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Chronic renal failure patients on long-term hemolysis are found to be under increased oxidative stress, caused by antioxidant deficiency, neutrophil activation during hemodialysis (HD), platelet activation and/or chronic inflammation. Increased levels of oxidants (e.g. malondialdehyde, 4-hydroxynonenal, hydrocarbons, lipohydroperoxides, oxycholesterols, carbonyls) in HD patients are thought to play an important role in the development of endothelial dysfunction, atherogenesis and cardiovascular disease, which is a frequent condition in end-stage renal disease. F_{2}-isoprostanes have been established as chemically stable, highly specific and reliable biomarkers of in vivo oxidative stress which can very sensitively measured by gas chromatography-mass spectrometry (Morrow et al. [17]). An up to 6-fold increase of plasma F_{2}-isoprostanes in HD patients is accompanied by an enhanced formation of indicators of inflammation (e.g. C-reactive protein) and decreases of endogenous antioxidants (e.g. ascorbate, α-tocopherol). In their esterified form _{2}-isoprostanes may be a useful criteria to evaluate the effectiveness of clinical interventions to diminish oxidant stress and associated inflammation. Furthermore, F_{2}-isoprostanes possess potent biological activities (e.g. 8-iso-PGF_{2α} is known as a renal vasoconstrictor) suggesting that they may also act as mediators of the cellular effects of oxidative stress and inflammation. [ABSTRACT FROM AUTHOR]

We have introduced two specific techniques for the quantitative measurement of monohydroxyeicosatetraenoic acids (HETEs) and F 2 -isoprostanes by gas chromatography-mass spectrometry/negative ion chemical ionization (GC-MS/NICI) to study lipid peroxidation in isolated rat brain mitochondria by iron/ascorbate. The analysis of HETEs involved hydrogenation, solid phase extraction on a C 18 -cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F 2 -isoprostanes, the analytical procedure was similar to that of HETEs except that the hydrogenation step was omitted. We found that HETE content (sum of 5-, 8-12-, and 15-isomers) in freshly prepared rat brain mitochondria was 220±40 pmol/mg protein. The corresponding content for the F 2 -isoprostane, 8-iso-PGF 2α , was 0.21±0.10 pmol/mg protein. HETEs and 8-iso-PGF 2α were predominantly present in the esterified form. The content of both HETEs and 8-iso-PGF 2α were increased in presence of iron/ascorbate as oxidation system. After 30 min incubation with Fe 2+ + ascorbate, the content of HETE isomers was increased about 6-fold compared with base-line levels whereas that for 8-iso-PGF 2α was elevated 100-fold. Formation of HETEs and F 2 -isoprostanes corresponded to the consumption of arachidonic acid (AA) and α-tocopherol, respectively. There were almost no changes in the content of free (non-esterified) HETEs and 8-iso-PGF 2α during the course of iron/ascorbate induced oxidation of the brain mitochondria. Our data provide the first direct evidence for the presence of HETEs and F 2 -isoprostanes in freshly isolated rat brain mitochondria and that esterified HETEs and 8-iso-PGF 2α are predominantly generated during iron/ascorbate induced lipid peroxidation. Sensitive quantification of these products of non-enzymatic lipid peroxidation as indicators of oxidant injury opens new areas of investigation regarding the role of free radicals in the pathogenesis of human diseases. In addition, HETEs and F 2 -isoprostanes may be important mediators for mitochondrial functions. [ABSTRACT FROM AUTHOR]

Summary Extracorporeal photoimmunotherapy (photopheresis) is a highly effective therapy in the treatment of various disorders. Although extracorporeal photoimmunotherapy has been successfully used for more than 10 y, its mechanism of action is still unclear. The formation of reactive oxygen species have been implicated in extracorporeal photoimmunotherapy, but malonyl dialdehyde as a marker of systemic lipid peroxidation did not increase significantly during treatment. To investigate further the involvement of reactive oxygen species in extracorporeal photoimmunotherapy, we have introduced a highly sensitive negative ion gas chromatography-mass spectrometry based method for quantitating oxygenated arachidonic acid isomers (hydroxyeicosatetraenoic acids) in plasma samples of patients treated with extracorporeal photoimmunotherapy. In the plasma of healthy volunteers pmole amounts of 2-, 3-, 5-, 8–12-, and 15-hydroxyeicosatetraenoic acid were detected and we observed a dose-dependent augmentation in these metabolites when the blood was irradiated with increasing doses of ultraviolet A in the presence of the photosensitizer 8-methoxypsoralen. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed a characteristic increase in total hydroxyeicosatetraenoic acid levels, particularly of 5-hydroxyeicosatetraenoic acid which contributed 80% to the sum of all hydroxyeicosatetraenoic acid isomers. Chiral phase high-performance liquid chromatography indicated almost equal amounts of 5S- and 5R-hydroxyeicosatetraenoic acid suggesting that the majority of lipid peroxidation products are formed via nonenzymatic oxidation reactions. [ABSTRACT FROM AUTHOR]