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

Abstract

F 2 -isoprostanes are prostaglandin-like products of nonenzymatic lipid peroxidation. Measurement of levels of endogenous unmetabolized F 2 -isoprostanes has proven to be a valuable approach to assess oxidative stress in vivo. However, measurement of levels of urinary metabolites of F 2 -isoprostanes in timed urine collections offers an advantage over measuring unmetabolized F 2 -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 F 2 -isoprostanes produced, 8-iso-prostaglandin F 2␣ (8-iso-PGF 2␣ ). 20 Ci of tritiated 8-iso-PGF 2␣ 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 g of unlabeled 8-iso-PGF 2␣ . 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 F 2␣ . The identification of 2,3-dinor-5,6-dihydro-prostaglandin F 2␣ as the major urinary metabolite of 8-iso-prostaglandin F 2␣ 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.Free radicals have been implicated in the pathogenesis of a wide variety of human disorders (1-4). 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) 1 F 2 -like compounds (F 2 -isoprostanes (F 2 -IPs)) are produced in vivo as products of the free radical-catalyzed peroxidation of arachidonic acid (5). 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 F 2 -IPs. The endoperoxides also undergo rearrangement in vivo to form D-and E-ring IPs (6). 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 (7). 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 (8), one compound that would be expected to be formed would be 8-iso-PGF 2␣ . Recently we demonstrated that 8-iso-PGF 2␣ is in fact one of the more abundant F 2 -IPs produced in vivo (9). There has been considerable interest in this molecule, because it exerts biological activity, e.g. it is a potent vasoconstri...

Oxidized derivatives of ω-3 fatty acids: identification of IPF3α-VI in human urine

“…Others, too, have been unable to measure nP formation in the urine of AD patients (20). The metabolism argument presented here is strengthened by the finding that iPF 2a -III is metabolized in vivo, with a major (29%) urinary metabolite being the dinor dihydro derivative (37). We and others have also studied this metabolic step (38)(39)(40).…”

Alcohol-induced generation of lipid peroxidation products in humans

“…Thus, oxidant stress in vivo was increased in patients with acute alcoholic hepatitis and in patients with cirrhosis and a history of alcohol abuse, and was provoked by alcohol administration to healthy volunteers. In addition to the increment in urinary iPF 2α -III, we report that excretion of 2,3-dinor-5,6-dihydro-iPF 2α -III, which may be formed as a metabolite of iPF 2α -III (27), is also increased in chronic ALD. This suggests that the increment in urinary iPF 2α -III reflects an increase in its generation in vivo, rather than reduced oxidative metabolism due to impairment of hepatic function.…”

“…Three consecutive 24-hour urine collections were obtained for iPF 2α -III. Given that little information is available on the metabolism of iPs and that the liver might be a site of metabolite formation, we developed methods to analyze a putative 2,3-dinor-5,6-dihydro-iPF 2α -III metabolite of iPF 2α -III (27,28) in a subset (n = 20) of study subjects to determine if an increment in iPF 2α -III might reflect reduced metabolism, rather than increased generation, of the compound. Urinary iPF 2α -VI was also measured in patients with ALD, and in age-and sex-matched controls.…”

“…Serum was heated with thiobarbituric acid at a pH of less than 5.0 for measurement of TBARS. The resulting malondialdehyde adduct was measured by its absorbance at 532 nm, as described previously (27).…”

Phase 2 study of imexon, a prooxidant molecule, in relapsed and refractory B-cell non-Hodgkin lymphoma

“…Urinary levels of 8-iso-prostaglandin F2alpha (8-isoP) were measured (Eicosanoid Core Laboratory, Vanderbilt University, Nashville TN) as previously described 20 and normalized to creatinine levels in the samples.…”