Evaluation of Lipophilic Antioxidant Efficacy in Vivo by the Biomarkers Hydroxyoctadecadienoic Acid and Isoprostane

Abstract

The evaluation of antioxidant activity in vivo is difficult. In this study, the effects of dietary natural and synthetic antioxidants on the lipid peroxidation in mice were assessed using a biomarker, total hydroxyoctadecadienoic acid (tHODE). Biological samples such as plasma, erythrocytes, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to tHODE. Subsequently, the absolute concentration of tHODE and its stereoisomer ratio, [9- and 13-(Z,E)-HODE)/[9- and 13-(E,E)-HODE], which is a measure of the hydrogen donor capacity of antioxidants, were determined by gas chromatography-mass spectrometry (GC-MS) analyses. These were then compared with total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)) which was also assessed after reduction and saponification. Remarkable increases in tHODE and t8-iso-PGF(2alpha) levels were observed in the plasma, erythrocytes, liver, and brain of mice that were fed an alpha-tocopherol (alphaT)-stripped (E-free) diet for 1 month when compared with those of mice that were fed a standard diet (alphaT = 0.002 wt%). When mice were fed for 1 month on an E-free diet supplemented with a lipophilic antioxidant (0.04 wt%), namely, alphaT, alpha-tocotrienol (alphaT3), gamma-tocopherol (gammaT), or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653), a potent synthetic antioxidant, the increases of tHODE and t8-iso-PGF(2alpha) in the plasma, erythrocytes, liver, and brain were suppressed to the levels lower than those of mice fed a standard diet. The (Z,E/E,E) HODE ratio was decreased in the plasma and erythrocytes of mice fed the E-free diet when compared with that in mice fed the standard diet. This stereo-isomeric ratio was significantly recovered by the addition of alphaT and BO-653. These results show that the tHODE level and the (Z,E/E,E) HODE ratio are useful biomarkers for the assessment of antioxidant capacity in vivo and that the antioxidant capacity decreased in the order: BO-653 > alphaT3 >or= alphaT, gammaT, as assessed by tHODE levels from blood, liver, and brain.