Folia Psychiatrica et Neurologica Japonica, Vol. 33, No, 1, 1979
Effects of Acute and Chronic Ethanol Administration on Amino Acid Metabolism in Rabbit Brain and Blood Yoshiaki Okamoto, M.D., Touhei Murayama, M.D. and Motoi Ogata, M.D. Department of Neuropsychiatry, Sapporo Medical College, Sapporo
INTRODUCTION
Studies on the aberrant biochemical process of alcoholism in humans have been made with the aid of laboratory animals. However, the studies have been concerned mostly with lipid and carbohydrate metabolism, and less with amino acid as well as protein metabolism. Most of the investigations on the effects of ethanol on amino acid metabolism in the brain are related to acute administration of ethanol whereas observations on the effects of chronic administration, probably presenting a neurochemical basis for a better understanding in some aspects of alcoholism, are scanty. Also, there is few literature regarding the effects of both acute and chronic administrations of ethanol in vivo on amino acids in the blood. Unfortunately, the results of these experiments have been mostly inconsistent and the reason for this discrepancy has not been completely clarified as yet. On the other hand, electrophysiological evidence has suggested that functionally different areas of the brain are affected to various degrees by ethanol."' It has been also shown that a different distribution of the free amino acid composition appears when the brain is divided into different parts,"" and that some amino acids not only are precursors of proteins or nucleic acids but also function as neurotransmitters.12 It may be therefore necessary to examine the Keceived for publication Sept. 6, 1978.
regional changes in order to assess the effects of ethanol on amino acid metabolism in the brain. Furthermore, recent reports have shown several features of interorganal relationships in amino acid metabolism in vivo, indicating that some amino acids in the blood plasma act as "nitrogen carriers" from organ to organ.1U'' Although the role of red blood cells in interorganal transport of amino acids must be considered,':' by examining the changes in the plasma concentration of amino acids after ethanol administration, it may be therefore possible to know whether there are disorders of amino acid metabolism in the organs caused by ethanol. In this present study we have investigated the effects of both acute and chronic administrations of ethanol in vivo on the contents of free amino acids and related compounds in the cerebral cortex and arterial blood plasma of rabbits. MATERIALS AND METHODS
Animals and Treatment Fifteen male white rabbits weighing 2.02.8 kg at the beginning of this experiment were used. All animals were fed on a standard laboratory diet for 100 days and terminated by decapitation. Acute Ethanol Experiments The animals in both acute ethanol-treated and control groups were given ordinary tap
112
Y. Okamoto er
water ad libitum for 100 days. The animals were injected intraperitoneally with 2596 ( v / v ) saline solution of 99.5% (v/v) ethanol at a dose of 1.6g,/kg body wt. Two control groups were used, one consisting of rabbits injected intraperitoneally with the same volume of saline and the other rabbits with no saline. The animals were sacrificed 30 min after the injection in order to obtain the highest level of blood ethanol." Chronic Ethanol Experiments The rabbits were given a 25% (v/v) aqueous solution of 99% (v/v) ethanol ad libitum as the only source of liquid for 100 days. In the case of animals receiving chronic pretreatment with ethanol, there was an apparent difference in the blood ethanol level between morning and afternoon since the animals drank mainly at night.":' Also, circadian rhythm in the contents of the plasma amino acids was reported for animals as well as humans."* All rabbits in each series of the experiments were therefore decapitated at 2 p.m. to preclude the influence of the diurnal variation. Preparation of Cerebrocortical Extracts The procedures employed were those we have previously described,"' except that tissue samples of the cerebral cortex with an added amount of norleucine solution as an internal standard were homogenized in 1 ml of ice-cold 4% (w/v) HClO, per 100 mg and that the homogenate was centrifuged for 10 min at 21,000 g and 4OC. In all of the experiments, the cerebrocortical tissue samples from the single brains, viz., unpooled specimens, were either chromatographed immediately, or were stored at - 20°C until analyzed, but never for longer than 24 hr. The acidic and neutral amino acids in the samples were analyzed. Preparation of Arterial Blood Plasma Precautions were taken to assure reliable analysis of plasma amino acids as commented by Perry et al.45 Arterial blood
a!.
samples were collected in heparinized beakers at the time of death. The blood specimens were rapidly centrifuged for 10 min at 1,580 g. The supernatant plasma was removed with a Pasteur pipette, taking care not to aspirate any of the buffy coat. The plasma was then deprotenized within 30 min of the blood collection by adding 30 mg of solid sulfosalicylic acid per ml, shaking to mix, and recentrifuging. The deprotenized plasma supernatants were stored at - 20°C until the acidic, neutral, and basic amino acids were analyzed, but never for longer than 4 days. No significant alterations were revealed for this duration of storage. Analytical Procedures of Amino Acids and Related Compounds The free amino acids and other ninhydrin positive compounds in the samples were determined by the accelerated two-column chromatographic method on a Hitachi liquid chromatograph. Chromatography was carried out with a minor modification of the use of a sodium citrate buffer elution system, details of which have been presented elsewhere.2i In this modified procedure, ammonia and y-aminobutyric acid (GABA) were eluted separately soon after phenylalanine in the analysis of the acidic and neutral amino acids, and the separation of tyrosine and norleucine (added as an internal standard) was achieved in the analysis of the basic amino acids. Ninhydrin reagent was prepared in the usual manner."" Four ml aliquots of the samples were placed on the column by using a Hitachi single sample injector. Standard calibration mixtures (Takara Kosan Co.), to which other amino acids were added, were measured for each new preparation of ninhydrin reagent to insure standardization of color. The constants thus obtained for individual amino acids were used to calculate the test samples. Quantitative calculations were made from the height and width of each peak."" If this was not possible because of incomplete resolution,
Effects of Ethanol on Amino Acid Metabolism the absorbance method was used.'") Cystathionine was expressed in terms of norleucine equivalents. Glutamine and asparagine, and taurine and urea were referred to collectively as glutamine and taurine respectively. Determination of Blood and Brain Ethanol Level Ethanol level of arterial blood and cerebrocortical tissue taken from the animals at the time of sacrifice were measured with alcohol dehydrogenase by the method of Bucher and Redetzki.l" The student t-test was used to determine the statistical significance of the difference between the means in the experimental and control groups. RESULTS Acute Ethanol Experiments The ethanol level of the arterial blood and the cerebral cortex were 1.37 -+ 0.28 mg/ml and 1.31 I+ 0.41 mg/g respectively. The injection of ethanol caused some un-
Table I :
1 I3
steadiness but had no other obvious effect on the movements of the animals, except for a slight ataxic gait. The average daily diet intake per rabbit was approximately 150 g in both the acute ethanol-treated and the control groups. The average body weight of the animals in both groups was 3.6 kg at the time of sacrifice. The two control groups contained the contents of amino acids to a very similar extent, and therefore the control values were pooled. The effects of this dose of ethanol on the concentration of amino acids and related compounds in the rabbit cerebral cortex are summarized in Table 1. The cerebrocortical contents of glutamic acid, glutamine, aspartic acid, alanine, serine, and phosphoethanolamine were significantly higher than those in the control group. There were no significant changes in the levels of GABA and glycine, although they seemed to increase. No notable alterations occurred in the contents of the other compounds. In contrast to changes in the cerebral cortex, there were no significant increases in the contents of amino acids and related
Etfects of Ethanol on Levels of Free Amino Acids and Related Compounds in Rabbit Cerebral Cortex Administration Control
Phosphcethanola mine
Taurinet Aspartic acid Threonine Serine
Glataminett Glutarnic acid Glycine Alanine GARA Cystathioninettt Ammonia
2.23f0.16 1.46f0.3 2.24f0.21 0.19f0.04 0.41 50.07 2.95f0 . 6 7 . 5 f0.62 0.54f0.08 0.54*0.07 1.39fO. 18 0.10*0.05
6.19f 1.43
Acute 2.68f0.14"" 1.62f0.22 2.68f0.26""" 0 ; 2 f0.03 0.54f0.08""" 3.94f0.57""" 9.75f0.22" 0.65f0.08 0.67&0.07*** 1.51 k 0 . 0 3 0.18f0.07 7.05 f0.67
Chronic 2.07 f0.43 1.56k0.32 1.97f0.31 0.19f0.09
0.50f0.07 3.65 k0.45 8.51 f0.88 0.52fO. 11 0.55f0.04 I .22&0.04 0.12f0.02 6.96k2.21
Values are means +- S.D. HS pn:olcs/g of wet wt. Number of animals is 5 in all cnses. t Sum o f taurine and wen, I{Sum of g:utnmine and nspsmgine, i t f Values Lire expresszd in tcmis of norleucine equivalents. Sibmificnntly diff?rent from contrn! v i u o ( * P < 0.001, *"P < 0.01. ***P < 0 . 0 5 )
114
Y. Okamoto et al.
compounds in the rabbit arterial plasma (Table 2 ) . The acute administration of ethanol decreased significantly the plasma contents of threonine, alanine, valine, isoleucine, leucine, phenylalanine, arginine, and phosphoethanolamine in comparison to those in the control group. Chronic Ethanol Experiments The chronic administration of ethanol in the drinking water of rabbits gave the ethanol level of the arterial blood of 0.23 s+ 0.11 mg/ml and that of the cerebral cortex
of 1.38 k 0.41 mg/g when the animals were sacrificed at 2 p.m. There was no apparent difference between the experimental and the control animals in their gross behavior. The mean daily food intake of the ethanol-treated animals was only half as much as in the control group. At the time of sacrifice, the ethanol-treated animals weighed about 20% less than the controls. The average daily consumption per rabbit of the 25% (v/v) aqueous solution was 100 ml in the first half of the experimental period and 160 ml in the second half. This dose of ethanol re-
Table 2: Effects of Ethanol on Levels of Free Amino Acids and Related Compounds in Rabbit Arterial Plasma Administration Control
Acute
Chronic
Phosphoethanolamine Taurinet Aspartic acid Threonine Serine Glutaminett Proline Glutamic acid Citrulline Glycine Alanine a-Aminobutyric acid Valine Cystine Cystathioninettt Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Ornithine Histidine Lysine Tryptophan Arginine ~
-
- __
-
~
0.1I f0.02 0.24f0.03 0.02f0.01 0.20f0.03 0.19f0.05 0.57f0.29 0.36f0.13
0.06fO.01 0.05f0.02 0.79fO. 32 0.51f0.12
Effects of Acute and Chronic Ethanol Administration on Amino Acid Metabolism in Rabbit Brain and Blood Yoshiaki Okamoto, M.D., Touhei Murayama, M.D. and Motoi Ogata, M.D. Department of Neuropsychiatry, Sapporo Medical College, Sapporo
INTRODUCTION
Studies on the aberrant biochemical process of alcoholism in humans have been made with the aid of laboratory animals. However, the studies have been concerned mostly with lipid and carbohydrate metabolism, and less with amino acid as well as protein metabolism. Most of the investigations on the effects of ethanol on amino acid metabolism in the brain are related to acute administration of ethanol whereas observations on the effects of chronic administration, probably presenting a neurochemical basis for a better understanding in some aspects of alcoholism, are scanty. Also, there is few literature regarding the effects of both acute and chronic administrations of ethanol in vivo on amino acids in the blood. Unfortunately, the results of these experiments have been mostly inconsistent and the reason for this discrepancy has not been completely clarified as yet. On the other hand, electrophysiological evidence has suggested that functionally different areas of the brain are affected to various degrees by ethanol."' It has been also shown that a different distribution of the free amino acid composition appears when the brain is divided into different parts,"" and that some amino acids not only are precursors of proteins or nucleic acids but also function as neurotransmitters.12 It may be therefore necessary to examine the Keceived for publication Sept. 6, 1978.
regional changes in order to assess the effects of ethanol on amino acid metabolism in the brain. Furthermore, recent reports have shown several features of interorganal relationships in amino acid metabolism in vivo, indicating that some amino acids in the blood plasma act as "nitrogen carriers" from organ to organ.1U'' Although the role of red blood cells in interorganal transport of amino acids must be considered,':' by examining the changes in the plasma concentration of amino acids after ethanol administration, it may be therefore possible to know whether there are disorders of amino acid metabolism in the organs caused by ethanol. In this present study we have investigated the effects of both acute and chronic administrations of ethanol in vivo on the contents of free amino acids and related compounds in the cerebral cortex and arterial blood plasma of rabbits. MATERIALS AND METHODS
Animals and Treatment Fifteen male white rabbits weighing 2.02.8 kg at the beginning of this experiment were used. All animals were fed on a standard laboratory diet for 100 days and terminated by decapitation. Acute Ethanol Experiments The animals in both acute ethanol-treated and control groups were given ordinary tap
112
Y. Okamoto er
water ad libitum for 100 days. The animals were injected intraperitoneally with 2596 ( v / v ) saline solution of 99.5% (v/v) ethanol at a dose of 1.6g,/kg body wt. Two control groups were used, one consisting of rabbits injected intraperitoneally with the same volume of saline and the other rabbits with no saline. The animals were sacrificed 30 min after the injection in order to obtain the highest level of blood ethanol." Chronic Ethanol Experiments The rabbits were given a 25% (v/v) aqueous solution of 99% (v/v) ethanol ad libitum as the only source of liquid for 100 days. In the case of animals receiving chronic pretreatment with ethanol, there was an apparent difference in the blood ethanol level between morning and afternoon since the animals drank mainly at night.":' Also, circadian rhythm in the contents of the plasma amino acids was reported for animals as well as humans."* All rabbits in each series of the experiments were therefore decapitated at 2 p.m. to preclude the influence of the diurnal variation. Preparation of Cerebrocortical Extracts The procedures employed were those we have previously described,"' except that tissue samples of the cerebral cortex with an added amount of norleucine solution as an internal standard were homogenized in 1 ml of ice-cold 4% (w/v) HClO, per 100 mg and that the homogenate was centrifuged for 10 min at 21,000 g and 4OC. In all of the experiments, the cerebrocortical tissue samples from the single brains, viz., unpooled specimens, were either chromatographed immediately, or were stored at - 20°C until analyzed, but never for longer than 24 hr. The acidic and neutral amino acids in the samples were analyzed. Preparation of Arterial Blood Plasma Precautions were taken to assure reliable analysis of plasma amino acids as commented by Perry et al.45 Arterial blood
a!.
samples were collected in heparinized beakers at the time of death. The blood specimens were rapidly centrifuged for 10 min at 1,580 g. The supernatant plasma was removed with a Pasteur pipette, taking care not to aspirate any of the buffy coat. The plasma was then deprotenized within 30 min of the blood collection by adding 30 mg of solid sulfosalicylic acid per ml, shaking to mix, and recentrifuging. The deprotenized plasma supernatants were stored at - 20°C until the acidic, neutral, and basic amino acids were analyzed, but never for longer than 4 days. No significant alterations were revealed for this duration of storage. Analytical Procedures of Amino Acids and Related Compounds The free amino acids and other ninhydrin positive compounds in the samples were determined by the accelerated two-column chromatographic method on a Hitachi liquid chromatograph. Chromatography was carried out with a minor modification of the use of a sodium citrate buffer elution system, details of which have been presented elsewhere.2i In this modified procedure, ammonia and y-aminobutyric acid (GABA) were eluted separately soon after phenylalanine in the analysis of the acidic and neutral amino acids, and the separation of tyrosine and norleucine (added as an internal standard) was achieved in the analysis of the basic amino acids. Ninhydrin reagent was prepared in the usual manner."" Four ml aliquots of the samples were placed on the column by using a Hitachi single sample injector. Standard calibration mixtures (Takara Kosan Co.), to which other amino acids were added, were measured for each new preparation of ninhydrin reagent to insure standardization of color. The constants thus obtained for individual amino acids were used to calculate the test samples. Quantitative calculations were made from the height and width of each peak."" If this was not possible because of incomplete resolution,
Effects of Ethanol on Amino Acid Metabolism the absorbance method was used.'") Cystathionine was expressed in terms of norleucine equivalents. Glutamine and asparagine, and taurine and urea were referred to collectively as glutamine and taurine respectively. Determination of Blood and Brain Ethanol Level Ethanol level of arterial blood and cerebrocortical tissue taken from the animals at the time of sacrifice were measured with alcohol dehydrogenase by the method of Bucher and Redetzki.l" The student t-test was used to determine the statistical significance of the difference between the means in the experimental and control groups. RESULTS Acute Ethanol Experiments The ethanol level of the arterial blood and the cerebral cortex were 1.37 -+ 0.28 mg/ml and 1.31 I+ 0.41 mg/g respectively. The injection of ethanol caused some un-
Table I :
1 I3
steadiness but had no other obvious effect on the movements of the animals, except for a slight ataxic gait. The average daily diet intake per rabbit was approximately 150 g in both the acute ethanol-treated and the control groups. The average body weight of the animals in both groups was 3.6 kg at the time of sacrifice. The two control groups contained the contents of amino acids to a very similar extent, and therefore the control values were pooled. The effects of this dose of ethanol on the concentration of amino acids and related compounds in the rabbit cerebral cortex are summarized in Table 1. The cerebrocortical contents of glutamic acid, glutamine, aspartic acid, alanine, serine, and phosphoethanolamine were significantly higher than those in the control group. There were no significant changes in the levels of GABA and glycine, although they seemed to increase. No notable alterations occurred in the contents of the other compounds. In contrast to changes in the cerebral cortex, there were no significant increases in the contents of amino acids and related
Etfects of Ethanol on Levels of Free Amino Acids and Related Compounds in Rabbit Cerebral Cortex Administration Control
Phosphcethanola mine
Taurinet Aspartic acid Threonine Serine
Glataminett Glutarnic acid Glycine Alanine GARA Cystathioninettt Ammonia
2.23f0.16 1.46f0.3 2.24f0.21 0.19f0.04 0.41 50.07 2.95f0 . 6 7 . 5 f0.62 0.54f0.08 0.54*0.07 1.39fO. 18 0.10*0.05
6.19f 1.43
Acute 2.68f0.14"" 1.62f0.22 2.68f0.26""" 0 ; 2 f0.03 0.54f0.08""" 3.94f0.57""" 9.75f0.22" 0.65f0.08 0.67&0.07*** 1.51 k 0 . 0 3 0.18f0.07 7.05 f0.67
Chronic 2.07 f0.43 1.56k0.32 1.97f0.31 0.19f0.09
0.50f0.07 3.65 k0.45 8.51 f0.88 0.52fO. 11 0.55f0.04 I .22&0.04 0.12f0.02 6.96k2.21
Values are means +- S.D. HS pn:olcs/g of wet wt. Number of animals is 5 in all cnses. t Sum o f taurine and wen, I{Sum of g:utnmine and nspsmgine, i t f Values Lire expresszd in tcmis of norleucine equivalents. Sibmificnntly diff?rent from contrn! v i u o ( * P < 0.001, *"P < 0.01. ***P < 0 . 0 5 )
114
Y. Okamoto et al.
compounds in the rabbit arterial plasma (Table 2 ) . The acute administration of ethanol decreased significantly the plasma contents of threonine, alanine, valine, isoleucine, leucine, phenylalanine, arginine, and phosphoethanolamine in comparison to those in the control group. Chronic Ethanol Experiments The chronic administration of ethanol in the drinking water of rabbits gave the ethanol level of the arterial blood of 0.23 s+ 0.11 mg/ml and that of the cerebral cortex
of 1.38 k 0.41 mg/g when the animals were sacrificed at 2 p.m. There was no apparent difference between the experimental and the control animals in their gross behavior. The mean daily food intake of the ethanol-treated animals was only half as much as in the control group. At the time of sacrifice, the ethanol-treated animals weighed about 20% less than the controls. The average daily consumption per rabbit of the 25% (v/v) aqueous solution was 100 ml in the first half of the experimental period and 160 ml in the second half. This dose of ethanol re-
Table 2: Effects of Ethanol on Levels of Free Amino Acids and Related Compounds in Rabbit Arterial Plasma Administration Control
Acute
Chronic
Phosphoethanolamine Taurinet Aspartic acid Threonine Serine Glutaminett Proline Glutamic acid Citrulline Glycine Alanine a-Aminobutyric acid Valine Cystine Cystathioninettt Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Ornithine Histidine Lysine Tryptophan Arginine ~
-
- __
-
~
0.1I f0.02 0.24f0.03 0.02f0.01 0.20f0.03 0.19f0.05 0.57f0.29 0.36f0.13
0.06fO.01 0.05f0.02 0.79fO. 32 0.51f0.12
