Isoprostanes: Formation, Analysis and Use As Indices of Lipid Peroxidation in Vivo

arachidonic acid gas chromatography mass spectrometry prostaglandin isoprostanes polyunsaturated fatty acids eicosapentaenoic acid docosahexaenoic acid solid phase extraction cyclooxygenase liquid chromatography enzyme immunoassay radioimmunoassay low density lipoprotein thromboxane A2 receptor thromboxane The formation of prostaglandin-like structures as a product of arachidonic acid (AA)1 peroxidation in vitro was first reported by Mihelich and others (1Mihelich E.D. J. Am. Chem. Soc. 1980; 102: 7141-7143Crossref Scopus (68) Google Scholar, 2O'Connor D.E. Mihelich E.D. Coleman M.C. J. Am. Chem. Soc. 1981; 103: 223-224Crossref Scopus (62) Google Scholar, 3Nugteren D.H. Vonkeman H. Van Dorp D.A. Recl. Trav. Chim. Pays-Bas Belg. 1967; 86: 1237-1245Crossref Scopus (108) Google Scholar, 4Porter N.A. Funk M.O. J. Org. Chem. 1975; 40: 3614-3615Crossref PubMed Scopus (114) Google Scholar). However, it was Morrow, Roberts, and co-workers (5Wendelborn D.F. Seibert K. Roberts L.J., II Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 304-308Crossref PubMed Scopus (38) Google Scholar, 6Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts L.J., II Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10721-10725Crossref PubMed Scopus (672) Google Scholar) who characterized interfering peaks observed in a GC/MS assay for 9α,11β-PGF2α, a metabolite of PGD2 in urine, as isomers of PGF2α. These compounds, termed F2-isoprostanes (F2-iPs), possess a 1,3-dihydroxycyclopentane ring (PGF ring) with hydroxyls mainly in the syn configuration and are formed from arachidonic acid by a free radical mechanism (5Wendelborn D.F. Seibert K. Roberts L.J., II Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 304-308Crossref PubMed Scopus (38) Google Scholar). Depending upon which of the labile hydrogen atoms is first abstracted by free radical attack, up to 64 isomers in four structural classes can be generated (6Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts L.J., II Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10721-10725Crossref PubMed Scopus (672) Google Scholar). Compounds analogous to the F2-iPs may be formed from other fatty acid substrates (7Nourooz-Zadeh J. Halliwell B. Anggard E.E. Biochem. Biophys. Res. Commun. 1997; 236: 467-472Crossref PubMed Scopus (87) Google Scholar,8Roberts L.J., II Montine T.J. Markesbery W.R. Tapper A.R. Hardy P. Chemtob S. Dettbarn W.D. Morrow J.D. J. Biol. Chem. 1998; 273: 13605-13612Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Similarly, free radical-derived isomers of other prostaglandins, leukotrienes, and epoxyeicosatrienoic acids have been reported (9Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts L.J., II J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar, 10Morrow J.D. Awad J.A. Wu A. Zackert W.E. Daniel V.C. Roberts L.J., II J. Biol. Chem. 1996; 271: 23185-23190Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Harrison K.A. Murphy R.C. J. Biol. Chem. 1995; 270: 17273-17278Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Mallat Z. Nakamura T. Ohan J. Lesecher G. Tecgui A. Maclouf J. Murphy R.C. J. Clin. Invest. 1999; 103: 421-427Crossref PubMed Scopus (165) Google Scholar). A new classification system for isoprostanes, based on the ω system of counting PUFA double bonds (13Rokach J. Khanapure S.P. Hwang S-W. Adiyaman M. Lawson J.A. FitzGerald G.A. Prostaglandins. 1997; 54: 853-873Crossref PubMed Scopus (130) Google Scholar), accounts for variations in chain length and the position of double bonds (Fig.1). PUFA, with a higher and a lower number of skipped double bonds than AA, can also be accommodated by this system (13Rokach J. Khanapure S.P. Hwang S-W. Adiyaman M. Lawson J.A. FitzGerald G.A. Prostaglandins. 1997; 54: 853-873Crossref PubMed Scopus (130) Google Scholar, 14Rokach J. Khanapure S.P. Hwang S-W. Adiyaman M. Schio L. FitzGerald G.A. Synthesis. 1998; 1: 569-580Crossref Scopus (54) Google Scholar). Selection of the ω system of counting the double bonds is designed to mimic the biochemical system by which the PUFA double bonds are generated at ω3, ω6, ω9, etc., ω being the terminal carbon of the PUFA. All ω3 PUFAs lead to iPs with identical lower side chains. Differences in chain length and the number of double bonds (n) in the original PUFA are reflected only in the upper side chains. For example, the iPs derived from EPA (an ω3 n-5 PUFA) and DHA (an ω3 n-6 PUFA), a major PUFA in the brain, have their lower side chains identical. The extra double bond in DHA leads to an additional two classes of iPs, Type VII and Type VIII. Alternatively, linolenic acid is a C18 ω3 n-3 PUFA with double bonds at ω3, ω6, and ω9. It can give rise to two classes of iPs, namely I and II, with lower side chains identical to Class I and Class II iPs from EPA and DHA. The upper side chains of these linolenic acid-derived iPs will have two carbons less than EPA and four carbons less than DHA (Group I and Group II). Finally, γ-linolenic acid, an ω6 n-3 PUFA, will yield two groups of iPs, namely Group III and Group IV, with identical lower side chains to Group III and Group IV derived from AA. The differences will again be in the upper side chains, which will have one less cis double bond and will be two carbons shorter. Isoprostanes may be formed by either of two routes of peroxidation (14Rokach J. Khanapure S.P. Hwang S-W. Adiyaman M. Schio L. FitzGerald G.A. Synthesis. 1998; 1: 569-580Crossref Scopus (54) Google Scholar,15Rokach J. Khanapure S.P. Hwang S.W. Adiyaman M. Lawson J.A. FitzGerald G.A. Prostaglandins. 1997; 54: 823-851Crossref PubMed Scopus (101) Google Scholar), an endoperoxide mechanism (Fig. 2) or a dioxetane/endoperoxide mechanism (Fig.3). In the former, the first oxygen molecule is incorporated into the endoperoxide ring to form the two hydroxyl groups on the PGF ring. In the latter, by contrast, it is the second oxygen molecule that is incorporated into the PGF ring. Also 5- and 15-hydroperoxy radicals can only form Groups VI and III by the dioxetane/endoperoxide mechanism. The radical at position 10 of arachidonic acid, by contrast, can yield iPs by both mechanisms. Thus, hydroperoxy radicals formed at 8 and 12 have the option to proceed to form a dioxetane ring (Fig. 3) or a dioxypentane ring (Fig. 2) on a competitive basis, although it is not yet clear which is favored. Recent attention (see below) has focused upon Group VI iPs. These compounds may be derived from a 9-hydroperoxy radical by the endoperoxide mechanism or from a 5-hydroperoxy radical by the dioxetane/endoperoxide mechanism. However, both are derived from an initial hydrogen atom abstraction at position 7 of arachidonic acid. Abstraction at carbon 13 can give rise to 11- and 15-hydroperoxy radicals, yielding only one series (Group III) of iPs. A radical at position 10 of arachidonic acid gives a radical at 8 and 12, which yields groups V and IV, respectively. If the dioxetane mechanism is operative, the same 8- and 12-hydroperoxy radicals will yield Groups IV and V, respectively.Figure 3Dioxetane/endoperoxide mechanism for the formation of isoprostanes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Isoprostanes are formed in a free radical-dependent manner and are chemically stable. They are generated initially in cell membranes at the site of free radical attack from which they are cleaved, presumably by phospholipases, circulate, and are excreted in urine. They have also been reported in body fluids besides blood and urine, such as pericardial fluid (16Mallat Z. Philip I. Lebret M. Chatel D. Maclouf J. Tedgui A. Circulation. 1998; 97: 1536-1539Crossref PubMed Scopus (391) Google Scholar), bile (17Leo M.A. Aleynik S.I. Siegel J.H. Kasmin F.E. Aleynik M.K. Lieber C.S. Am. J. Gastroenterol. 1997; 92: 2069-2072PubMed Google Scholar, 18Praticò D. Rossi E. Merli M. Riggio O. FitzGerald G.A. Violi F. J. Investig. Med. 1998; 46: 1-5Google Scholar), lung condensates (19Montuschi P. Ciabattoni G. Paredi P. Pantelidis P. duBois R.M. Kharitonov S.A. Barnes P.J. Am. J. Respir. Crit. Care Med. 1998; 158: 1524-1527Crossref PubMed Scopus (238) Google Scholar), and cerebrospinal fluid (20Praticò D. Trojanowski J. Lee V. Rokach J. FitzGerald G.A. FASEB J. 1998; 12: 1777-1783Crossref PubMed Scopus (384) Google Scholar, 21Montine T.J. Markesbery W.R. Morrow J.D. Roberts L.J., II Ann. Neurol. 1998; 44: 410-413Crossref PubMed Scopus (228) Google Scholar). These have been based on the sensitive and specific capillary GC/negative ion electron capture chemical ionization MS technique. Derivatizing the carboxylic acid, common to all eicosanoids, to the pentafluorobenzyl ester is the key to its sensitivity. Bombardment of a moderating gas by an electron beam produces low energy thermal electrons, which are captured by the electrophilic pentafluorobenzyl moiety. This then cleaves, leaving the carboxylate anion which remains to a large degree intact, yielding a spectrum that is dominated by a single ion and is therefore well suited to selected ion monitoring. The closer the homology between analyte(s) and the stable isotope-labeled internal standard, the more reliable the assay. The original assay of Morrow et al. (22Morrow J.D. Harris R. Roberts L.J., II Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (427) Google Scholar) utilized 2H79α,11β-PGF2α as an internal standard. It was formalized and amended to use commercially available 2H4PGF2α (23Nakamura T. Bratton D.L. Murphy R.C. J. Mass Spectrom. 1997; 323: 888-896Crossref Scopus (98) Google Scholar) and is still the most widely used reference technique. It requires two solid phase extraction (SPE) steps, two thin layer chromatography (TLC) steps, and two derivatization steps. A large number of overlapping peaks were observed, and one of them (shown to be resolved from the enzymatic isomers known to be present in urine and itself clearly composed of multiple isomers) was chosen for integration and comparison with the internal standard. The only synthetic F2-iP available at the time, iPF2α-III (also known as 8-iso-PGF2α), eluted as one component of the chosen peak. However, because the target compound and the internal standard were heterologous, differences in recovery in any of the four purification steps, especially the TLC, could cause differential recovery of one or more analytes, relative to the internal standard. The identity, number, or TLC retention characteristics of the analytes were not known. A simplified version eliminated the TLC steps (24Gopaul N.K. Nourooz-Zadeh J. Mallet A.I. Änggard E.E. Biochem. Biophys. Res. Commun. 1994; 200: 338-343Crossref PubMed Scopus (65) Google Scholar). This minimized loss of isomers due to differential recovery during purification. However, this method also cannot resolve iPF2α-III from the other isomers. Indeed, application of this and the earlier assay led to the conclusion that iPF2α-III was a major F2-iP isomer (25Gopaul N.K. Nourooz-Zadeh J. Mallet A.I. Änggard E.E. FEBS Lett. 1994; 348: 297-300Crossref PubMed Scopus (58) Google Scholar), which turned out not to be the case. Such minimal purification may also confound the sensitivity and reliability of the GC/MS. Our approach has been to synthesize homologous standards (26Hwang S.W. Adiyaman M. Khanapure S. Schio L. Rokach J. J. Am. Chem. Soc. 1994; 116: 10829-10830Crossref Scopus (69) Google Scholar, 27Pudukulathan Z. Manna S. Hwang S.W. Khanpure S.P. Lawson J.A. FitzGerald G.A. Rokach J. J. Am. Chem. Soc. 1998; 120: 11953-11961Crossref Scopus (45) Google Scholar) and to develop assay conditions that permit the quantitation of a single isomer. Given reports that iPF2α-III was a prominent F2-iP and that it had bioactivity in vitro and in vivo (28Banerjee M. Kang K.H. Morrow J.D. Roberts L.J., II Newman J.H. Am. J. Physiol. 1992; 263: H660-H663Crossref PubMed Google Scholar, 29Takahashi K. Nammour T.M. Fukunaga M. Ebert J. Morrow J.D. Roberts L.J., II Hoover R.L. Badr K.F. J. Clin. Invest. 1992; 90: 136-141Crossref PubMed Scopus (498) Google Scholar), we focused initially on this compound. We developed an assay that seemed to measure a single isomer by synthesizing 18O2iPF2α-III and improving the GC/MS characteristics by using the tert-butyldimethylsilyl ether, instead of the trimethylsilyl ether (30Pratico D. Lawson J.A. FitzGerald G.A. J. Biol. Chem. 1995; 270: 9800-9808Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In reality, the assay (one SPE step, two TLC steps, and two derivatizations) was technically demanding. However, we found that iPF2α-III, unlike other F2-iPs, could be formed by either COX-1 or COX-2 (30Pratico D. Lawson J.A. FitzGerald G.A. J. Biol. Chem. 1995; 270: 9800-9808Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 31Praticó D. FitzGerald G.A. J. Biol. Chem. 1996; 271: 8919-8924Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), potentially undermining its value as an index of lipid peroxidation in vitro. Therefore, we focused on iPF2α-VI (formerly known as IPF2α-I) (32Adiyaman M. Lawson J.A. Huang S.-W. Khanapure H. FitzGerald G.A. Rokach J. Tetrahedron Lett. 1996; 37: 4849-4852Crossref Scopus (48) Google Scholar), which had promise as a target analyte because it could be easily converted to a cyclic lactone, enabling facile separation from F2-iPs of classes III, IV, and V. It is present in urine at concentrations higher than iPF2α-III and is not subject to COX-dependent formation (33Praticò D. Barry O.P. Lawson J.A. Adiyaman M. Hwang S-W. Khanapure S.P. Iuliano L. Rokach J. FitzGerald G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3449-3454Crossref PubMed Scopus (211) Google Scholar). The application of this technique to iP analysis has been pioneered by Murphy and colleagues (11Harrison K.A. Murphy R.C. J. Biol. Chem. 1995; 270: 17273-17278Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Nakamura T. Bratton D.L. Murphy R.C. J. Mass Spectrom. 1997; 323: 888-896Crossref Scopus (98) Google Scholar, 34Waugh R.J. Morrow J.D. Roberts L.J., II Murphy R.C. Free Radical Biol. Med. 1997; 23: 943-954Crossref PubMed Scopus (113) Google Scholar, 35Waugh R.J. Murphy R.C. J. Mass Spectrom. 1996; 7: 490-499Crossref Scopus (54) Google Scholar). Using this approach, we have shown 8,12-iso-iPF2α-VI and 5-epi-8,12-iso-iPF2α-VI to be the most abundant F2-iPs in human urine (36Lawson J. Rokach J. Hongwei L. Adiyaman M. Hwang S-W. Khanapure S.P. FitzGerald G.A. J. Biol. Chem. 1998; 273: 29295-29301Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Recent advances in electrospray ionization have rendered LC/MS a practical alternative to GC/MS. Because all F2-iPs are isomeric and LC is unable to resolve completely all the isomers, the result is unsatisfactory. However, tandem MS adds another level of selectivity (34Waugh R.J. Morrow J.D. Roberts L.J., II Murphy R.C. Free Radical Biol. Med. 1997; 23: 943-954Crossref PubMed Scopus (113) Google Scholar, 35Waugh R.J. Murphy R.C. J. Mass Spectrom. 1996; 7: 490-499Crossref Scopus (54) Google Scholar, 36Lawson J. Rokach J. Hongwei L. Adiyaman M. Hwang S-W. Khanapure S.P. FitzGerald G.A. J. Biol. Chem. 1998; 273: 29295-29301Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), permitting virtual separation of the four classes of F2-iPs (Fig. 4 A) as well as an increased signal-to-noise ratio. LC/MS is still 2 or 3 orders of magnitude less sensitive than GC/MS. However, sample preparation can consist of a single SPE step, with no derivatization required, so analyte recovery may be an order of magnitude higher. The method is presently sensitive enough to quantitate a single F2-iP isomer from 1 ml of urine (Fig. 4 B). As tandem MS instrumentation becomes more common, more sensitive, and less expensive, it will likely become an important method for F2-iP analysis. GC/MS/MS has not contributed significantly to F2-iP analysis, with only one such assay being published (37Schweer R.H. Watzer B. Seyberth H.W. Nusing R.M. J. Mass Spectrom. 1997; 32: 1362-1370Crossref PubMed Scopus (52) Google Scholar). Although in principle the method can have specificity for the four F2-iP classes (34Waugh R.J. Morrow J.D. Roberts L.J., II Murphy R.C. Free Radical Biol. Med. 1997; 23: 943-954Crossref PubMed Scopus (113) Google Scholar, 35Waugh R.J. Murphy R.C. J. Mass Spectrom. 1996; 7: 490-499Crossref Scopus (54) Google Scholar) using electron impact ionization, the sensitivity of the negative ion electron capture MS technique is sacrificed. When the more sensitive negative ion method is used, the initial ionization yields the carboxylate anion (m/z 353), and the collision-induced dissociation ions monitored in the second quadrupole originate from the loss of (CH3)3SiOH groups, common to all isomers and therefore devoid of any structural information. No distinct advantage in F2-iP quantitation is obvious. However, any spurious contribution to the F2-iP peak by non-iP impurities would be minimized. Specific quantitation of iPF2α-III requires off-line high pressure liquid chromatography purification (37Schweer R.H. Watzer B. Seyberth H.W. Nusing R.M. J. Mass Spectrom. 1997; 32: 1362-1370Crossref PubMed Scopus (52) Google Scholar), incompatible with routine sample preparation. An assay for iPF2α-III has been reported (38Bachi A. Zuccato E. Baraldi M. Fanelli R. Chiabrando C. Free Radical Biol. Med. 1996; 20: 619-624Crossref PubMed Scopus (115) Google Scholar). The analyte is selectively extracted on an immunoaffinity column, derivatized, and analyzed by GC/MS. The immunoaffinity column effectively replaces the SPE and TLC steps, with significantly more specificity. The columns must be reused, so sample carryover must be monitored, and the columns have a finite lifespan, requiring a constant supply of antibody. Adaptation of EIA and RIA from prostaglandin to iP analysis is complex. Traditionally, the used for analysis have been for with the other major The degree of has been low because of the major differences in distinct When to F2-iPs, all of the 64 isomers the same ring It is that is the so the of F2-iPs may be Given that may also be present in than the compounds, the becomes more complex. This not the use of in F2-iP analysis, it that to have not been have in have the for with all other They are of to be iPF2α-III Z. Ciabattoni G. C. Lawson J.A. FitzGerald G.A. C. Maclouf J. J. 1995; Google Scholar). because the degree of can from to of from be with because of sample preparation quantitation have also been the of iP analysis in For example, the endoperoxide of iPs, analogous to (22Morrow J.D. Harris R. Roberts L.J., II Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (427) Google Scholar), can to or be to F2-iPs (9Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts L.J., II J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar). Thus, of F2-iPs not only the peroxidation of arachidonic acid also the of the in which peroxidation with identical of peroxidation in F2-iP An assay which an F2-iP and a for such differences in they A more is that iPF2α-III can be formed by either in vitro and vivo (30Pratico D. Lawson J.A. FitzGerald G.A. J. Biol. Chem. 1995; 270: 9800-9808Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 31Praticó D. FitzGerald G.A. J. Biol. Chem. 1996; 271: 8919-8924Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, R.H. Watzer B. Seyberth H.W. Nusing R.M. J. Mass Spectrom. 1997; 32: 1362-1370Crossref PubMed Scopus (52) Google P. G. G. A. M. Maclouf J. Ciabattoni G. C. J. 1996; PubMed Scopus Google Scholar), and and F. L. FitzGerald G.A. Circulation. 1988; PubMed Scopus Google Scholar, J. J.A. FitzGerald G.A. Circulation. PubMed Scopus Google Scholar). 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A. 1998; 95: 3449-3454Crossref PubMed Scopus (211) Google Scholar, Morrow J.D. Roberts L.J., II B. J. Clin. Invest. 1994; PubMed Scopus Google Scholar). However, it is of either in vitro or to in Isoprostanes are present in human D. J. A. S. Lawson J. Rokach J. Maclouf J. Violi F. FitzGerald G.A. J. Clin. Invest. 1997; PubMed Scopus Google Scholar, C. Morrow J.D. Roberts L.J., II H. Biol. 1997; PubMed Scopus Google Scholar), as are which to be more abundant Z. Nakamura T. Ohan J. Lesecher G. Tecgui A. Maclouf J. Murphy R.C. J. Clin. Invest. 1999; 103: 421-427Crossref PubMed Scopus (165) Google Scholar). Isoprostanes in increased in the of with M. D. G. E. D. S. Rokach J. Lawson J. FitzGerald G.A. Circulation. 1998; PubMed Scopus Google Scholar). iPs are also increased in with and to with the in M. D. G. E. D. S. Rokach J. Lawson J. FitzGerald G.A. Circulation. 1998; PubMed Scopus Google Scholar). the that iPs be used for we the that on a FitzGerald G.A. 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Given the of their iPs may also to in conditions of these it is to the concentrations of iPs used to in vitro to in a of is formed conditions of and to have on single isomers. these compounds may be subject to from Finally, the and of their are are that they have in A is more at in than of formation by the FitzGerald G.A. Proc. Natl. Acad. Sci. U. S. A. 86: PubMed Scopus (165) Google Scholar). This of the by a distinct from and iPF2α-III is known to during in this M. Lawson J.A. J. F. FitzGerald G.A. Circulation. 1997; 95: PubMed Scopus Google Scholar). Finally, not only iPF2α-III in with also the of a as by et al. G. Ciabattoni G. A. A. A. S. E. E. T. F. F. C. Circulation. 1999; PubMed Scopus Google Scholar), iPs, such as iPF2α-III, to the in these of F2-iPs has as a approach to the of lipid peroxidation in Given the of the iP it to upon sensitive of specific isomers and their The of iPs as remains less although the that they a of and remains