1nt.J. Eiochem. Vol. 17,No.2.p~. 195-201.1985 0020-711X/85 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright @ 1985 Pergamon Press Ltd zyxwvutsrq ETHANOL AND LEUCINE OXIDATION-II. LEUCINE OXIDATION BY RAT TISSUE IN zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP VITRO LEIGH C. WARD, GRANT A. RAMM, STEVEN MASON and Ross DALY Department of Biochemistry, University of Queensland, St Lucia. Brisbane 4067, Australia. [Tel. 07-377-I 1111 (Received 15 March 1984) Abstract-l. The effect of ethanol upon leucine oxidation by rat tissues in vitro is reported. 2. The activities of branched chain amino acid aminotransferase and 2-0~0 acid dehydrogenase were decreased by chronic administration of ethanol (20% v/v solution as drinking water for 3.5 d) in muscle and kidney but were increased, although not significantly, in liver. 3. Acute administration of ethanol (8 g kg-’ body-weight0.73) did not affect enzyme activities. 4. Tissue NAD+:NADH ratios, calculated from lactate:pyruvate ratios, were significantly decreased in the liver and kidney of rats receiving ethanol acutely. 5. These data are consistent with the view that ethanol decreases leucine oxidation by decreasing availa- bility of NAD+ when given acutely and by decreasing enzyme activity when administered chronically. INTRODUCTION The branched chain amino acids (BCAA; leucine, isoleucine and valine) are extensively catabolised not only in the liver but also in other tissues, particularly kidney and muscle (Smith and Elia, 1983). In rats branched chain aminotransferase (BCAT) activity is high in kidney and muscle relative to that observed in liver (Sketcher et al.. 1974; Dohm et al., 1976; Shin- nick and Harper, 1976). In contrast the branched chain 2-0x0 acid dehydrogenase complex (OADH) is predominantly located in the liver, where its activity is variously reported as being lower (Sketcher et al., 1974), similar (Dohm et al., 1976) or higher (Shinnick and Harper, 1976) relative to that of BCAT. This relative distribution of enzyme activities may vary with species (Mason and Ward, 1981). This differential tissue distribution of enzyme activities allows BCAA to circumvent metabolism in the liver after absorption from the intestinal tract and to be stored in muscle protein. Here they are availa- ble for subsequent use as oxidisable substrates via transamination in muscle and decarboxylation of the 2-0x0 acids thus produced in the liver. The regulation of the plasma concentrations of BCAA and their cor- responding 2-0~0 acids thus represents a complex co- ordination of hepatic and extra-hepatic enzyme activities (Eriksson and Wharen, 1982). Further- more, the activities of these enzymes is highly depen- dent upon hormonal and dietary conditions (Adibi et al., 1975; Hutson et al., 1980). It has long been observed (Siegel et al., 1964) that the concentrations of BCAA, and leucine in particu- lar, in plasma are markedly affected by exposure to ethanol, given either acutely or chronically. Ward et al. (1984) reported that the oxidation of leucine by the rat in vivo was markedly depressed by acute doses of ethanol, although no such inhibition was observed when ethanol was administered chronically. The mechanism by which such despression occurs is not yet know but appears to be independent of a more general depression of oxidative metabolism induced by exposure to ethanol (Ward et al., 1985). In view of the capacity exhibited by animals for adaptive change in the oxidative metabolism of BCAA (Sketcher et al., 1974; Amen and Yoshimura, 1981) the present study was conducted to determine whether the observed decrease in whole-body leucine oxidation resulted from such an adaptive change in the activities of BCAT and OADH. The data have in part, been previously presented in abstract form elsewhere (Ramm and Ward, 1983). EXPERIMENTAL PROCEDURE Animals Male Wistar rats, weighing between 180 and 250 g, were used and housed as previously described (Ward eral.. 1985). M aterials Radiochemicals. L-[l-‘4C]leucine (54.7 $Zi pmol-‘) and [l-‘4C]ethanol (53.4 PCi prnol-‘) were obtained from Amersham International Ltd., Amersham, U.K. and used without further purification. Liquid scintillation counting chemicals. 2.5 diphenyloxazole, (PPO) and his-2,4-(methyl-5phenoxyl)- benzene, (POPOP), were purchased from Packard Instru- ment Company Inc., Downers Grove, Illinois. Toluene. low-suphur grade, was obtained from May and Baker Ltd, Victoria. 2-Methoxyethanol was purchased from BDH Ltd. Poole. O ther chemicals. Pyridoxal phosphate. malic acid, thiamine pyrophosphate, 2-oxoglutarate, NAD’ and ATP were products of Sigma Chemical Co., St Louis. Coenzyme A was obtained from Boehringer Mannheim Australia Pty Ltd, Victoria. Ethanolamine was purchased from Ajax chemicals, Sydney and redistilled and stored under nitrogen before use. All other chemicals were obtained from BDH Ltd. Poole and of analytical reagent grade whenever possi- ble. Experimental treatment of animals The effects of ethanol administered acutely and chroni- cally were both studied. The mode of administration of 195