IPF 2 a-I : An index of lipid peroxidation in humans

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2a, an isomer of the PGGyH synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2a (PGF2a), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2a-I, (IPF2a-I) a class I isomer (8-isoPGF2a is class IV), using gas chromatographyymass spectrometry. IPF2a-I is severalfold as abundant in human urine as 8-iso-PGF2a, with mean values of 737 6 20.6 pgymg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2a-I, unlike 8-iso-PGF2a, is not formed in a COXdependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2a-I. Neither serum IPF2a-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2a-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2a formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2a-I is elevated in cigarette smokers compared with controls (1525 6 180 versus 740 6 40 pgymg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2a (r 5 0.9; P < 0.001). Urinary IPF2a-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radicalbut not COX-dependent manner. Although 8-iso-PGF2a may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers. Isoprostanes are chemically stable prostaglandin (PG)-like compounds that are produced independent of the cyclooxygenase (COX) enzyme by free radical-catalyzed peroxidation of arachidonic acid (AA) in situ in membrane phospholipids (1). They are cleaved by phospholipases, circulate in plasma, and are excreted in urine (2). F2-isoprostanes are isomers of PGF2a, a natural product derived from COX and PGF synthase. Depending on the site of the original arachidonyl radical formed, four groups of regioisomers of the F2-isoprostanes may be produced upon rearrangement, oxygenation, and reduction, each of which may comprise 16 isomers. Thus, in theory, up to 64 different F2isoprostanes can be generated (3, 4). Semiquantitative estimates of their generation have been based on enzyme immunoassay and gas chromatographyymass spectrometry (GCyMS), the latter using deuterated PGF2a as internal standard (5, 6). We have previously developed a specific method to measure a class IV F2-isoprostane, 8-isoPGF2a, using an O2-labeled internal standard (7). Although this compound is produced in vitro and in vivo via free radical peroxidation of AA, we have shown that 8-iso-PGF2a is also a minor product of platelet COX-1 and monocyte COX-2 dependent metabolism of AA in vitro (7, 8). It may also be formed in a COX-dependent manner in serum ex vivo (4, 9). However, COX-dependent formation seems to contribute little, if at all, to levels of 8-iso-PGF2a measured in the urine of cigarette smokers (9), a syndrome of COX-1 activation (10). Given that our original data suggest that 8-iso-PGF2a, but not other F2-isoprostanes, might be formed also by a COXdependent mechanism (7), we have developed a specific GCyMS assay for IPF2a-I, a structurally distinct (class I) F2-isoprostane (11). We demonstrate that IPF2a-I is formed coincidental with conventional markers of lipid peroxidation in low density lipoprotein (LDL) oxidized by copper in vitro. Although antioxidants prevent formation of either isoprostane under these conditions, IPF2a-I, unlike 8-iso-PGF2a, is not formed in a COX-dependent manner by aggregating platelets in vitro or in serum ex vivo. IPF2a-I is excreted in human urine, where it is more abundant than 8-iso-PGF2a. Furthermore, COX inhibition in vivo, by either low dose aspirin or indomethacin, fails to suppress urinary levels of either isoprostane. These results suggest that measurement of urinary IPF2a-I represents a novel approach to the noninvasive assessment of oxidant stress in humans. These data also support the notion that COX activity contributes trivially, if at all, to urinary 8-iso-PGF2a under physiological conditions. MATERIALS AND METHODS Reagents. O2-labeled 8-iso-PGF2a was prepared as previously described by Pickett and Murphy (12). IPF2a-I was synthesized as previously described (11). The tetradeutero derivative, 17,17,18,18-teradeuterated IPF2a-I, was prepared by a modified synthesis (unpublished results). N,Ndiisopropylethylamine, pentafluorobenzyl (PFB) bromide, dodecane, N,N9-dicyclohexyl-carbodiimide, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide methiodide, copper sulfate (CuSO4), and butylated hydroxytoluene (BHT) were purchased from Sigma. Bis(trimethylsilyl)trif luoroacetamide was purchased from Supelco. Pyridine was purchased from Mallinckrodt. All other solvents were obtained from Burdick and Jackson. Isoprostane Extraction and Purification. Urine samples, 0.1 ml, were spiked with 300 pg of tetradeuterated IPF2a-I and then mixed well and allowed to equilibrate for 15 min at room temperature. The volume was brought up to 1 ml with phosphate The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y953449-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: PG, prostaglandin; COX, cyclooxygenase; SPE, solid phase extraction; GCyMS, gas chromatographyymass spectrometry; AA, arachidonic acid; Tx, thromboxane; LDL, low density lipoprotein; PFB, pentafluorobenzyl; BHT, butylated hydroxytoluene; TMS, trimethylsilyl. †These authors contributed equally to this work. §Present address: Department of Internal Medicine, University of Rome ‘‘La Sapienza’’ Rome, Italy. ¶To whom reprint requests should be addressed at: Center for Experimental Therapeutics, 905 Stellar Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 191046100. e-mail: garret@spirit.gcrc. upenn.edu.