Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-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-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-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 radical- but not COX-dependent manner. Although 8-iso-PGF2alpha 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 (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF 2␣-F2-iPs-in urine has reflected lipid peroxidation in humans. However, up to 64 F 2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F 2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F 2-iPs, using liquid chromatography͞tandem mass spectrometry (LC͞MS͞MS), in which sample preparation is minimized. Authentic standards of F 2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF 2␣-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F 2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F 2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC͞MS and LC͞MS͞MS of iPF 2␣-VI in hypercholesterolemia and of 8,12-iso-iPF 2␣-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.
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