J. Clin. Biochem. Nutr., 16, 151-159, 1994

Stimulation of Ethanol Induced by and 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 , 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 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 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- 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: , , , 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. AND ETHANOL METABOLISM IN RATS 153

Table 1. Composition of the experimental .

*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. Blood samples were collected from a tail vein at 15 min, 30 min, 1, 2, 4, 6, 9, and 12 h after the oral administration of the ethanol, and the blood ethanol concentration was then determined. Each value is the average of three rats. A: EtOH group compared with the control group.

Vol. 16, No. 3, 1994 156 S.-C. YANG et al. and the blood ethanol disappeared by 4 h, which was sooner than in the other groups. Figure 2 shows changes in the blood acetaldehyde of each group. In the control group, the maximum acetaldehyde concentration was observed at 15 min, and decreased immediately. For rats chronically administered ethanol, the maxi- mum level in the EtOH group was approximately three times that of the control group at 30 min and 1 h. Higher acetaldehyde levels remained at 12 h, whereas, in the control group, the blood acetaldehyde had disappeared completely even by 9 h after the administration. In the EtOH/pro and EtOH/Pro + Lys groups, the blood acetaldehyde was lower than in the control group at 15 min, 30 min, and 1 h, but increased and remained at high levels by 12 h after the administration. In the case of the ethanol-administered rats that consumed lysine-containing water, a rise in blood acetaldehyde was not observed in the initial period after administration, and these animals showed the lowest acetaldehyde level throughout the observa- tion period of any of the prolonged ethanol-administered groups.

A B

C D

Fig. 2. Effects of proline and lysine on blood acetaldehyde levels following ethanol administration to prolonged ethanol-administered rats. See legend of Fig. 1 for details. A: EtOH group compared with the control group.

J. Clin. Biochem. Nuts. AMINO ACID AND ETHANOL METABOLISM IN RATS 157

DISCUSSION

Ethanol is mainly metabolized by alcohol dehydrogenase (ADH) into acetal- dehyde, which is then oxidized to acetate by acetaldehyde dehydrogenase (ALDH). Compared with the control group, the EtOH group showed no change in blood ethanol levels, but did have a higher blood acetaldehyde level. According to the results of the present study, we may suppose that ADH of prolonged ethanol-administered rats acted properly but that the function of ALDH was reduced greatly (cf., Figs. 1 and 2, EtOH group). Decreases in ADH and ALDH activities were reported in rats that had ingested alcohol for a long time [13]. In that study, the accumulation of alcohol dehydrogenase and acetaldehyde dehy- drogenase inhibitors was found in the liver tissues of rats with chronic alcohol intoxication, and the inhibition of ALDH was more evident when compared with the inhibition of ADH. For prolonged ethanol-administered rats on proline-containing solution, higher blood ethanol levels continued. However, higher blood acetaldehyde was not observed, which was the opposite to the case for the EtOH group (cf., Figs. 1 and 2, EtOH/Pro group). The possible reason for this phenomenon is that the metabolism of proline into glutamate in the liver is limited by the rate of reoxida- tion of mitochondrial NADH in the respiratory chain, and the rate of ethanol oxidation is also determined by the supply of NAD+, an important coenzyme for ADH [14-16]. Because of the competition for NAD+ between proline metabolism and ethanol oxidation, the higher blood ethanol levels were found in the EtOH/ Pro group. For prolonged ethanol-administered rats on lysine-containing water, the plasma GOT activity was like that in the control group (Table 4). The lowering of blood ethanol and acetaldehyde levels was also observed (Figs. 1 and 2, EtOH/Lys group). Dorato et al. reported that lysine did not lower the blood ethanol level when the ethanol was administered intraperitoneally, but did do so after oral administration of the alcohol [17, 18]. The results in this study show good agreement with Dorato's report. It can be considered that either poor absorption or increased elimination might be a factor in lowering the ethanol level in the blood. As for the action of lysine on lowering blood ethanol and acetalde- hyde levels, another hypothesis is that lysine increases the capability of mitochon- dria to produce additional ADH and ALDH or directly affects the activity of ADH and ALDH [19]. For prolonged ethanol-administered rats on both proline and lysine, the effect on the blood ethanol level was similar to that of rats on lysine only, and the action on blood acetaldehyde was like that in the EtOH/Pro group (Figs. 1 and 2, EtOH/Pro + Lys group). This suggests that the influence of proline on the alcohol metabolism is different from that of lysine. Acetaldehyde accumulation in the blood leads to several responses such as flushing of the face, headache, loss of appetite, vomiting, increase in heart rate, and changes in blood pressure [20]. With respect to the role of acetaldehyde in the

Vol. 16, No. 3, 1994 158 S.-C. YANG et al. action of ethanol, Truitt and Walsh reported that the most important effect of acetaldehyde was its influence on the biogenic metabolism of the brain [21]. It was reported that a higher acetaldehyde output suggests a greater inhibi- tory influence on the metabolism of the brain, which could decisively affect behavior with respect to ethanol preference [22]. In the present study, the lower acetaldehyde levels encountered during the ethanol oxidation in the EtOH/Pro and EtOH/Lys groups as compared with that of the EtOH group could explain the nutritional and physiological meanings of the high preference for proline and lysine in prolonged ethanol-administered rats [1]. That is to say, the preference for ethanol is associated with the blood acetaldehyde level after ethanol intake.

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