Prevention of Effects of Ethanol on Amino Acid Concentrations in Plasma and Tissues

Transcription

1 Prevention of ffects of thanol on Amino Acid Concentrations in Plasma and Tissues by Hepatic Lipotropic Factors in Rats RONALD T. STANKO, M.D., MIL L. MORS, B.S., and SIAMAK A. ADIBI, M.D., Ph.D. Gastrointestinal and Nutrition Unit of Montefiore Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania The authors' previous studies have shown that hepatic steatosis of chronic ethanol ingestion in rats can be prevented by adding pyruvate, dihydroxyacetone, and riboflavin to their diet. In this study, the authors investigated the effect of chronic ethanol ingestion, with or without addition of the above metabolites to the diet, on protein and amino acid concentrations in tissues. Rats (12 g) were divided into three groups and fed isoca1rically one of the following diets for 3 days: control diet (28"lo fat, 15"lo protein, and 57"lo carbohydrate), ethanol diet (28"lo fat, 15"lo protein, 23"lo carbohydrate, and 34"lo ethanol), and metabolite diet (ethanol diet plus pyruvate, dihydroxyacetone, and riboflavin). Chronic ethanol ingestion reduced growth of muscle and intestinal mucosa without affecting that of liver and kidney. Among the 15 amino acids measured, chronic ethanol ingestion had the most consistent effect on plasma and tissue concentrations of leucine, alanine and a-amino-n-butyrate. The concentration of leucine was increased in muscle, liver, and plasma; that of a-amino-n-butyrate was increased in muscle and plasma, whereas that of alanine was decreased in plasma and liver. Addition of pyruvate, dihydroxyacetone, and riboflavin to the ethanol diet either totally or partially prevented ethanol-induced changes in plasma and tissue concentrations of amino acids despite similarity in plasma ethano11eve1s. Although these metabolites prevented the inhibition of the growth of intestinal mucosa, they were Received February 23, Accepted July 24, Address requests for reprints to: Dr. S. A. Adibi, Montefiore Hospital, Pittsburgh, Pennsylvania A portion of this study was supported by a grant (AM 15855) from the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, U.S. Public Health Service by the American Gastroenterological Association ineffective in blunting the effect of ethanol on the skeletal muscle. This latter observation suggests that the mechanism of ethanol-induced inhibition of tissue growth is not the same for these tissues. Comparative studies of plasma and tissue amino acid concentrations have provided insight into changes in protein and amino acid metabolism induced by dietary alterationsy There are indications that such studies might be useful for a better understanding of the metabolic effects of ethanol. Siegel et al." have shown both alterations in the plasma amino acid pattern of alcoholics in the fasting state and acute changes in this metabolic parameter after ethanol loading in alcoholic and nonalcoholic subjects. However, the effect of chronic ethanol ingestion on plasma and tissue amino acid profiles in the face of adequate dietary intake has yet to be established. The first aim of the present investigation was to determine the effect of chronic ethanol ingestion on concentrations of amino acids in plasma and tissues such as liver, muscle, renal cortex, and intestinal mucosa in control and ethanol-fed rats. Both groups of rats consumed the same amount of calories from isocaloric diets with the same protein composition. In a previous study,4 after investigating the hepatic lipotropic potential of a variety of natural dietary and metabolic substances, the authors found that ethanol-induced accumulation of fatty acids in the liver can be prevented by addition of pyruvate, dihydroxyacetone, and riboflavin to the diet. These observations raised the possibility that this prevention of hepatic steatosis might be accompanied by other metabolic alterations such as changes in tissue and plasma amino acid concentrations. Therefore, the second aim of the present investigation was to compare plasma and tissue concentrations of amino acids of rats fed ethanol alone and those fed

2 January 1979 PRVNTION OF FFCTS OF THANOL ON AMINO ACID CONCNTRATIONS 133 ethanol together with the combination of pyruvate, dihydroxyacetone, and riboflavin. Shaw et al.,5 from studies in ambulatory and hospitalized alcoholics as well as in baboons fed alcohol along with an adequate diet, reported an elevation of plasma ratio of O'-amino-n-butyric acid to leucine (AIL). From these observations, they suggested that the plasma AIL ratio may be used as an objective, empirical marker for the detection and assessment of alcoholism. Although Morgan et al. 6 have subsequently questioned the sensitivity of plasma AIL ratio in the diagnosis of long-term ethanol abuse, they nevertheless have found an elevation of this ratio in patients with both ethanol-related and nonethanol-related liver disease. The third aim of the present investigation was to provide further insight into this observation by determining AIL ratio in plasma of control and ethanol-fed rats and of rats whose ethanol-induced hepatic steatosis is prevented by the addition of pyruvate, dihydroxyacetone, and riboflavin to the diet. Methods Male Sprague-Dawley rats weighing g were fed for 3 days with one of the following diets: Diet C. Lieber-DeCarli diet containing 28% fat, 15% protein, and 57% carbohydrate (Bio-Mix no. 711, Bio Serv, Inc., Frenchtown, N.J.). The percent composition refers to percent of total calories. Diet. Partial isocaloric substitution of carbohydrate content of diet C with 95% ethanol (62 mlj5 ml of diet), which resulted in the following caloric composition: 28% fat, 15% protein, 23% carbohydrate, and 34% ethanol. Diet M. To 5 ml of diet were added 22 g each of pyruvate and dihydroxyacetone and 2.2 g of riboflavin. Dextrin was added to diets C and to make them isocaloric with diet M. The rats used in this experiment consisted of series of eight rats; each was divided into three groups (C,, M) with two, three, and three rats in each group, respectively. The three rats in group were given diet and their caloric intake for 24 h was determined and averaged. On the following day this amount of calories was fed to the rats in the other groups. This feeding pattern, which is similar to one previously used" was continued for 3 days. The total intake (calories, mean ± SM) during the 3-day feeding period was as follows: group C, 2246 ± 3, n = 11; group, 226 ± 42, n = 17; group M, 215 ± 6, n = 16. There was no significant difference between the caloric intakes of these groups. The rats had access to food and water until 1 hr before they were killed. This was done to avoid the effects of starvation on tissue protein and amino acid concentrations" and to simulate the experimental conditions of the authors' previous study.4 On the morning after the 3th day of feeding, the rats were killed by decapitation between 8: and 1: AM. Blood was collected from the cervical stump into a heparin-containing test tube and plasma was separated by centrifugation. One milliliter of 6% sulfosalicylic acid was added to 1 ml plasma and centrifuged at 6 g for 1 min to obtain supernatant for amino acid analysis. The liver, gastrocnemius muscle (left), and kidney (left) were removed. The small intestine from the ligament of Treitz to the ileocecal valve was removed, washed with saline, and opened along its longitudinal axis, and the mucosa was scraped with a glass slide. Approximately.4 g of liver, muscle, mucosa, or kidney cortex was immediately frozen in liquid nitrogen, weighed, and homogenized in an all-glass homogenizer in 6% sulfosalicylic acid (1 mlj.1 g tissue). All supernatants obtained in the above preparations were stored at -2 C until analyzed for amino acid concentration on a Beckman amino acid analyzer, model 12C (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.)." 2 A portion of each of the above tissues was weighed and analyzed for protein concentration according to the method of Lowry et aj.7 On another portion of each tissue, wet weight was determined, and after 24 h of drying at 1 C, dry weight was measured. The difference between these two weights was used to calculate the percent water content of each organ. The remainder of the liver, kidney, gastrocnemius muscle, and intestinal mucosa was weighed for determination of total organ weight. The concentration of ethanol in plasma was measured by using Sigma ethyl alcohol test kit (Sigma Chemical Co., St. Louis, Mo.). Student's t-test was used for the statistical evaluation of the data. B Results ffect on Protein Metabolism Despite identical caloric intake by control and ethanol-fed rats, chronic ethanol ingestion for 3 days resulted in a significantly smaller gain in body weight (Table 1). Although there was no significant difference between either the liver or kidney weights of control and ethanol-fed rats when they were killed, the muscle and intestinal mucosal weights of ethanol-fed rats were significantly smaller (Table 1). The protein concentration and water content of liver, muscle, and kidney were not significantly affected by ethanol, but the protein concentration in intestinal mucosa was significantly reduced (Table 1). The total protein contents (weight of organ X protein concentration) of liver and kidney were not significantly affected by ethanol, but they were significantly reduced in the muscle and intestinal mucosa (Table 1). When pyruvate, dihydroxyacetone, and riboflavin were added to the diet to prevent ethanol-induced fatty liver,4 the body weight gain and weights of liver and muscle remained similar to those of eth-

3 134 ST ANKO T AL. GASTRONTROLOGY Vol. 76. No. 1 Table 1. Organ Weight and Composition (Mean ± SM) co Gain in body weight (g) 29. ± ± 12.9" ± 6.6 (l1)d (17) (16) Liver W eight (g) 1.91 ± ± ±.3 (8) (15) (1) (mg/g) 241 ±6 239 ±4 231 ±3 (7) (11) (8) Total protein (g) 2.64 ± ± ±.1 (7) (11) (8) Water (%) 68.1 ± ± ±.7 (3) (9) (6) Muscle W eight (g) 1.72 ± ±.8" 1.27 ±.4 (1) (12) (14) (mg/g) 221 ±5 223 ±6 232 ±4 (1) (12) (13) Total protein (g).38 ±.1.31 ±.2'.3 ±.1 (1) (12) (13) Water (%) 75.4 ± ± ±.3 R (3) (9) (6) Kidney W eight (g) 1.7 ± ± ±.3' (8) (14) (1) (mg/g) 184 ±6 199 ±5 193 ±4 (8) (15) (1) Total protein (g).2 ±.1.2 ±.1.23 ±O.Ol R (8) (14) (1) Water(%) 76. ± ± ± 1. (3) (9) (6) Intestinal mucosa W eight (g) 4.9 ± ±.28 g 4.94 ±.2' (5) (6) (6) (mg/g) 145 ±3 132 ± ±2 (5) (6) (6) Total protein (g).71 ±.3.52 ±.3".64 ±.2" (5) (6) (6) Water (%) 79.8 ± ± ±.7 (6) (6) (6) o Animals fed control diet. b Animals fed diet containing ethanol. C Animals fed diet containing ethanol + metabolites. d Numbers inparentheses denote numbers of animals in each group. e p <.1. 'p <.2. R P <.5. P values were determined between groups C and and groups M and. anol-fed rats, but there were significant increases in the weights of both kidney and intestinal mucosa (Table 1). Furthermore, the total protein contents of liver and skeletal muscle remained similar to those of ethanol-fed rats, but there were significant increases in the protein contents of kidney and intestinal mucosa (Table 1). thanol was not detected in plasma of control rats (n = 6), and there was no significant difference between ethanol concentrations (grams/l ml plasma) in rats fed either ethanol (.11 ±.2, n = 7) or ethanol together with metabolites and riboflavin (.8 ±.2, n = 6). ffects on Plasma and Tissue Amino Acid Concentrations Among the 15 amino acids studied (aspartic acid, threonine, serine, asparagine + glutamine, glutamic acid, glycine, alanine, a-amino-n-butyric acid, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine), chronic ethanol ingestion had the most consistent and significant effect on plasma and tissue concentrations of three amino acids (leucine, alanine, and a-amino-n-butyric acid). Furthermore, as in previous studies' 2 with dietary alterations, changes in plasma amino acid concentrations more closely reflected changes in amino acid concentrations of liver and muscle than those of renal cortex and intestinal mucosa. thanol feeding significantly increased plasma, muscle, and liver concentrations of leucine (Figure la). There were similar trends in plasma and tissue concentrations of the other branched-chain amino acids (isoleucine and valine), but the increases reached statistical significance only in the muscle for valine and in plasma for isoleucine (Table 2). In contrast to leucine, plasma and liver concentrations of alanine were markedly decreased while the muscle concentration remained unaffected in the ethanol-fed rats (Figure lb). thanol ingestion significantly increased the concentrations of a-amino-n-butyric acid in both plasma and muscle without affecting the concentration of this amino acid in the liver (Figure lc). In addition to changes described above, chronic ethanol ingestion caused significant increases in concentrations of glycine and phenylalanine in the muscle and significant increases in concentrations of glutamic acid, glutamine + asparagine, and phenylalanine in the liver (Figure ld and Table 2). With the addition of pyruvate, dihydroxyacetone, and riboflavin to the diet of ethanol-fed rats, the ethanol-induced changes in concentrations of leucine, alanine, and a-amino-n-butyric acids were either totally or partially prevented (Figure la-c). Moreover, there were significant decreases in the plasma, muscle, and liver concentrations of glutamic acid (Figure ld) and significant increases in the plasma, muscle, and liver concentrations of serine in these rats (Figure l). thanol caused complex changes in amino acid concentrations in the renal cortex (Table 3): Some were increased (aspartate, threonine, asparagine +

4 January 1979 PRVNTION OF FFCTS OF THANOL ON AMINO ACID CONCNTRATIONS 135 LUCIN (A) ALANIN (B) 2B LIvR PLASMA MUSCL LIVR PLASMA MUSCL o IB o IB 4 o I Lb.; 14 rn I : 18 :: 56 " " " 22 " o o 1 o II W I!? OOll. n o 11. n OOII. n I ool. n 2F. C M C M C M C M C M C M p<o 5 p<o 5 p<o 5 p<o 1 P<OOI P 'NS p< 1 P<O 1 P<O 1 p'ns p'ns p'ns a-amino-n -BUTYRAT IC) GLUTAMAT () LIVR PLASMA MUSCL o I I LIVR PLASMA MUSCL I 16 "- <?.;.3.; 12 :: 6 :: 7 : " " " W I 1 1. n 41. n T W dj OII. D C M C M C M C M C M C M P -NS pons P<O 1 P<O 1 P<O 1 P<O 1 p<5 p < OOI p'ns P < 1. p 'NS p < OOI SRIN I) LIVR PLASMA MUSCL C> : I "- :: 55 " II " iw 1. n OOI. n OOI. n C M C M C M p'ns p<o 1 p ans P<.5 pons P<.5 Figure. 1. Concentrations (mean ± SM) of leucine (A), alanine (B), a-amino-n-butyrate (C), glutamate (D), and serine () in liver, muscle, and plasma. Bar graphs represent groups fed control diet (solid bar, C), ethanol diet (hatched bar, ), and metabolite diet (shaded bar, M). P values were determined between groups C and and groups and M. The number of rats used in each group is as in Table 2. glutamine, a-amino-n-butyrate); some were decreased (serine, alanine, methionine, and phenylalanine); and some remained unchanged (glutamate, glycine, valine, isoleucine, leucine, and tyrosine). Among these changes, only the changes in alanine and methionine were prevented by the addition of pyruvate, dihydroxyacetone, and riboflavin to the ethanol diet (Table 3). thanol significantly increased concentrations of most amino acids in the intestinal mucosa (Table 3). There was a general diminution of this effect by the addition of metabolites and riboflavin to the ethanol diet (Table 3). ffect on AIL Ratio Chronic ethanol ingestion significantly increased the AIL ratio (mean ± SM) in plasma (.178 ±.22 in seven rats vs..247 ±.21 in 17 rats, P <.5). Addition of pyruvate, dihydroxyacetone, and riboflavin to the diet prevented this increase (.179 ±.22 in 13 rats, P = NS as compared with control value). Discussion The results of the present studies show that chronic ethanol ingestion in rats has a varied effect

5 Table 2. Amino Acid Concentrations (Mean ± SM) in Liver, Muscle, and Plasma of Rats Fed Specified Diets Liver Plasma Muscle C M C M C M (n = 8) (n = 17) (n = 6) (n = 7) (n = 17) (n = 13) (n = 6) (n = 16) (n = 6) JLmol/g JLmol/ml JLmol/g Aspartate 1.38 ± ± ±.1.3 ±.4.3 ±.OO2.3 ±.2.56 ± ±.5.68 ±.6 Threonine.5 ±.7.42 ±.4.49 ±.8.36 ±.3.32 ±.2.4 ±.2b.85 ±,8.94 ± ±.6 Asparagine + glutamine 6.97 ± ±.39 b 9.51 ± ±.4.9 ±.5.87 ± ± ± ±.14 Glycine 1.8 ± ± ± ±.2.26 ±.1.28 ± ± ± ±.3 Valine.22 ±.2.27 ±.2.22 ±.2.22 ±.1.21 ±.1.19 ±.1.2 ±.2.28 ±O.Ol b.24 ±.2 Methionine.6 ±.OO5.6 ±.1.4 ±.OO5.6 ±.OO3.5 ±.OO3.6 ±.OO3.7 ±.4.8 ±.4.7 ±.1 Isoleucine.13 ±.1.16 ±.1.13 ±.2.9 ±.OO3.1 ±.OO4.8 ±.OO3 b.11 ±.1.13 ±.OO5.11 ±.1 Tyrosine.8 ±.1.1 ±.1.8 ±.1.9 ±.1.8 ±.OO3.8 ±.OO5.13 ±.1.13 ±.OO5.14 ±.1 Phenylalanine.8 ±.4.1 ±.4.9 ±.5.7 ±.2.6 ±.3.6 ±.3.6 :7.2.9 ±.OO4b.8 ±.1 P <.5. b P<.1. C.. and M as in Table 1.

6 January 1979 PRVNTION OF FFCTS OF THANOL ON AMINO ACID CONCNTRATIONS 137 Table 3. Amino Acid Concentrations (Mean ± SM) in Kidney Cortex and Intestinal Mucosa of Rats Fed Specified Diets Aspartate Threonine Serine Asparagine + glutamine Glutamate Glycine Alanine a-amino-n-butyrate Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine C.. and M as in Table 1. o p <.1. b p<.2. c P <.5. Kidney cortex C (n = 5) (n = 7).52 ±.3.89 ±.4.52 ±.4.57 ±.4 c.88 ±.4.59 ±.2" 1.52 ± ±.18 c 7.77 ± ± ± ± ±.9.85 ±.5.11 ±.1.19 ±.O2.25 ±.2.24 ±.2.19 ±.1.12 ±.1.11 ±.5.11 ±.4.19 ±.1.19 ±.1.15 ±.9.13 ±O.Ol.18 ±.1.12 ±.1 Intestinal mucosa M C M (n= 7) (n=5) (n = 8) (n = 5) I'mol/g 1. ± ±.8.78 ±.5 b 1.23 ± ±.5.54 ±.2.95 ±.5.92 ±.1.57 ±.5.91 ±.5 'l.15 ±.8 c.82 ± O.l1C 2.31 ± ± ± ± ± ± ± ±.37 b 4.5 ± ± ± ± ± ± ± ± ±.4.2 ±.5.3 ±.5.5 ±O.Ol b.24 ±.2.58 ±.2.77 ±.4.54 ±.5.19 ±.2.31 ±.1.3 ±.3.2 ±.2 b.13 ±.2.41 ±.2.57 ±.3.38 ±.4.22 ±.1.59 ±.4.78 ±.5.54 ±.7.14 ±.1.42 ±.2.45 ±.4.37 ±.4.1 ±.1.32 ±.3.34 ±.3.25 ±.O4 on protein metabolism in tissues. Although tissue growth, as assessed by total protein content, is not affected in the liver and kidney, it is reduced in the muscle and intestinal mucosa (Table 1). In view of the fact that muscle mass accounts for 45% of body weight,9 the impairment in muscle growth appears to be a significant component of the smaller body weight gain by ethanol-fed rats as compared with control rats (Table 1). This impression receives further support by the observation that the failure to restore the muscle growth by the addition of pyruvate, dihydroxyacetone, and riboflavin to the ethanol diet was also accompanied by a similar smaller gain in body weight (Table 1). In addition to reduced growth, intestinal mucosa of ethanol-fed rats displayed a decrease in its protein concentration (Table 1) and general increases in its amino acid concentrations (Table 3), results indicating a greater ethanol effect on protein metabolism in this tissue. This is in keeping with a known biologic characteristic of the intestinal mucosal protein, its greater sensitivity to change because of its rapid turnover.lo The results of the present studies also show that chronic ethanol ingestion increases the concentrations of several amino acids in plasma, liver, and skeletal muscle. Among these increases, those of branched-chain amino acids are most prominent. Although the mechanism of these alterations is not yet apparent, the increases in plasma and tissue concentrations of branched-chain amino acids in starved 1 and diabetic" rats has been attributed to increased muscle protein breakdown!2 Plasma and liver concentrations of alanine were uniquely decreased by chronic ethanol ingestion (Figure 1 B). A fall in plasma and liver concentrations of alanine such as that demonstrated in starvation and diabetes is usually indicative of enhanced gluconeogenesis. However, implication of enhanced gluconeogenesis as a mechanism would be at variance with the conclusion derived from previous studies that have shown ethanol to inhibit gluconeogenesis from alanine.13 Therefore, other explanations must be considered. Pyruvate, the transamination product of alanine, acts both as a substrate for gluconeogenesis and as a reducing agent by conversion to lactate. In the face of increasing conversion of NAD to NADH by ethanol,14 the reduction in alanine concentration, as revealed in the results of the present experiments, may represent increased conversion of alanine to lactate. Indeed, previous studies in man have shown that ethanol stimulates this conversion!5 The conversion of alanine to pyruvate is usually coupled with transamination of a-ketoglutarate to glutamate. This may have accounted for the increase in the concentration of glutamate in the liver of ethanol-fed rats (Figure 1D). The authors' studies with ethanol-fed rats when pyruvate, dihydroxyacetone, and riboflavin were added to the diet demonstrate that such dietary intervention cannot prevent the inhibitory effect of ethanol on protein metabolism in the skeletal muscle, but it can blunt this effect in intestinal mucosa (Table 1). Furthermore, alteration in plasma and tissue amino acid profile was prevented totally

7 138 STANKO T AL. GASTRONTROLOGY Vol. 76, No. 1 as far as branched-chain amino acids and a-aminobutyrate were concerned, and partially as far as alanine was concerned (Figure 1). These observations establish that prevention of ethanol-induced hepatic steatosis by pyruvate, dihydroxyacetone, and riboflavin is accompanied by normalization of growth in some tissues (intestine) and of concentrations of some amino acids (leucine and a-aminobutyrate); furthermore, these differences are not due to differences between plasma ethanol levels. The increases in alanine and serine concentrations in plasma and tissues of rats fed ethanol together with metabolites and riboflavin were probably the result of addition of pyruvate and dihydroxyacetone to the diet, because alanine and serine can be readily synthesized from these substrates. Glutamate appeared as the donor of the amino group for these transamination reactions, because the concentration of glutamate in plasma, liver, and skeletal muscle was sharply reduced when the metabolites were added to the diet of ethanol-fed rats. Finally, chronic ethanol ingestion in rats, like that in baboons and human subjects,s resulted in an elevation of plasma AIL ratio. The increase of this ratio was due to a relatively greater increase of a-aminobutyrate than of leucine (Figure 1). Muscle appeared to be the tissue largely responsible for the elevation of a-amino butyrate. The liver concentration of this amino acid was not altered by chronic ethanol ingestion, although there was a 18% increase in the concentration of a-aminobutyrate in the muscle (Figure 1e). In a recently published abstract, Shaw and Lieber'6 reported that chronic ethanol feeding increased the concentration of a-amino butyrate in rat liver. Because the details of this study have not yet been published, the authors are uncertain of an explanation for the varied results. Nonetheless, an interesting observation that emerged from the studies of AIL ratio in the present experiments was the finding that ethanol-induced alterations in this ratio were entirely prevented by the addition of pyruvate, dihydroxyacetone, and riboflavin to the diet of ethanol-fed rats. References 1. Adibi SA: Interrelationships between level of amino acids in plasma and tissues during starvation. Am J Physiol 221: Adibi SA. Modesto T A. Morse L. et al: Amino acid levels in plasma. liver. and skeletal muscle during protein deprivation. Am J Physiol 225: Siegel FL. Roach MK, Pomeroy LR: Plasma amino acid patterns in alcoholism: the effects of ethanol loading. Proc Nat! Acad Sci USA 51: Stanko RT. Mendelow H. Shinozuka H. et al.: Prevention of alcohol-induced fatty liver by natural metabolites and riboflavin. J Lab Clin Med 91: Shaw S. Stimmel B, Lieber CS: Plasma alpha amino-n-butyric acid to leucine ratio: an empirical biochemical marker of alcoholism. Science 194: Morgan MY. Milsom JP. Sherlock S: Ratio of plasma alpha amino-n-butyric acid to leucine as an empirical marker of alcoholism: diagnostic value. Science 197: Lowry OH. Rosebrough NJ. Farr AL. et al.: Protein measurement with the folin phenol reagent. J Bioi Chem 193: Dixon WJ. Massey. FJ. Jr. : Introduction to Statistical Analysis. Third edition. McGraw-Hill. Inc. New York Munro HN: volution of protein metabolism in mammals. In: Mammalian Protein Metabolism. Vol. III. dited by HN Munro. New York. Academic Press. Inc p Neuberger A. Richards FF: Protein biosynthesis in mammalian tissues. II. Studies on turnover in the whole animal. In: Mammalian Protein Metabolism. Vol. I. dited by HN Munro and JB Allison. New York. Academic Press. Inc p Blackshear PJ. Alberti KGMM: Sequential amino acid measurements during experimental diabetic ketoacidosis. Am J Physiol 228: Adibi SA: Metabolism of branched-chain amino acids in altered nutrition. Metabolism 25: Freinkel N. Arky RA. Singer DL. et al.: Alcohol hypoglycemia. IV. Current concepts of its pathogenesis. Diabetes 14: Forsander O. Riiihii N. Suomalainen H: Alkoholoxydation und Bildung von Acetoacetat in normaler und glykogenarmer intakter Rattenleber. Hoppe-Seyler's Z Physiol Chem 312: Kreisberg RA. Siegal AM. Owen WC: Alanine and gluconeogenesis in man: effect of ethanol. J Clin ndocrinol Metab 34: Shaw S. Lieber CS: Increased hepatic production of a-aminon-butyric acid after chronic alcohol consumption (abstr). Gastroenterology 73: