CLINICAL NUTRITION (1987) 6: 241-245 Cl Longman Group UK Ltd zyxwvutsrq Branched-Chain Amino-acids and Albumin Synthesis. Study With Hepatocytes of Normal and Stressed Rats zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA S. Schwartz, M. Farriol, A. Montoya, M.J. Gbmez Lechdn,J. V. Castell Department of Biochemistry, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Ciudad Sanitaria Vall d’hebron, Barcelona and Department of Experimental Immunology, Ciudad Sanitaria La Fe, Valencia (Spain). (Reprint requests to S.S.) ABSTRACT To evaluate the incorporation of branched-chain amino-acids (BCAA) in de novo synthesised albumin, a series of experiments was performed with cultured hepatocytes from normal (Group 1) and stressed rats (groups 2 and 3). Stress was induced by keeping the rats immobilised in a cold room for 6-8 h and assessed by the presence of bleeding ulcers in the stomach. The cells were cultured in a commercial synthetic medium (Ham F-12) poor in BCAA (5.74(& of total) increasing amounts of BCAA were added to plates in groups 1 and 2 and equivalent quantities of nitrogen in the form of glycine were added to plates in group 3. In group 1, the increase in BCAA concentration from 22.74-210.8 mg/ml was followed by an increase in the albumin synthesis rate from 0.4-0.95 ug/mg cell prot/ h. In group 2, the rise in BCAA was followed by a far greater increase from 0.3-5 ug/mg cell prot/h (at least 6 times normal levels). In group 3, only 20y0 of the increase in albumin synthesis obtained in group 2 was observed. These results suggest that in stress the liver increases the use of BCAA for protein synthesis, but the increase of nitrogen in the culture medium respresents only a low percentage of this effect. INTRODUCTION Reports from several authors on clinical [l-4] and ex- perimental observations indicate that branched-chain amino-acids (BCAA) play a key role in muscle protein metabolism. They also emphasise the importance of these amino-acids in the field of nutrition [5]. Experiments with isolated rat diaphragm showed that protein synthesis was stimulated, and protein catabolism was inhibited, when BCAA were added to the incubation medium [6]. Moreover, a possible regu- latory role of leucine in the protein turnover in muscle was reported [ 71. The protein-sparing effect of BCAA may be due essentially to leucine. This effect has been attributed to anabolic effects on protein metabolism and inhibition of urea synthesis by leucine. The effect on the urea cycle is associated with the stimulation of glutamate dehydro- genase activity by leucine [8]. The effects of BCAA on hepatic protein synthesis have been studied, but because experimental conditions vary widely, it is still premature to obtain definite con- clusions. Experimental works with perfused rat livers have shown that six amino-acids could have an inhibitory effect on intracellular proteolysis. When tests individu- ally, leucine was the strongest inhibitor [9]. Reports with rat models [ 10, 1 l] have indicated that BCAA reduce protein catabolism in surgical trauma. The study of identical clinical situations with com- parative formulae differing only in BCAA concentra- tions is of prime importance. These considerations have led us to evaluate hepatic branched-chain amino-acid utilisation in normal and stressed cultured rat hepato- cytes. MA TERIA LS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Animals. Male Sprague-Dawley rats weighing 200 +Z 10 g, fed ‘ad libitum’, were used in the experiments and were divided into two groups: normal and stressed. Stress was induced by immobilisation at 4°C for 6-S h and confirmed by the presence of bleeding ulcers in the stomach [ 12-141. Isolation and culture of hepatocytes. The hepato- cytes were obtained by perfusion of the liver with colla- genase as described [15-171. The cellular viability was estimated by the dye-exclusion test with 0.027% trypan blue in saline. Only suspensions with viabilities over 241