Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F 2 -isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F 2 -isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F 2 -isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F 2 -isoprostanes by ϳ80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

Increased CSF F2-isoprostanes are not an inevitable consequence of neurodegeneration and suggest that increased brain oxidative damage may occur early in the course of AD.

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F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the none-nzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

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...