exercise reduces systemic lipid peroxidation levels only when accompanied by relatively marked gains in aerobic fitness. Cornish et al. [96] examined the effects of 5 weeks of resistance exercise on a 24-h urine sample obtained before and immediately after the last training session. They reported no significant effects despite the fact that the levels of F2-IsoP increased by 17%. However, considering that the ‘‘training” urine sample was collected immediately after the last exercise session, at least part of this increase may be due to the acute effects of the last training session. Devries et al. [97] investigated the effects of 12 weeks of endurance chronic exercise on F2-IsoP in a 24-h urine sample. The protocol commenced with two 15-min cycling sessions at 50% VO2max per week during the first week and increased to three 60-min cycling sessions at 65% VO2max per week during the final 2 weeks of training. Chronic exercise lowered the levels of urine F2-IsoP by 38%. Margonis et al. [98] examined the response of urine F2-IsoP to a chronic resistance exercise protocol of progressively increased and decreased training load. The protocol was designed to elicit an overtraining state and to cause severe muscle damage. In other words, the protocol was very demanding to induce performance deterioration instead of enhancement. Twelve males participated in a 12-week resistance training consisting of five 3-week periods (of progressively increased intensity and volume), followed by a 3week period of complete rest. Urine samples were collected at baseline and 3 days following the last training session of each period. F2-IsoP increased following light exercise (2.4-fold), intense exercise (4-fold), and overtraining exercise (7-fold), whereas normalized after the recovery. It is worth mentioning that F2-IsoP was the only oxidative stress marker (along with the reduced-tooxidized glutathione ratio from the total of 9 markers) that positively correlated with performance drop (from r = 0.77 to 0.93 in four different performance tests) and training volume increase (r = 0.81) during the overtraining state. This study indicated that exercise-induced overtraining elicits a significant increase in urine F2-IsoP in humans, which was proportional to the training load im-

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posed on the subjects, indicating that F2-IsoP may be a promising overtraining marker. Mori et al. [99] examined the effects of 8 weeks of aerobic exercise (55–65% VO2max 3 days per week) on urine F2-IsoP in untrained, dyslipidemic and non-insulin-dependent diabetes mellitus patients. In addition, all subjects consumed diets that supplied less than 30% of total energy as fat. The 24-h urine collected at baseline and 2 days after the final exercise period at completion of the intervention. The exercise and nutritional intervention did not alter the levels of F2-IsoP. Moreover, and contrary to Campbell et al. [95], there was no significant correlation between F2-IsoP and aerobic fitness (i.e., VO2max) either at baseline or post-exercise. Schmitz et al. [100] explored the effect of 15 weeks of aerobic exercise on F2-IsoP in 24-h urine collected 2 days after the last exercise session. The 5 weekly exercise training sessions included 30 min of treadmill or elliptical exercise. Exercise intensity was 70–75%, 75–80% and 80–85% of maximal heart rate for weeks 1– 5, weeks 6–10 and week 11 to end of intervention, respectively. The long aerobic training resulted in a 34% decrease in urine F2IsoP. Wang et al. [101] studied the effects of chronic exercise on urine F2-IsoP. The exercise training consisted of running on a treadmill, at an intensity of about 70% VO2max for 60 min per session for 5 days per week for 10 weeks. To avoid the potential effects of acute exercise, rats were killed 2 days after the last training session. The trained group had 2.7-fold lower urinary excretion of F2-IsoP than the control sedentary group. Based on these data, it seems that the levels of urine F2-IsoP are decreased with chronic exercise in most of the cases [95,97,100,101], while some studies have supported no changes [96,99]. More interestingly, chronic exercise may rarely result in increased urine F2-IsoP levels. Indeed, Margonis et al. [98] observed that when the training volume was exceptionally high, then urine F2-IsoP levels may actually increase. Certainly, valid conclusions based solely on a single study cannot be drawn and further studies are needed to confirm or not these findings. To our knowledge, no study has investigated the influence of chronic exercise on the levels of F2-IsoP in skeletal muscle.

7. Formation of F2-IsoP in skeletal muscle and plasma lipids: influence of arachidonic acid content For a sound interpretation of the changes in F2-IsoP levels that appear during exercise it is essential to appreciate the basic chemistry and biology behind F2-IsoP formation and metabolism. F2-IsoP are a series of compounds formed as a result of lipid peroxidation of arachidonic acid initiated by reactive species. To our opinion, a frequently reproduced view, which is rarely being sufficiently supported, is that F2-IsoP are formed only in situ in esterified arachidonic acid. However, there is no apparent restriction for the formation of F2-IsoP to also occur in the non-esterified (i.e., ‘‘free”) arachidonic acid. Even if this possibility is actual, though, the content of non-esterified arachidonic acid in tissues, plasma and urine is miniscule, and as a result, the vast majority of F2-IsoP are apparently bound to be formed in esterified arachidonic acid. Indeed, non-esterified arachidonic acid in skeletal muscle made up about 0.0042% of total lipids in rats [102]. Similarly, non-esterified arachidonic acid in plasma made up about 1.2% of total lipids in humans [103]. However, it is worth mentioning that concentration of arachidonic acid is almost doubled after acute exercise in the non-esterified fraction of plasma [104,105]. Formation of F2-IsoP in urine is also regarded as negligible, since the total lipid content in urine is minor (total lipids in urine are about 11 nmol/L [106], which is about three order of magnitude lower than that of plasma [104] and skeletal muscle [107]). In fact, the concentration of ara-

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chidonic acid in urine (apparently non-esterified) reported to be below the detection limit of 50 nmol/L [108]. Having confirmed the miniscule content of non-esterified arachidonic acid in tissues and fluids, it is important now to deal with another frequently encountered statement in the relevant literature, namely F2-IsoP are formed by oxidation of arachidonic acid esterified only in phospholipids. Despite this undoubtedly account for a considerable amount of F2-IsoP formed in skeletal muscle and plasma, neutral lipids (primarily monoacylglycerols and cholesterol esters) may also account for a significant amount of F2-IsoP generation. Regarding skeletal muscle, the content of arachidonic acid esterified in monoacylglycerols of muscle is approximately 61.7% of total arachidonic acid, whereas the content of arachidonic acid esterified in phospholipids of skeletal muscle is approximately 19.1% of total arachidonic acid [102]. On the other hand, in plasma, about 32.5% of arachidonic acid is esterified to neutral lipids [29.8 in cholesterol esters (i.e., cholesteryl arachidonate) and 2.8% in triacylglycerols (i.e., a triacylglycerol with one arachidonoyl group)], 48.9% in phospholipids and the remaining 18.6% in other lipids (mainly non-esterified fatty acids, monoacylglycerols and diacylglycerols) [103]. No relevant data could be found for the arachidonic acid content in urine, but based on the very low lipid content of urine [106], potential differences in esterification degree of arachidonic acid between neutral lipids and phospholipids is rendered meaningless. Noteworthy, it has been shown that the amount of arachidonic acid alone could not account for the different F2-IsoP levels detected in LDL and HDL of plasma [109]. The above suggests that in addition to abundant parent lipid, other factors, such as the peroxidizability of non-esterified arachidonic acid compared to esterified arachidonic acid, may also affect the formation of F2-IsoP. Indeed, non-esterified fatty acids, the only lipid class in plasma not transported in lipoproteins but bound to albumin, were found to be preserved from peroxidative damage compared to esterified fatty acids, most likely due to site-specific antioxidant protection by albumin-bound bilirubin and possibly by albumin itself [110]. On the other hand, there is no doubt that the content of arachidonic acid affects to some extent the rate of F2-IsoP formation [22]. Based on this analysis, it seems that (i) arachidonic acid (i.e., the parent lipid of F2-IsoP) is located in significant quantities in both neutral lipids and phospholipids in skeletal muscle and plasma, (ii) arachidonic acid is found in different esterification ratios in skeletal muscle and plasma lipids, and as a result, the phospholipid-derived arachidonic acid may contribute to a higher degree to F2-IsoP formation in plasma than in skeletal muscle and (iii) theoretically, both skeletal muscle and plasma can independently produce F2-IsoP.

8. Metabolism of F2-IsoP First, we have to stress that the metabolism of F2-IsoP has received scant attention and that the relevant information presented herein is not derived from studies in skeletal muscle. Most of the studies that dealt with the metabolic fate of F2-IsoP measured a single F2-IsoP isomer (mostly 15-F2t-IsoP) and its major metabolites in plasma and/or urine of animals and humans [74,111– 117]. In addition, two of these studies determined the major metabolites of F2-IsoP in hepatocyte cultures [112,118]. As a result, caution should be exercised in the extrapolation of findings from these studies to what is happening in vivo in skeletal muscle of animals and humans at rest or during exercise. Skeletal muscle is able to produce reactive species during increased contractile activity [119,120], thus providing the ‘‘spark” for lipid peroxidation. Most of the data available indicate that the production of intracellular reactive species is increased by 2- to 4-fold during skeletal muscle contractions [121]. The primary reac-

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tive species generated by resting and contracting skeletal muscle are the relatively poorly reactive nitric oxide (NO) and O2 , the latter dismutating rapidly to form H2O2 [122]. In the presence of catalytic transition metals H2O2 forms the considerably more reactive  OH, which can readily initiate peroxidation of arachidonic acid [122]. Following formation primarily from esterified arachidonic acid, F2-IsoP are presumably hydrolyzed into free form likely by the actions of phospholipase A2 enzymes (PLA2; [123]). Phospholipases A2 specifically hydrolyze the sn-2 ester bond of phospholipids [124] and are found in both skeletal muscle [125] and plasma [126]. This de-esterification step might perhaps be viewed as one of the rate-limiting steps for the release of free F2-IsoP in skeletal muscle and later on in the peripheral circulation and urine [127]. It is important to note that acute exercise has been found to increase PLA2 in rat skeletal muscle [125,128,129]. This fact indicates that the increased free F2-IsoP concentration in skeletal muscle, plasma and urine after exercise reported in many studies may have appeared in part due to increased activity of PLA2 in skeletal muscle and not exclusively due to increased reactive species generation. Moreover, this analysis suggests that when there is a need to control the contribution of PLA2 on free F2-IsoP levels in a tissue, it may be more appropriate that the esterified F2-IsoP rather than free F2-IsoP are measured, since concentration of the former is likely affected much less by PLA2 activity. This is because the levels of esterified F2-IsoP are about 5-fold higher than those of free F2IsoP in human plasma [130]. As presented in the previous section, considerable amount of arachidonic acid is esterified as cholesteryl esters and could serve as the substrate for F2-IsoP formation [131]. If this indeed happens, then an enzyme capable of hydrolyzing cholesteryl esters (i.e., a carboxyl ester lipase; [132]) should be available in skeletal muscle for facilitating the release of the F2IsoP esterified to a cholesteryl ester. Whether or not such an enzyme is important in cholesteryl arachidonate degradation in skeletal muscle remains to be examined. A crucial question that needs to be addressed is whether F2-IsoP produced in skeletal muscle are released in plasma and urine in their original form (i.e., unmetabolized). For a relatively small amount of F2-IsoP the answer is definitely yes. However, several major F2-IsoP metabolites have been detected either in plasma or urine. This indicates that F2-IsoP are extensively metabolized and, as a result, can escape continuous monitoring. This contrasts with the usual notion that F2-IsoP are thought to be chemically stable end-products and therefore superior analytical marker of lipid peroxidation compared to rapidly decomposing lipid hydroperoxides (e.g., [133]). The most informative (in quantitative terms) metabolic data were provided by Basu [74]. In this investigation, the labelled 15-F2t-IsoP administered intravenously to rabbits was eliminated rapidly from the circulation. Even at 1.5 min after administration, 19% of the plasma radioactivity represented by 15-keto-15-F2t-IsoP (a metabolite of 15-F2t-IsoP) and 13% represented by several b-oxidised products. At 20 min after administration plasma values were just 5% for 15-F2t-IsoP and 88% for boxidised products. In urine, at 20 min following administration only 2% of the total radioactivity represented unmetabolized 15F2t-IsoP. In rabbits, the major metabolite of 15-F2t-IsoP in the urine was a-tetranor-15-keto-13,14-dihydro-15-F2t-IsoP (a b-oxidised product). On the other hand, in monkeys and a human volunteer, Roberts et al. [115] reported that 2,3-dinor-5,6-dihydro-15-F2tIsoP (a b-oxidation product as well) was the major urinary metabolite, accounting for about 20% of total excreted products following infusion of the parent compound. In a second study from the same group, Morrow et al. [113] verified that 2,3-dinor-5,6-dihydro-15F2t-IsoP is the major urinary metabolite of the 15-F2t-IsoP in vivo in humans. In addition to confirming the presence of 2,3-dinor-5,6dihydro-15-F2t-IsoP reported by Morrow et al. [113] and Roberts

et al. [115], Chiabrando et al. [112] have detected another major b-oxidation product (2,3-dinor-15-F2t-IsoP) of similar abundance to be excreted under basal conditions in rat and human urine. Similarly, Schwedhelm et al. [116] have shown that the basal human urinary levels of 2,3-dinor-5,6-dihydro-15-F2t-IsoP were found to be more than 2-fold higher than those of the parent compound (i.e., 15-F2t-IsoP). Likewise, Nourooz-Zadeh et al. [114] reported that the levels of the two major 15-F2t-IsoP metabolites in urine were 15-fold higher (for 2,3-dinor-15-F2t-IsoP) and 6-fold higher (for 2,3-dinor-5,6-dihydro-15-F2t-IsoP) than the parent compound. In agreement to the previous study, Yan et al. [117] reported that the levels of 2,3-dinor-15-F2t-IsoP was 6 times higher than that of 15-F2t-IsoP. Collectively, it has been shown that 2,3-dinor-15F2t-IsoP is the major b-oxidation product present followed by 2,3-dinor-5,6-dihydro-15-F2t-IsoP whilst the parent compound (15-F2t-IsoP) is only a minor component in plasma and in urine. As a final note, it is worth mentioning that statistical correlations between the values of 15-F2t-IsoP and its metabolites were performed in three studies and that either moderate to high (r = 0.76; [117] and r = 0.86; [116]) or almost perfect correlation coefficients (r = 0.94–0.99; [114]) were reported. The strong correlation appeared between 15-F2t-IsoP and its metabolites can be explained on the basis of substrate-to-product relationship. It also indicates that 2,3-dinor-15-F2t-IsoP and 2,3-dinor-5,6-dihydro15-F2t-IsoP could be used as markers of F2-IsoP production at least as informative as the parent compound (i.e., 15-F2t-IsoP).

9. The origin of F2-IsoP detected in plasma The main aims of this section are to present evidence that formation of IsoP are likely to occur in blood plasma as in the tissues and originate from cholesteryl esters as from the phospholipids. The first objective seems straightforward, as all the necessary ‘‘ingredients” for IsoP formation are available in plasma. Indeed, reactive species are found in plasma mainly through reactions with metals [134]. In addition, the levels of reactive species in human plasma increase during exercise generally 2- to 3-fold compared to resting levels as detected by electron paramagnetic spectroscopy [119,135,136]. Regarding the oxidizable substrate, as shown previously, plasma contains significant amounts of arachidonic acid, which is mainly found in lipoproteins as phospholipid and cholesteryl esters [131,137]. Supporting the above hypothesis, it has been demonstrated that in vitro incubation of fresh human plasma with 2,2’-azobis(2-methylpropionamidine) dihydrochloride (a compound producing peroxyl radicals) increased F2-IsoP levels [138]. Similar results reported by Waugh and Murphy [139], who identified several IsoP isomers after autoxidation (using copper(I) chloride and H2O2 to generate OH) of synthetic arachidonic acid in vitro. Furthermore, Proudfoot et al. [109] detected F2-IsoP in isolated lipoproteins and found that HDL is the major lipoprotein carrier of F2-IsoP in human plasma. Based on the discussion above, it follows that the F2-IsoP detected in plasma may not have been necessarily produced inside skeletal muscle or inside any other tissue. Despite the fact that this appears a sound idea, it is often overlooked when data regarding exercise-induced oxidative stress/damage are interpreted in original research articles. Thus far, F2-IsoP have been reported to be derived from arachidonic acid esterified in phospholipids [9,140]. However, there is no apparent reason to believe that F2-IsoP will not be also formed from arachidonic acid esterified in other lipids, as shown by a mechanistic study performed in vitro [141]. Identification of oxidation products of cholesteryl arachidonate is not possible through conventional techniques (i.e., purification by chromatography and characterization by spectroscopy; [142]). In response to this

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numerous (if not all) tissues of the body or (part of them) are derived from F2-IsoP produced locally in the kidney. Even though there is no doubt that unmetabolized IsoP are excreted in urine, the quantitative dimensions of this excretion have been described by only one study [74]. Based on this study, F2-IsoP are quickly eliminated from the urine: at 20 min after intravenous infusion only 2% of the F2-IsoP isomer appeared in urine in its original form [74]. On the other hand, not surprisingly, evidence indicates that renal tubular epithelial cells and whole kidneys subjected to cold storage for few days were able to independently produce considerable amounts of F2-IsoP [154]. Based on this scarce evidence, kidneys can be considered as a site of both F2-IsoP production and clearance. Additional studies are required to reveal the exact quantitative contribution of kidneys in urine F2-IsoP levels either at rest and/or after exercise. As a general note, the effect of exercise on oxidative stress markers clearance has been unduly neglected; to the best of our knowledge, this issue has been addressed only by one study published almost two decades ago [155].

deficit, Yin et al. [141] developed a method for the analysis of complex mixtures of peroxide compounds based upon Ag+ coordination ion-spray mass spectrometry. These investigators managed to identify bicyclic endoperoxides from cholesteryl arachidonate. Formation of bicyclic endoperoxides has been implied in the non-enzymatic reactive species oxidation of arachidonic acid or its esters and these endoperoxides are presumed intermediates in the formation of IsoP [141]. Consequently, IsoP can be theoretically formed from sources other than arachidonic acid esterified to phospholipids. Based on the studies presented in the previous discussion, it cannot be concluded whether the F2-IsoP detected in blood plasma are generated in plasma and/or are derived from extra-vascular tissues. It is still possible that F2-IsoP are generated and released in the circulation from blood cells and/or vascular endothelium. Beyond doubt, the production of F2-IsoP in skeletal muscle is augmented after acute exercise [86] and increased contractile activity [41,42,89]. Moreover, considerable quantities of F2-IsoP have been detected in erythrocytes [143], neutrophils [144], lymphocytes [145] and platelets [146]. Furthermore, F2-IsoP have been detected in vascular endothelial cells [147], which some of them are in direct contact with the blood flow. Studies that will examine the contribution of blood cells and tissues to plasma F2-IsoP levels and reveal their complex interactions are warranted. Special attention should be paid to the potential role of hemolysis in the increased plasma F2-IsoP levels after exercise. Increased short-lived (up to few hours post-exercise) hemolysis after nonmuscle-damaging exercise is a well-described phenomenon in the relevant literature [148]. Moreover, marked and long-lived (up to 4 days post-exercise) increases in hemolysis have been reported after muscle-damaging exercise [149]. Taking into account that the levels of F2-IsoP in erythrocytes are 10- to 18-fold higher compared to those in plasma [143,150], moderate hemolysis could produce considerable increases in plasma F2-IsoP. Furthermore, lipid peroxidation may be indirectly facilitated by the release of the high polyunsaturated fatty acid and iron content of erythrocytes into the plasma [151]. Independently of exercise, hemolysis also appears unavoidably during plasma separation due to the contact of blood to foreign surfaces and due to centrifugation [152]. To partially control the influence of erythrocyte lysis on the levels of F2IsoP measured in plasma, hemolysis markers (such as plasma hemoglobin or haptoglobin) may also be determined. In a recent study, Dreissigacker et al. [153] offered a partial support to the above argument reporting a weak positive correlation (r = 0.34) between 15-F2t-IsoP and hemoglobin concentration in plasma under resting and exercise conditions.

11. Do F2-IsoP measured in plasma or urine after exercise reflect changes in skeletal muscle? One of the central assumptions in studies measuring oxidative stress markers in plasma or urine after exercise is that these markers reliably reflect the tissue redox status of interest (mostly skeletal muscle in exercise studies). To test whether changes in plasma or urine F2-IsoP levels reflect alterations in tissue F2-IsoP levels, F2IsoP concentration has to be determined in both fluids and tissues after implementing a treatment known to affect redox status (e.g., exercise or antioxidant administration). To our knowledge, no study has investigated whether F2-IsoP measured in plasma or urine adequately depict oxidative stress in tissues after exercise. Nevertheless, useful information can be derived by examining the trend of change in F2-IsoP measured in plasma or urine and tissues after implementing treatments known to affect oxidative stress. As Table 1 shows, most of the relevant studies reported changes toward the same direction in F2-IsoP measured simultaneously in plasma or urine and tissues after various experimental interventions [75,156–163]. For example, Awad et al. [156] examined the effect of diquat (a herbicide that generates O2 in vivo) on plasma and tissue F2-IsoP levels. F2-IsoP increased 2- to 9-fold in plasma, liver, kidney and lung in rats received diquat. Similarly, Pratico et al. [160] compared the F2-IsoP levels in plasma, urine and vascular tissue from mice deficient in apolipoprotein E and after supplementation with vitamin E. Deficiency in apolipoprotein E resulted in increased F2-IsoP in plasma, urine and vascular tissue. In addition, supplementation with vitamin E reduced F2-IsoP levels in all tissues. It is worth mentioning that F2-IsoP levels in the brain seem to resist to changes in redox status [75,157,159,161]. Indeed, no alterations in brain F2-IsoP levels appeared in rats fed a diet deficient in a-tocopherol and selenium [157], in mice fed a high fat

10. The origin of F2-IsoP detected in urine F2-IsoP have been very frequently determined in urine. Two possibilities exist regarding the origin of F2-IsoP detected in urine: they are either derived from F2-IsoP generated systemically from

Table 1 Fold-changes in F2-IsoP levels compared to the control condition after various experimental treatments. Study

Experimental treatment

Plasma

Urine

Erythrocyte

Muscle

Heart

Liver

Kidney

Lung

Aorta

Brain

Awad et al. [157] Yoshida et al. [162] Yoshida et al. [163] Pratico et al. [160] Ding et al. [159] Awad et al. [156] Morrow et al. [75] Wong et al. [161] Choksi et al. [158]

Diet deficient in a-tocopherol and selenium Diet deficient in choline Diet deficient in a-tocopherol a-Tocopherol administration Diet rich in fats Diquat administration Carbon tetrachloride administration Aging Aging

2.0 3.3 2.4 2.3 2.0 2.2 18.8 1.5 1.1

– – – 2.2 – – – – –

– – 1.8 – – – – – –

12.5 – – – – – 1.8 – –

3.5 – – – 1.7 – 1.7 3.4 –

1.9 4.7 1.4 – 1.5 2.7 75.4 4.4 2.1

2.7 – – – – 2.9 15.8 2.1 –

3.0 – – – – 8.3 7.9 – –

– – – 2.0 1.9 – – – –

0.9 – 1.5 – 1.1 – 1.6 1.2 –

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diet [159], in rats administered carbon tetrachloride [75] or in aging rats fed a normal diet [161]. These findings suggest that several experimental treatments may have, at least qualitatively, similar effects on F2-IsoP levels as measured in plasma or urine and tissues (except for the brain). This is in agreement with the single study found to have statistically evaluated (i.e., performed correlations between selected markers in blood and tissues) the relationship between several oxidative stress markers in blood and tissues after exercise and reported that blood provides a reasonable picture of the general redox status [164]. Noteworthy, the fact that several oxidative stress stimuli affect the concentration of F2-IsoP in plasma or urine toward the same direction does not necessarily indicates that F2-IsoP detected in these fluids have been oxidized inside tissues [134]. Future studies should examine whether measurement of F2-IsoP in plasma and/or urine (currently a common practice in biomedical research) adequately describes the lipid peroxidation levels of skeletal muscle and tissues. 12. Do changes in F2-IsoP after exercise correlate with changes in other markers of lipid peroxidation?

icant Pearson’s or Spearman’s correlation coefficients [171] or very low to good coefficients (0.20 [170], 0.63 [34] and 0.86 [33]) between the two methods. Furthermore, almost all these ‘‘negative” studies consistently reported divergent values as well as both fixed and proportional bias between the two methods. On the bright side, Wang et al. [172] compared the values of F2-IsoP in urine obtained by EIA and GC–MS and reported an excellent correlation coefficient between the two methods (r = 0.99) as well as good sensitivity and specificity. However, no analysis for bias was performed. Moreover, Devaraj et al. [173] evaluated an EIA method for urinary F2-IsoP against a GC–MS method. A good correlation coefficient was obtained between the two methods (r = 0.80) of F2-IsoP measurement. Moreover, the median F2-IsoP concentrations for the EIA and GC–MS methods were very similar. However, these authors also did not analyze the methods in terms of fixed or proportional bias. Despite most of the studies suggest that GC–MS and EIA do not measure the same compounds, more comprehensive validation studies are needed to decipher if comparison of results using GC–MS and EIA should be avoided or not. 14. F2-IsoP are not simply markers of oxidative stress

It is important to be aware whether it is valid to compare studies that measured F2-IsoP to those measured other markers of lipid peroxidation. In general, most of the relevant studies reported that plasma levels of F2-IsoP, malondialdehyde, thiobarbituric acid reactive substances (TBARS) or lipid hydroperoxides were changed in a similar manner after acute exercise [45,56,58,59,63,65,70,71,98]. On the other hand, several other studies reported divergent effects of acute exercise on plasma levels of F2-IsoP, TBARS or lipid hydroperoxides [46,52–55]. In addition, most of the studies reported no significant correlations between F2-IsoP and other markers of lipid peroxidation in plasma [52,54,55,58], whereas others reported moderate correlation between F2-IsoP and lipid hydroperoxides (r = 0.44; [59]) and between F2-IsoP and malondialdehyde (r = 0.48–0.75; [70]). Based on this incomplete evidence, it seems that F2-IsoP can be compared to other markers of lipid peroxidation qualitatively but not quantitatively. 13. Do EIA and MS assays provide comparable results? Many of the studies presented in this review determined F2-IsoP using commercially available EIA kits. This is also the case in many translational studies. The main reason for selecting EIA kits is the limited access of most of the exercise physiology laboratories in the more sophisticated GC/HPLC-MS equipment. Remarkable, a laborious sample pretreatment in EIA is still required. Due to the wide use of EIA kits it is important to know whether EIA kits validly measure F2-IsoP. Before examining this issue, it is essential to consider whether the many different MS-based methods provide comparable results. The major concern stems from the absence of a consensus on a protocol that reliably measures a single F2-IsoP isomer or a sum of specific F2-IsoP isomers. This is exemplified by the large number of available methods to analyze F2-IsoP, which include chromatographic separation involving solid-phase extraction or affinity chromatography with or without thin-layer chromatography followed by final determination with GC–MS, HPLC-MS or tandem MS [9,99,117,162,165–168]. This may result in measuring a mixture of different F2-IsoP and/or other IsoP isomers and related metabolites [169]. Even though there is extensive use of F2-IsoP EIAs, relatively little work has been done to validate them in comparison to the reference MS methods. In general, four out of the six available studies indicated that the measurements of F2-IsoP by the means of EIA and GC–MS or LC–MS/MS do not produce equivalent data [33,34,170,171]. All four studies reported either virtually no signif-

In all exercise studies presented so far, F2-IsoP were measured as markers of lipid peroxidation. However, at least some of these oxidized lipids, appear to be biologically active. This is not surprising, as it has long been shown that lipid peroxidation products exert various biological effects either directly by reacting with proteins, enzymes and nucleic acids or indirectly through receptor-mediated pathways [174]. The first action of F2-IsoP to be revealed was the vasoconstriction of renal glomerular arterioles, as demonstrated by the direct infusion of 15-F2t-IsoP (the most tested isomer) into the renal artery [175]. Other biological effects of 15F2t-IsoP are those on smooth muscle cells [175,176] and on endothelial cells [177] in which DNA synthesis and cell proliferation is stimulated. Moreover, 15-F2t-IsoP seems to mediate the increased production of transforming growth factor-b1 in kidney mesangial and glomerular cells exposed to high glucose levels [178]. Other effects of F2-IsoP include platelet activation [179], stimulation of proliferative responses in fibroblasts [180] and induction of hypertrophy in ventricular myocytes [181]. The aforementioned effects of F2-IsoP can be provoked either directly by modulating the activity of other molecules or indirectly by modulating the properties of the membranes they are in. Unfortunately, no studies relevant to exercise and/or skeletal muscle have been performed. Infusion of F2-IsoP into exercising animals and or contracting muscles may be a worthwhile future research target. It is critical to realize that the increased F2-IsoP levels observed in most of the studies after acute exercise may not exclusively reflect byproducts of metabolism but may also represent regulators of the crucial responses and adaptations that take place during and after exercise. Importantly, in order to act as mediators of cellular functions, the formation of F2-IsoP must be strictly controlled and programmed. Nevertheless, it seems difficult to imagine how the cell can control and program the non-enzymatic generation of F2-IsoP. For an excellent discussion on the biological effects of F2-IsoP the interested reader is referred to Comporti et al. [10]. 15. Methodological considerations for measuring F2-IsoP

15.1. Controlling ex vivo oxidation Arachidonic acid in plasma and tissue samples is susceptible to reactive species oxidation and thus prevention of the ex vivo oxidation is crucial in the processing and storage of the samples. This

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is especially relevant for plasma and tissue samples that contain abundant arachidonic acid. For example, the concentration of arachidonic acid in total lipids in plasma is about 90 lg/mL whereas the concentration of free F2-IsoP in plasma is about 0.08 ng/mL [182]. The vast arachidonic acid to free F2-IsoP ratio (1  106:1) indicates that slight ex vivo oxidation of arachidonic acid can lead to large increases in free F2-IsoP concentration. As a result, the samples should be snap-frozen in liquid nitrogen immediately on collection and not thawed until analysis [32]. Moreover, addition of butylated hydroxyl toluene and triphenylphosphine are advisable to be added to prevent ex vivo oxidation during the extraction of lipids [32]. 15.2. The choice of matrix: plasma or urine? This question pertains mainly to human studies where access to tissues is limited. Does measurements of F2-IsoP in plasma correlate with those in urine? Despite the large number of studies that measured F2-IsoP in plasma and urine after exercise only two of them measured F2-IsoP in both plasma and urine. McAnulty et al. [68] reported a trend for increased levels of F2-IsoP in plasma but no change in urine F2-IsoP after a 160-km run lasting about 26 h. On the other hand, Nieman et al. [69] reported the reverse: increases in urinary F2-IsoP in urine and a trend for increase in plasma F2-IsoP after a triathlon lasting 12 h. In addition, idiopathic pulmonary fibrosis patients (a progressive, fibrotic lung disease) exhibited increased levels of F2-IsoP immediately after acute cycling lasting approximately 4 min in urine but not in plasma [183]. Similarly, a lack of correlation between plasma and urinary F2-IsoP concentration was observed in dengue fever patients and in smokers compared to healthy individuals and non-smokers, respectively [184,185]. Since the elimination half-life of F2-IsoP in the circulation is relatively short (less than 20 min; [74]), measurement of F2-IsoP in plasma may be suitable as a marker of lipid peroxidation during and after an acute bout of exercise. This is supported by the vague effects of acute exercise on F2-IsoP in urine, which is in sharp contrast to the clear increases observed after acute exercise on F2-IsoP in plasma. On the contrary, in studies investigating the effects of chronic exercise on F2-IsoP levels urine samples collected during 24 h or in the morning may be more suitable for getting an integrated picture of lipid peroxidation. Lastly, it is to be remembered that determination of F2-IsoP in either plasma or urine does not necessarily provide information on skeletal muscle lipid peroxidation levels during exercise. For a more detailed presentation on this topic the reader is referred to Halliwell and Lee [169]. 15.3. The choice of F2-IsoP isomer A key issue in IsoP analysis is to determine which IsoP should be measured, among the 192 different IsoP that could be generated from arachidonic acid. Despite the fact that 15-F2t-IsoP is by far the most studied isomer, this probably largely reflects the commercial availability of the assay kit rather than the superiority of 15-F2t-IsoP as a lipid peroxidation marker over the other IsoP isomers. In fact, other F2-IsoP isomers, such as 5-F2t-IsoP and 5-F2cIsoP, are about 4–20 times more abundant in urine than 15-F2t-IsoP [31,186]. It is also possible that determining more than one isomer may provide a more integrated picture of lipid peroxidation. 15.4. The choice of lipid class: free, esterified or total? This question is valid in plasma and tissues since the quantity of esterified F2-IsoP in urine is negligible. Most of the exercise studies reviewed in the present paper, measured free F2-IsoP in plasma and no study compared the effects of exercise on free, esterified

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or total F2-IsoP. Despite this lack of data, esterified F2-IsoP may be more long-lasting markers of lipid peroxidation than free F2IsoP and may enable the site of endogenous oxidative modification to be identified. On the other hand, measurement of free F2-IsoP may better reflect systemic changes in lipid peroxidation. In addition, if metabolism of free F2-IsoP in plasma is more rapid than that of esterified F2-IsoP, it may be more suitable to measure esterified F2-IsoP in plasma after exercise. No matter what it is measured, though, it is imperative to clearly mention it in the paper, as this is not usually done. 15.5. Expressing F2-IsoP concentration To allow meaningful comparison of urinary F2-IsoP levels among subjects and conditions, it is recommended that urine samples are normalized for creatinine content or other comparable marker. This is may not be necessary if 24 h urine collections are carried out [169]. Regarding plasma, though, the situation is more complicated. Almost all studies present data per plasma volume, even though correcting F2-IsoP levels per parent compound (i.e., arachidonic acid) may lead to different results [169]. Indeed, post-prandial levels of F2-IsoP rise following a fatty meal, demonstrating the confounding effect of diet [187]. However, when measurements were corrected for arachidonic acid levels, there was no longer a post-prandial difference. On the contrary, others reported that expressing the plasma levels of F2-IsoP per arachidonic acid produces similar results [188]. Considering that acute exercise increases the levels of non-esterified (i.e., free) arachidonic acid [104], it would be interesting to investigate if normalizing the exercise F2-IsoP concentration for arachidonic acid affect the results. Detailed discussion on this issue can be found in Halliwell and Lee [169]. 16. Conclusions and perspectives It has long been known that acute exercise can increase lipid peroxidation levels in many tissues [189–191]. This was largely based on studies measuring non-specific markers of lipid peroxidation, such as TBARS and conjugated dienes. The last years more and more studies employed F2-IsoP, a lipid peroxidation marker now considered the reference method in the field. From the analysis of the relevant literature in the preceding sections, the following effects of exercise have emerged: i. Acute exercise clearly increases F2-IsoP levels in plasma and this effect is generally short-lived. ii. Acute exercise and increased contractile activity markedly increase F2-IsoP levels in skeletal muscle. iii. Chronic exercise exhibits trend for decreased F2-IsoP levels in urine but further research is needed. No valid conclusions (mainly due to limited studies) can be drawn regarding the effects of acute and chronic exercise on F2IsoP levels in urine and plasma, respectively. In addition, no study has investigated the effect of chronic exercise on F2-IsoP levels in skeletal muscle. Equally important to have described the effects of exercise on F2-IsoP levels, this review tried to present a conceptual framework integrating the physiology with biochemical data as an approach for better interpretation of changes in F2-IsoP after any physiological stimulus. Based on the analysis of the available literature, the present review casts doubt on the frequently reproduced view that F2-IsoP are formed only in situ by oxidation of arachidonic acid esterified only in phospholipids. Theoretically, it seems that significant amounts of F2-IsoP can be produced from

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neutral lipids as well. A muscle-centric point of view is frequently adopted to explain F2-IsoP generation, thus obscuring the possibility that sources other than skeletal muscle may be also at work during exercise. In theory, plasma and blood cells can autonomously produce significant amounts of F2-IsoP at rest and during exercise. Smooth muscle cells and endothelial cells may also significantly contribute to the F2-IsoP detected in blood. Consequently, the origin of F2-IsoP detected in plasma and urine (as done by almost all studies in humans) remains controversial, as a multitude of tissues (including skeletal muscle and plasma) can independently produce F2-IsoP. Exercise is perhaps one of the most characteristic examples demonstrating that reactive species are not necessarily ‘‘harmful” entities, considering that the well-known benefits of regular exercise on muscle function and health are accompanied by repeated episodes of oxidative stress [192]. In view of the fact that several IsoP display a wide range of biological actions [193], it is tempting to suggest that the increased F2-IsoP levels observed in most of the studies after acute exercise may not exclusively reflect by-products of metabolism but may also represent regulators of the crucial responses and adaptations that take place during and after exercise. Redox biology of exercise, by nature multidisciplinary, has been characterized by a huge number of studies providing a purely descriptive analysis and not determining mechanistic relationships. For redox biology of exercise to provide more than a catalog of exercise-related changes in lipid peroxidation and redox status in general, the focus will be to understand the underlying biology of these phenomena, including their role in regulating muscle mass and as signaling molecules for organismal adaptation. Acknowledgment We would like to thank the three anonymous reviewers for their constructive comments. References [1] de Zwart LL, Meerman JH, Commandeur JN, Vermeulen NP. Biomarkers of free radical damage applications in experimental animals and in humans. Free Radic Biol Med 1999;26(1–2):202–26. [2] Pryor WA. On the detection of lipid hydroperoxides in biological samples. Free Radic Biol Med 1989;7(2):177–8. [3] Yin H. New techniques to detect oxidative stress markers: mass spectrometry-based methods to detect isoprostanes as the gold standard for oxidative stress in vivo. Biofactors 2008;34(2):109–24. [4] Giustarini D, Dalle-Donne I, Tsikas D, Rossi R. Oxidative stress and human diseases: origin, link, measurement, mechanisms, and biomarkers. Crit Rev Clin Lab Sci 2009;46(5–6):241–81. [5] Halliwell B. The wanderings of a free radical. Free Radic Biol Med 2009;46(5):531–42. [6] Montuschi P, Barnes P, Roberts 2nd LJ. Insights into oxidative stress: the isoprostanes. Curr Med Chem 2007;14(6):703–17. [7] Nourooz-Zadeh J. Key issues in F2-isoprostane analysis. Biochem Soc Trans 2008;36(Pt 5):1060–5. [8] Yin H, Davis T, Porter NA. Simultaneous analysis of multiple lipid oxidation products in vivo by liquid chromatographic–mass spectrometry (LC–MS). Methods Mol Biol 2010;610:375–86. [9] Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts 2nd LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a noncyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci USA 1990;87(23):9383–7. [10] Comporti M, Signorini C, Arezzini B, Vecchio D, Monaco B, Gardi C. F2isoprostanes are not just markers of oxidative stress. Free Radic Biol Med 2008;44(3):247–56. [11] Crankshaw DJ, Rangachari PK. Isoprostanes: more than just mere markers. Mol Cell Biochem 2003;253(1–2):125–30. [12] Nikolaidis MG, Jamurtas AZ, Paschalis V, Kostaropoulos IA, Kladi-Skandali A, Balamitsi V, et al. Exercise-induced oxidative stress in G6PD-deficient individuals. Med Sci Sports Exerc 2006;38(8):1443–50. [13] Nikolaidis MG, Paschalis V, Giakas G, Fatouros IG, Koutedakis Y, Kouretas D, et al. Decreased blood oxidative stress after repeated muscle-damaging exercise. Med Sci Sports Exerc 2007;39(7):1080–9. [14] Hellsten Y, Sjodin B, Richter EA, Bangsbo J. Urate uptake and lowered ATP levels in human muscle after high-intensity intermittent exercise. Am J Physiol 1998;274(4 Pt 1):E600–6.

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