Identification of the Major Urinary Metabolite of the F2-isoprostane 8-Iso-prostaglandin F2α in Humans

Abstract

F2-isoprostanes are prostaglandin-like products of nonenzymatic lipid peroxidation. Measurement of levels of endogenous unmetabolized F2-isoprostanes has proven to be a valuable approach to assess oxidative stress in vivo. However, measurement of levels of urinary metabolites of F2-isoprostanes in timed urine collections offers an advantage over measuring unmetabolized F2-isoprostanes, e.g. in a plasma sample, in that it can provide an integrated index of isoprostane production over time. Therefore, we sought to identify the major urinary metabolite in humans of one of the more abundant F2-isoprostanes produced, 8-iso-prostaglandin F2α (8-iso-PGF2α). 20 µCi of tritiated 8-iso-PGF2α was infused over 1 h into a male volunteer. 75% of the infused radioactivity was excreted into the urine during the following 4.5 h and was combined with urine collected for 4 h from a rhesus monkey following infusion of 500 µg of unlabeled 8-iso-PGF2α. Urinary metabolites were isolated and purified by adsorption chromatography and high pressure liquid chromatography. The major urinary metabolite, representing 29% of the total extractable recovered radioactivity in the urine, was structurally identified by gas chromatography and mass spectrometry as 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2α. The identification of 2,3-dinor-5,6-dihydro-prostaglandin F2α as the major urinary metabolite of 8-iso-prostaglandin F2α provides the basis for the development of methods of assay for its quantification as a means to obtain an integrated assessment of oxidative stress status in humans. F2-isoprostanes are prostaglandin-like products of nonenzymatic lipid peroxidation. Measurement of levels of endogenous unmetabolized F2-isoprostanes has proven to be a valuable approach to assess oxidative stress in vivo. However, measurement of levels of urinary metabolites of F2-isoprostanes in timed urine collections offers an advantage over measuring unmetabolized F2-isoprostanes, e.g. in a plasma sample, in that it can provide an integrated index of isoprostane production over time. Therefore, we sought to identify the major urinary metabolite in humans of one of the more abundant F2-isoprostanes produced, 8-iso-prostaglandin F2α (8-iso-PGF2α). 20 µCi of tritiated 8-iso-PGF2α was infused over 1 h into a male volunteer. 75% of the infused radioactivity was excreted into the urine during the following 4.5 h and was combined with urine collected for 4 h from a rhesus monkey following infusion of 500 µg of unlabeled 8-iso-PGF2α. Urinary metabolites were isolated and purified by adsorption chromatography and high pressure liquid chromatography. The major urinary metabolite, representing 29% of the total extractable recovered radioactivity in the urine, was structurally identified by gas chromatography and mass spectrometry as 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2α. The identification of 2,3-dinor-5,6-dihydro-prostaglandin F2α as the major urinary metabolite of 8-iso-prostaglandin F2α provides the basis for the development of methods of assay for its quantification as a means to obtain an integrated assessment of oxidative stress status in humans. INTRODUCTIONFree radicals have been implicated in the pathogenesis of a wide variety of human disorders (1Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar, 2Southorn P.A. Powis G. Mayo. Clin. Proc. 1988; 63: 390-408Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 3Ames B.N. Science. 1983; 221: 1256-1264Crossref PubMed Scopus (2729) Google Scholar, 4Harman D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7124-7128Crossref PubMed Scopus (1564) Google Scholar). One of the major targets of free radical injury are lipids, which undergo peroxidation. Previously, we reported the discovery that a series of prostaglandin (PG) 1The abbreviations used are: PGprostaglandinF2-IPF2-isoprostaneGCgas chromatographyMSmass spectrometryHPLChigh pressure liquid chromatography. F2-like compounds (F2-isoprostanes (F2-IPs)) are produced in vivo as products of the free radical-catalyzed peroxidation of arachidonic acid (5Morrow J.D. Hill K.E. Burk R.F. Nammour T.M. Badr K.F. Roberts II, L.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9383-9387Crossref PubMed Scopus (1707) Google Scholar). Formation of these compounds occurs independently of the cyclooxygenase enzyme and proceeds through intermediates comprising arachidonoyl peroxyl radical isomers of arachidonic acid, which undergo endocyclization to form bicyclic endoperoxides. The endoperoxides are then reduced to yield F2-IPs. The endoperoxides also undergo rearrangement in vivo to form D- and E-ring IPs (6Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts II, L.J. J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar). Four positional isomers of IPs are formed, each of which can comprise eight racemic diastereomers. IPs are initially formed esterified to phospholipids and subsequently released preformed (7Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts II, L.J. Proc. Nat. Acad. Sci. U. S. A. 1992; 89: 10721-10725Crossref PubMed Scopus (664) Google Scholar). Based on the mechanism of formation of IPs, i.e. the formation of compounds with the side chains oriented cis in relation to the cyclopentane ring are highly favored (8Morrow J.D. Harris T.M. Roberts L.J.I. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar), one compound that would be expected to be formed would be 8-iso-PGF2α. Recently we demonstrated that 8-iso-PGF2α is in fact one of the more abundant F2-IPs produced in vivo (9Morrow J.D. Minton T.A. Badr K.F. Roberts II, L.J. Biochim. Biophys. Acta. 1994; 1210: 244-248Crossref PubMed Scopus (161) Google Scholar). There has been considerable interest in this molecule, because it exerts biological activity, e.g. it is a potent vasoconstrictor in the lung and kidney (10Takahashi K. Nammour T.K. Fukunaga M. Ebert J. Morrow J.D. Roberts II, L.J. Hoover R.L. Badr K.F. J. Clin. Invest. 1992; 90: 136-141Crossref PubMed Scopus (496) Google Scholar, 11Banerjee M. Kang K.H. Morrow J.D. Roberts L.J.I. Newman J.H. Am. J. Physiol. 1992; 263: H660-H663Crossref PubMed Google Scholar). Furthermore, it has been suggested that the biological effects of 8-iso-PGF2α may result from an interaction with a unique receptor (12Fukunaga M. Makita N. Roberts II, L.J. Morrow J.D. Takahashi K. Badr K.F. Am. J. Physiol. 1993; 264: C1619-C1624Crossref PubMed Google Scholar).It has been recognized that one of the greatest impediments in the field of free radical research has been the lack of reliable methods to assess oxidant stress status in humans (13Gutteridge J.M.C. Halliwell B. Trends. Biol. Sci. 1990; 15: 129-135Abstract Full Text PDF PubMed Scopus (1077) Google Scholar). A considerable body of evidence has accumulated indicating that measurement of F2-IPs provides a valuable and reliable approach to assess oxidant stress in vivo both in animal models of oxidant injury and in humans (14Morrow J.D. Awad J.A. Kato T. Takahashi K. Badr K.F. Roberts II, L.J. Burk R.F. J. Clin. Invest. 1992; 90: 2502-2507Crossref PubMed Scopus (298) Google Scholar, 15Morrow J.D. Frei B. Longmire A.W. Gaziano M. Lynch S.M. Shyr Y. Strauss W.E. Oates J.A. Roberts II, L.J. N. Engl. J. Med. 1995; 332: 1198-1203Crossref PubMed Scopus (1260) Google Scholar). In this regard, however, quantification of unmetabolized IPs has certain limitations. First, F2-IPs can be artifactually generated ex vivo, e.g. in plasma, by auto-oxidation of plasma arachidonic acid if appropriate precautions are not taken (8Morrow J.D. Harris T.M. Roberts L.J.I. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar). In addition, quantification of F2-IPs esterified in tissues or circulating in plasma only provides information at a single point in time rather than an integrated index of IP production. Having a means to obtain an integrated index of oxidant stress status would be very valuable in situations in which the level of oxidant stress fluctuates over time. In this regard, analogous to quantification of urinary metabolites of cyclooxygenase-derived prostanoids (16Roberts II, L.J. Willis A.L. Handbook of Eicosanoids: Prostaglandins and Related Lipids, Chemical and Biochemical Aspects, Part A. CRC Press Inc., Boca Raton, FL1987: 233Google Scholar), measurement of the urinary excretion of F2-IPs should provide a reliable and integrated index of oxidative stress status in vivo.We have previously identified urinary metabolites of F2-IPs that copurify through a mass spectrometric assay developed for quantification of the major urinary metabolite of cyclooxygenase-derived PGD2 (17Awad J.A. Morrow J.D. Takahashi K. Roberts II, L.J. J. Biol. Chem. 1993; 268: 4161-4169Abstract Full Text PDF PubMed Google Scholar, 18Morrow J.D. Prakash C. Awad J.A. Duckworth T.A. Zackert W.E. Blair II, A. Oates J.A. Roberts II, L.J. Anal. Biochem. 1991; 193: 142-148Crossref PubMed Scopus (33) Google Scholar). However, we do not know the parent compounds from which these F2-IP metabolites derive. Furthermore, we have found that a metabolite of cyclooxygenase-derived PGF2α, 9α,11α-dihydroxy-15-oxo-13,14-dihydro-2,3,18, 19-tetranorprost-1,20-dioic acid, cochromatographs on capillary gas chromatography (GC) with these F2-IP metabolites. 2L. J. Roberts II, K. P. Moore, W. E. Zackert, J. A. Oates, and J. D. Morrow, unpublished data. This latter finding confounds an interpretation as to whether an increase in the intensity of these peaks when analyzed by GC and mass spectrometry (MS) represents overproduction of F2-IPs or PGF2α. Thus, we undertook a study to identify the major urinary metabolite of 8-iso-PGF2α in humans as a basis for the development of methods of assay for its quantification to assess oxidative stress status in humans. INTRODUCTIONFree radicals have been implicated in the pathogenesis of a wide variety of human disorders (1Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar, 2Southorn P.A. Powis G. Mayo. Clin. Proc. 1988; 63: 390-408Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 3Ames B.N. Science. 1983; 221: 1256-1264Crossref PubMed Scopus (2729) Google Scholar, 4Harman D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7124-7128Crossref PubMed Scopus (1564) Google Scholar). One of the major targets of free radical injury are lipids, which undergo peroxidation. Previously, we reported the discovery that a series of prostaglandin (PG) 1The abbreviations used are: PGprostaglandinF2-IPF2-isoprostaneGCgas chromatographyMSmass spectrometryHPLChigh pressure liquid chromatography. F2-like compounds (F2-isoprostanes (F2-IPs)) are produced in vivo as products of the free radical-catalyzed peroxidation of arachidonic acid (5Morrow J.D. Hill K.E. Burk R.F. Nammour T.M. Badr K.F. Roberts II, L.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9383-9387Crossref PubMed Scopus (1707) Google Scholar). Formation of these compounds occurs independently of the cyclooxygenase enzyme and proceeds through intermediates comprising arachidonoyl peroxyl radical isomers of arachidonic acid, which undergo endocyclization to form bicyclic endoperoxides. The endoperoxides are then reduced to yield F2-IPs. The endoperoxides also undergo rearrangement in vivo to form D- and E-ring IPs (6Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts II, L.J. J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar). Four positional isomers of IPs are formed, each of which can comprise eight racemic diastereomers. IPs are initially formed esterified to phospholipids and subsequently released preformed (7Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts II, L.J. Proc. Nat. Acad. Sci. U. S. A. 1992; 89: 10721-10725Crossref PubMed Scopus (664) Google Scholar). Based on the mechanism of formation of IPs, i.e. the formation of compounds with the side chains oriented cis in relation to the cyclopentane ring are highly favored (8Morrow J.D. Harris T.M. Roberts L.J.I. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar), one compound that would be expected to be formed would be 8-iso-PGF2α. Recently we demonstrated that 8-iso-PGF2α is in fact one of the more abundant F2-IPs produced in vivo (9Morrow J.D. Minton T.A. Badr K.F. Roberts II, L.J. Biochim. Biophys. Acta. 1994; 1210: 244-248Crossref PubMed Scopus (161) Google Scholar). There has been considerable interest in this molecule, because it exerts biological activity, e.g. it is a potent vasoconstrictor in the lung and kidney (10Takahashi K. Nammour T.K. Fukunaga M. Ebert J. Morrow J.D. Roberts II, L.J. Hoover R.L. Badr K.F. J. Clin. Invest. 1992; 90: 136-141Crossref PubMed Scopus (496) Google Scholar, 11Banerjee M. Kang K.H. Morrow J.D. Roberts L.J.I. Newman J.H. Am. J. Physiol. 1992; 263: H660-H663Crossref PubMed Google Scholar). Furthermore, it has been suggested that the biological effects of 8-iso-PGF2α may result from an interaction with a unique receptor (12Fukunaga M. Makita N. Roberts II, L.J. Morrow J.D. Takahashi K. Badr K.F. Am. J. Physiol. 1993; 264: C1619-C1624Crossref PubMed Google Scholar).It has been recognized that one of the greatest impediments in the field of free radical research has been the lack of reliable methods to assess oxidant stress status in humans (13Gutteridge J.M.C. Halliwell B. Trends. Biol. Sci. 1990; 15: 129-135Abstract Full Text PDF PubMed Scopus (1077) Google Scholar). A considerable body of evidence has accumulated indicating that measurement of F2-IPs provides a valuable and reliable approach to assess oxidant stress in vivo both in animal models of oxidant injury and in humans (14Morrow J.D. Awad J.A. Kato T. Takahashi K. Badr K.F. Roberts II, L.J. Burk R.F. J. Clin. Invest. 1992; 90: 2502-2507Crossref PubMed Scopus (298) Google Scholar, 15Morrow J.D. Frei B. Longmire A.W. Gaziano M. Lynch S.M. Shyr Y. Strauss W.E. Oates J.A. Roberts II, L.J. N. Engl. J. Med. 1995; 332: 1198-1203Crossref PubMed Scopus (1260) Google Scholar). In this regard, however, quantification of unmetabolized IPs has certain limitations. First, F2-IPs can be artifactually generated ex vivo, e.g. in plasma, by auto-oxidation of plasma arachidonic acid if appropriate precautions are not taken (8Morrow J.D. Harris T.M. Roberts L.J.I. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar). In addition, quantification of F2-IPs esterified in tissues or circulating in plasma only provides information at a single point in time rather than an integrated index of IP production. Having a means to obtain an integrated index of oxidant stress status would be very valuable in situations in which the level of oxidant stress fluctuates over time. In this regard, analogous to quantification of urinary metabolites of cyclooxygenase-derived prostanoids (16Roberts II, L.J. Willis A.L. Handbook of Eicosanoids: Prostaglandins and Related Lipids, Chemical and Biochemical Aspects, Part A. CRC Press Inc., Boca Raton, FL1987: 233Google Scholar), measurement of the urinary excretion of F2-IPs should provide a reliable and integrated index of oxidative stress status in vivo.We have previously identified urinary metabolites of F2-IPs that copurify through a mass spectrometric assay developed for quantification of the major urinary metabolite of cyclooxygenase-derived PGD2 (17Awad J.A. Morrow J.D. Takahashi K. Roberts II, L.J. J. Biol. Chem. 1993; 268: 4161-4169Abstract Full Text PDF PubMed Google Scholar, 18Morrow J.D. Prakash C. Awad J.A. Duckworth T.A. Zackert W.E. Blair II, A. Oates J.A. Roberts II, L.J. Anal. Biochem. 1991; 193: 142-148Crossref PubMed Scopus (33) Google Scholar). However, we do not know the parent compounds from which these F2-IP metabolites derive. Furthermore, we have found that a metabolite of cyclooxygenase-derived PGF2α, 9α,11α-dihydroxy-15-oxo-13,14-dihydro-2,3,18, 19-tetranorprost-1,20-dioic acid, cochromatographs on capillary gas chromatography (GC) with these F2-IP metabolites. 2L. J. Roberts II, K. P. Moore, W. E. Zackert, J. A. Oates, and J. D. Morrow, unpublished data. This latter finding confounds an interpretation as to whether an increase in the intensity of these peaks when analyzed by GC and mass spectrometry (MS) represents overproduction of F2-IPs or PGF2α. Thus, we undertook a study to identify the major urinary metabolite of 8-iso-PGF2α in humans as a basis for the development of methods of assay for its quantification to assess oxidative stress status in humans.