Analysis of monohydroxyeicosatetraenoic acids and F2-isoprostanes as markers of lipid peroxidation in rat brain mitochondria.
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-PGF2alpha, was 0.21 +/-+/- 0.10 pmol/mg protein. HETEs and 8-iso-PGF2alpha were predominantly present in the esterified form. The content of both HETEs and 8-iso-PGF2alpha were increased in presence of iron/ascorbate as oxidation system. After 30 min incubation with Fe2+ ascorbate, the content of HETE isomers was increased about 6-fold compared with baseline levels whereas that for 8-iso-PGF2alpha was elevated 100-fold. Formation of HETEs and F2-isoprostanes corresponded to the consumption of arachidonic acid (AA) and alpha-tocopherol, respectively. There were almost no changes in the content of free (non-esterified) HETEs and 8-iso-PGF2alpha 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-PGF2alpha 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.
Wiswedel
Ingrid
I
Department of Pathobiochemistry, Medical Faculty,Otto-von-Guericke-University, Magdeburg, Germany. ingrid.wiswedel@medizin.uni-magdeburg.de
Hirsch
Daniela
D
Nourooz-Zadeh
Jaffar
J
Flechsig
Antje
A
Lück-Lambrecht
Annett
A
Augustin
Wolfgang
W
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D015237:Dinoprost / Q000378:metabolism; D028441:F2-Isoprostanes / Q000032:analysis*; D005227:Fatty Acids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis*; D007501:Iron / Q000494:pharmacology; D015227:Lipid Peroxidation; D008928:Mitochondria / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D013997:Time Factors; D024502:alpha-Tocopherol / Q000378:metabolism
D028441:F2-Isoprostanes; D005227:Fatty Acids; D006893:Hydroxyeicosatetraenoic Acids; D017392:Thiobarbituric Acid Reactive Substances; D015237:Dinoprost; D007501:Iron; D024502:alpha-Tocopherol; D001205:Ascorbic Acid