Analysis of Monohydroxyeicosatetraenoic Acids and F2-isoprostanes as Markers of Lipid Peroxidation in Rat Brain Mitochondria

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Abstract
We have introduced two specific techniques for the quantitative measurement of monohydroxyeicosatetraenoic acids (HETEs) and F2-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 C18-cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F2-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 - 40pmol/mg protein. The corresponding content for the F2-isoprostane, 8-iso-PGF 2α, was 0.21 - 0.10pmol/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 30min incubation with Fe2+ + ascorbate, the content of HETE isomers was increased about 6-fold compared with base-line levels whereas that for 8-iso-PGF2α was elevated 100-fold. Formation of HETEs and F2-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-PGF2α 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 F2-isoprostanes in freshly isolated rat brain mitochondria and that esterified HETEs and 8-iso-PGF2α 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 F2-isoprostanes may be important mediators for mitochondrial functions.

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