J. Clin. Biochem. Nutr., 16, 151-159, 1994 Stimulation of Ethanol Metabolism Induced by Proline and Lysine Ingestion in Prolonged Ethanol-Administered Stroke-Prone Spontaneously Hypertensive Rats Suh-Ching YANG,1* Michiko ITO,1 Fumiki MORIMATSU,2 Slamet BUDIJANTO,1 Yuji FURUKAWA,1 and Shuichi KIMURA1 1 Department of Applied Biochemistry, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai 981, Japan 2 Research and Development Center, Nippon Meat Packers Inc., Haibara-gun, Shizuoka 421-03, Japan (Received September 25, 1993) Summary The influence of proline and lysine solution ingestion on ethanol metabolism after chronic oral ethanol administration was inves- tigated in stroke-prone spontaneously hypertensive rats (SHRSP). Compared with rats that were not administered ethanol (control group), the prolonged ethanol-administered rats (EtOH group) showed little change in their blood ethanol levels, but a remarkable increase and slow appearance in the blood acetaldehyde levels after the oral administration of ethanol. When prolonged ethanol-administered rats ingested the proline solution (EtOH/Pro group), higher levels of blood ethanol continued. However, the increase in blood acetaldehyde levels was not observed in the EtOH/Pro group. In the case of prolonged ethanol- administered rats that ingested the lysine-containing solution (EtOH/Lys group), blood ethanol levels decreased and disappeared 4 h after the oral administration. An effect of lowered blood acetaldehyde levels was also observed. When prolonged ethanol-administered rats ingested both proline and lysine (EtOH/Pro+Lys group), the effect on blood ethanol levels was similar to that of rats that had ingested lysine only, and the influence on blood acetaldehyde was like that of rats that had only ingested proline. These results suggest that proline and lysine can regulate ethanol metabolism and the influence of proline on ethanol metabolism is different from that of lysine. Key Words: proline, lysine, ethanol metabolism, oral ethanol adminis- tration, stroke-prone spontaneously hypertensive rats (SHRSP) *To whom correspondence should be addressed . 151 152 S.-C. YANG et al. There is increasing interest in the facts that determine ethanol preference in animals. It has previously been demonstrated in experimental animals that the preference for ethanol depends not only on genetic factors, but also on the nutritional status, i.e., dietary protein levels. For example, spontaneously hyperten- sive rats (SHR) showed a higher ethanol preference than other strains of rats (Wistar-Kyoto, Sprague-Dawley, and Wistar-sic) [I]. We also observed that stroke-prone spontaneously hypertensive rats (SHRSP) showed a high preference for ethanol, like the SHR [2]. Further, we previously showed that the intake of alcohol can be increased in SHRSP by the addition of amino acids to the ethanol solution. In particular, a large amount of intake of 5% ethanol solution containing 100 mM L-proline, 100 mM L-lysine, and 100 mM L-threonine was observed [3]. Mori et al. [4] reported that animals can seek and select specific nutrients in the case of deficiency or disease. Accordingly, it has been speculated that the selection of proline, lysine, threonine in rats that ingest chronic amounts of ethanol may have important nutritional and physiological value. The effects of various natural amino acids on ethanol oxidation in isolated hepatocytes of rats have been investigated [3]. It was also reported that the ethanol metabolism was accelerated in the presence of certain amino acids: glycine, serine, methionine, etc. [5-8]. Thus, for clarification of the nutritional and physiological value of preferred amino acids in rats with chronic ethanol intake, one method is to investigate the effects of amino acids on ethanol metabolism in these animals. To determine the role of amino acids in the intake of ethanol, the present study was undertaken to elucidate the influence of the amino acids proline and lysine on ethanol metabolism in SHRSP. SHRSP were chosen as experimental animals in this study because they showed a high ethanol preference in our previous studies [1 -3] . MATERIALS AND METHODS Experimental animals. Male SHRSP bred at Shimane Medical University and weighing approximately 195 g were used in the present study. They were housed individually in stainless steel cages at controlled temperature (25+ 1°C) and relative humidity (50%), with a 12-h light/dark cycle. Experimental groups and diets. The rats were divided into two groups, non ethanol-administered group (control group) and the ethanol-administered group. To provide a certain volume of ethanol, ethanol was orally administered to rats via a gastric probe. Rats were given 2 ml of 15% ethanol solution twice a day for 5 weeks. Thereafter, 3 ml of 18% ethanol solution was orally administered to the animals twice a day to increase the ethanol intake. The same volume of distilled water was administered to the control group. Furthermore, the rats in the ethanol- administered group were divided into four subgroups: the EtOH group (distilled water), the EtOH/Pro group (100 mM proline solution), the EtOH/Lys group (100 mM lysine solution), and the EtOH/Pro + Lys group (100 mM proline+ 100 mM J. Clin. Biochem. Nutr. AMINO ACID AND ETHANOL METABOLISM IN RATS 153 Table 1. Composition of the experimental diet. *Oriental Mixture , Oriental Yeast Co., Ltd., Tokyo. lysine solution), n = 6 per group. Drinking water was supplied ad libitum through- out the experimental period. Fifteen percent purified egg protein (PEP) diets were prepared for this study. The composition of the experimental diet is shown in Table 1. Rats were pair-fed in order to provide a similar consumption of energy. One gram of ethanol can provide 7.11 calories, and thus the amount of feed given to the control group was increased to compensate for their insufficient energy intake compared with that of the ethanol-administered group. The body weight of each group was measured once every three days, and the drinking water intake was recorded daily. Biochemical analysis. On day 42, plasma was prepared from the venous blood of the tail; and the transaminase activity, an index of hepatic function, was measured for each group with a Transaminase CII Test (Wako Pure Chemical Industries, Ltd., Osaka) [9, 10]. Experiment on alcohol metabolism. On day 85, after a 12-h fast, 3 ml of 18% ethanol solution was administered to all rats. Blood was collected from the tail vein at various intervals (15, 30 min, 1, 2, 4, 6, 9, and 12 h), and the ethanol and acetaldehyde concentrations of the blood samples were measured. Ethanol and acetaldehyde concentrations were determined by enzymatic methods (F kit: Ethanol and F kit: Acetaldehyde, Boehringer, Mannheim and Yamanouchi, Co., Tokyo) [11]. Statistical analysis. Data were analyzed by Student's t-test [12]. RESULTS Body weight and drinking water intake The final body weight and the body weight gain for each group are shown in Table 2. There was no significant difference between the five groups. The drinking water intake is shown in Table 3. The control and EtOH groups showed similar intakes of drinking water. When proline or lysine was added to the drinking water, the drinking water intake increased. The drinking water intake in the EtOH/Pro + Lys group also rose about 50%. Vol. 16, No. 3, 1994 154 S.-C. YANG et al. Table 2. Final body weight and body weight gain in the experimental groups. Each value represents the mean + SE. Table 3. Comparison of the effects of proline and lysine on drinking water intake. Each value represents the mean + SE. Table 4. Comparison of the effects of proline and lysine on plasma transaminase activity. Each value represents the mean + SE. TYansaminase activity Transaminase activity is shown in Table 4. The GOT activity was significantly higher in the EtOH group and the EtOH/Pro + Lys group than in the control group and the EtOH/Lys group. This result suggests that the plasma GOT activity was elevated after the prolonged intake of ethanol and the rise in plasma GOT activity was suppressed when prolonged ethanol-administered rats ingested lysine- containing water. Furthermore, there was no change in the GPT activity of any group. Changes in blood ethanol and acetaldehyde levels To investigate the effects on ethanol metabolism in prolonged ethanol- administered rats that ingested proline- and lysine-containing water daily, the same volumes of ethanol were administered to all rats fed the experimental diet for J. Clin. Biochem. Nutr. AMINO ACID AND ETHANOL METABOLISM IN RATS 155 12 weeks, and blood ethanol levels and acetaldehyde levels were measured at various intervals. Changes in blood ethanol concentrations are shown in Fig. 1. Blood ethanol levels in the control group, which had not been administered ethanol during the 12 weeks, reached a maximum level at 30 min, and then decreased rapidly and almost disappeared by 6 h after administration. In the EtOH group, results similar to those of the control group were observed, but the disappearance of blood ethanol was a little slower than that of the control group. The maximum level of blood ethanol in the EtOH/Pro group was higher than that of the control group, reaching 2 g/liter in 1 h. Then, the level decreased gradually to 0.5 g/liter at 6 h, and became undetectable by 12 h. In the EtOH/Lys and EtOH/Pro + Lys groups, the maximum concentration of blood ethanol was 60% that of the control group, A B C D Fig. 1. Effects of praline and lysine on blood ethanol levels following ethanol administra- tion to prolonged ethanol-administered rats. The rats were divided into five groups: control group, EtOH group, EtOH/Pro group, EtOH/Lys group, and EtOH/Pro + Lys group (n = 6 for each group). At 12 weeks, all rats were fasted for 12 h; and then ethanol (0.54 g) was administered with a gastric probe.