ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 339, No. 1, March 1, pp. 157–164, 1997 Article No. BB969856 Antioxidant Reactions of Vitamin E in the Perfused Rat Liver: Product Distribution and Effect of Dietary Vitamin E Supplementation Amy J. L. Ham 1 and Daniel C. Liebler 2 Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207 Received August 1, 1996, and in revised form December 9, 1996 dant reactions and antioxidant effects in an intact or- gan system during oxidative stress.  1997 Academic Press We have investigated the relationship between vita- min E (a-tocopherol, TH) oxidation and antioxidant protection in a perfused rat liver model. Perfusion of a male Sprague – Dawley rat liver with 2 mM tert-butyl- The lipid-soluble chain-breaking antioxidant TH 3 hydroperoxide (t-BuOOH) for 10 min resulted in lipid (Fig. 1) protects cell membranes and lipoproteins peroxidation and metabolic changes reflecting oxida- against oxidative damage [reviewed in (1)]. The fate of tive stress. Mitochondria isolated from the liver exhib- TH in radical scavenging reactions has been identified ited increases in state 3 and state 4 respiration and a previously in chemical model systems (2 – 6) and most decline in the respiratory control ratio. In livers from rats given supplementary vitamin E in the diet, TH recently in mitochondria in vitro (7). TH scavenges per- content was 7- to 10-fold higher than in controls and oxyl radicals by a two-reaction sequence. Initial hydro- lipid peroxidation and metabolic changes induced by gen transfer from TH to a peroxyl radical (Eq. [1]) is t-BuOOH were decreased. In mitochondria from these followed by reaction of the resulting tocopheroxyl radi- vitamin E-supplemented livers, the t-BuOOH-induced cal with another peroxyl radical to yield a nonradical increase in state 4 respiration was reduced and the product (Eq. [2]). respiratory control ratio was maintained. In livers from unsupplemented rats, t-BuOOH induced oxida- TH / ROOr r Tr / ROOH [1] tion of TH to a-tocopherolquinone, a-tocopherolhydro- quinone, 2,3-epoxy-a-tocopherolquinone, and 5,6- Tr / ROOr r nonradical products [2] epoxy-a-tocopherolquinone as determined by gas chromatography – mass spectrometry analysis. Yields Products formed by reaction [2] include both 8a-substi- of these products were approximately doubled by tuted tocopherones [e.g., 8a-(alkyldioxy)-tocopherones treatment of samples with dilute acid, which indicated and 8a-hydroxytocopherone] and isomeric epoxy-8a- the presence of tocopherone and epoxytocopherone (hydroperoxy)tocopherones (4, 5). The 8a-substituted precursors. Oxidation of TH in vitamin E-supple- tocopherones readily hydrolyze to TQ upon treatment mented livers yielded the same products and the rela- with dilute HCl, whereas the epoxytocopherones yield tive extent of TH oxidation appeared similar to that in TQE1 and TQE2 (5). TQ, TQE1, and TQE2 thus are unsupplemented livers. In livers from both unsupple- markers that indicate the consumption of TH through mented and vitamin E-supplemented animals, the dis- antioxidant reactions (8). tribution of oxidation products was similar in whole We recently reported that TQ, TQE1, and TQE2 are liver and isolated mitochondria. These data provide the principal products of TH oxidation in rat liver mito- the first simultaneous documentation of TH antioxi- chondria incubated in vitro with the free radical-gener- 1 Current address: Department of Pathology, 515 Brinkhouse-Bul- 3 Abbreviations used: t-BuOOH, tert-butylhydroperoxide; TBARS, thiobarbituric acid-reactive substances; TH, a-tocopherol; THQ, a- litt Bldg., CB #7525, University of North Carolina, Chapel Hill, NC 27599-7525. tocopherolhydroquinone; T(H)Q, sum of measured TQ and THQ; TQ, a-tocopherolquinone; TQE1, 5,6-epoxy-a-tocopherolquinone; TQE2, 2 To whom correspondence should be addressed. Fax: (520) 626- 2466. E-mail: liebler@tonic.pharm.arizona.edu. 2,3-epoxy-a-tocopherolquinone. 157 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.