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Aminoacid imbalance and malnutrition in liver cirrhosis
CLINICAL
Aminoacid Cirrhosis
NUTRITION
Imbalance and Malnutrition
(1985) 4: :’ t!I “i’i
in Liver
M. Merli*, 0. Riggio *, S. Iapichinot, P. Miazzoj-, and L. Capocacciat
*Institute of Medical Sciences, University of L’Aquila, Italy, and t2nd Gastroenterological Unit, 3rd Department of
Internal Medicine, University of Rome ‘La Sapienza’, Italy (Reprint requests to L.C.)
The reason branched chain aminoacids are decreased and aromatic aminoacids increased in chronic liver failure is unclear. Branched chain aminoacids are mainly catabolised in muscles, and it is known that protein energy malnutrition may decrease the concentration of these aminoacids in plasma. In this study we have evaluated the nutritional status of a group of cirrhotics and compared it with their plasma aminoacid imbalance. Fourteen patients were considered as well-nourished and nine as malnourished. Plasma levels of branched chain aminoacids were significantly decreased and the phenylalanine increased in the malnourished group. Arm muscle circumference was significantly correlated with branched chain aminoacids. In conclusion our data suggest that malnutrition may contribute to the low levels of these aminoacids in patients with liver cirrhosis.
ABSTRACT
INTRODUCTION
PATIENTS
AND METHODS
Plasma amino acid concentrations are known to be altered in different pathological situations [l-4] and in altered nutritional status [5-71. Several reports have shown that chronic liver failure is associated with typical changes in plasma amino acid pattern [8111: branched chain amino acids (BCAA) are reduced, while aromatic amino acids @AA) and methionine are increased. However, the pathogenesis of this abnormal amino acid profile has not been clarified. Aromatic amino acids and methionine are catabolised mainly in the liver [12, 131; therefore it has been suggested that their elevation might depend on the progressive liver dysfunction and portalsystemic shunting. On the other hand, branched chain amino acids escape hepatic metabolism and are muscle [13-l 51; catabolised mainly in skeletal therefore the reason for their decreased levels is still unclear. Interestingly, protein-energy malnutrition produces a similar decrease in branched chain amino acids [l&18]. Clinical signs of malnutrition are frequent in patients with chronic liver disease [19, 301, and the nutritional status could therefore contribute to the plasma amino acid imbalance in these patients. In order to evaluate the possible influence of the nutritional status on the plasma amino acid profile in patients with liver cirrhosis, we have studied different nutritional parameters, liver function tests, and plasma amino acid concentrations in well-nourished and malnourished cirrhotic patients.
Twenty-three patients with clinical and histological diagnosis of liver cirrhosis were investigated. They were 12 males and 11 females ranging in age from 45 to 74 years (mean =59+ 10 years). A history of alcohol abuse was present in six patients, while seven had post-necrotic and ten cryptogenic cirrhosis. Different liver function tests (bilirubin, GPT, prothrombine time, ammonia) and the galactose elimination capacity were performed in all subjects. Plasma proteins such as albumin, prealbumin and transferrin were determined as indices of protein synthesis. These proteins were not included in the nutritional assessment, as we have previously demonstrated that they are mainly influenced by the degree of liver damage. Oesophageal varices were present in 15 patients, and in three cases previous hematemesis had occurred. Mild ascites was present in eight subjects at the time of the study, as revealed by ultrasonography. Hepatic encephalopathy grade I according to the classification of Parson-Smith, et al. 1211 was present in four cases. None of the patients had overt diabetes or renal insufficiency as determined by serum creatinine and serum urea nitrogen. All subjects were in hospital at the time of the study and consumed a controlled diet provided 30 kcal/kg and 1 g/kg of proteins for 3 days prior to the study. The patients showing clinical signs of hepatic encephalopathy grade I were submitted to moderate protein restriction, 0.8 g/kg protein, and lactulose therapy. Patients were categorised as malnourished or well-nourished, according to their
“50
AMINOACID IMBALANCE AND MALNU’I‘RII‘ION IN LIVER CIRRHOSIS
nutritional status. For this purpose weight, height, weight loss, arm muscle circumference and triceps skinfold were regularly recorded. Malnourished patients were identified as those showing two or more anthropometric parameters below the standard values according to Jeliffe [22]. Patients showing no, or no more than one, altered nutritional parameter were considered well nourished. The mean values obtained for the nutritional parameters in the well-nourished (14 patients) and malnourished (9 patients) group are shown in Table 1. Serum bilirubin, glutamic pyruvic transaminase, prothrombine activity and albumin were determined by standard laboratory techniques. Plasma ammonia was determined enzymatically. The galactose elimination capacity (GEC) was assessed according to Tygstrup [23] after intravenous galactose injection. The results were expressed in milligrams per kilogram per min (N.V. 2 7). Prealbumin and transferrin were determined by radial immunodiffusion techniques using commercial kits (Behering, Scoppito, L’Aquila, Italy). Anthropometric measurements were performed as previously reported [24]. Arm muscle circumference (cm) was measured with a steel tape on the left relaxed arm at the mid point between the tip of the acromion and oleocranon process. Triceps skinfold (mm) was measured to the nearest mm with a Harpenden Skinfold Caliper (British Indicators, Ltd, St Albans, Herts, England) at the same site. The percentage weight loss was calculated from the observed weight subtracted from the usual weight as given by the patient. Blood samples for plasma amino acid determination were always taken in the morning in the post-
Table 1
absorbtive state. Plasma amino acids were measured by means of a Carlo Erba 3A29 Automatic Amino Analyzer using lithium citrate buffers. 1 ml of heparinized plasma was deproteinized by the addition of 0.5 ml of sulfosalicilic acid 5”,, (w/v) containing norleucine as internal standard. Analyses were performed on 0.2ml samples. Control values for plasma amino acids were assessed in 10 subjects similar in age (62k 14 years), with normal liver function, who were hospitalised mild for cardiovascular problems.
Statistical
analysis
Student’s t test for unpaired data was used for the comparison between malnourished and wellnourished patients. Correlation coefficients were determined by standard procedures. All data are presented as the mean k SEM.
RESULTS The well-nourished and the malnourished patients constitute two groups comparable in age (585 11 years vs. 605 10 years respectively). The ratio males vs. females was 6:8 in the well-nourished and 4:5 in malnourished group. The anthropometric the measurements for the two groups are shown in Table 1. Clinical signs, such as the presence of ascites or oesophageal varices or hepatic encephalopathy, were equally distributed in the two groups. Liver function tests in the two groups of patients are reported in Table 2. Malnourished patients tended to show a more severe liver insufficiency as documented by
Nutritional parameters in well-nourished and malnourished cirrhotic patients AMC (cm)
TSF (mm)
2.OkO.7
25.6iO.7
18.2f2.3
10.4L2.2”
21.5k0.5”
9.2+ 1.01”
IBW (%) Well-nourished patients (14)
WL (SC)
110.9k5.7 84.7k2.2”
Malnourished patients (9) “p < 0.05.
Table 2
Liver function tests in well-nourished and malnourished cirrhotic patients Bilirubin mg/dl
Well-nourished
patients (14)
Malnourished patients (9) “p < 0.05 at least.
GPT (mu/ml)
Prothrombin Activity (%)
Ammonia (fimolil)
GEC (mg/k/min)
1.11*0.1
42.9 k 10.4
71.8k3.5
78.6k7.4
4.41*
2.25 kO.6”
36.2*
62.9k4.6a
82.7k5.2
3.77 * 0.3
8.9
1.1
CLINICAL
serum bilirubin and prothrombin activity. However, the galactose elimination capacity taken as a global index of liver cell mass did not show a statistically significant difference between the two groups. All the plasma proteins tended to be more reduced in the malnourished vs. the well-nourished patients, ie., 2.9f0.2 vs. 3.8kO.l g/d1 @<0.05), albumin prealbumin 9.87 + 1.6 vs. 12.lf1.5mg/dl and transferrin 214 + 19 vs. 266+ 17 mg/dl respectively. Plasma amino acid concentrations in well-nourished and malnourished cirrhotic patients and in control subjects are reported in Table 3. Methionine, phenylalanine, tyrosine and serine were significantly increased in the cirrhotic patients when compared with controls, while the BCAA were significantly decreased only in the malnourished group. Plasma levels of valine, leucine and isoleucine were significantly reduced in malnourished patients when well-nourished patients. compared with Phenylalanine was significantly increased in the malnourished group, while tyrosine and methionine differences. did not show significant The BCAAIAAA ratio (which was significantly lower in both groups vs. controls) was consequently much more reduced in the malnourished group. muscle Arm circumference was the only anthropometric parameter which correlated
Table 3 Plasma amino acids (mmol/ml) cirrhotic patients and in controls
Taurine Aspartate Threonine Serine Glutamate Glutamine Proline Glycine Alanine Citrulline y-amino-butyrate Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine ZBCAAjZAAA
NUl‘RIl‘ION
251
significantly with leucine (p < O.Ol), valine (p < 0.05) and the sum of BCAA (p < 0.05). No correlation was found between AMC and tyrosine or phenylalanine plasma levels. The sum of AAA (tyrosine and phenylalanine) demonstrated a weak correlation with the galactose elimination capacity (t. = 0.42) (0.1 >p > 0.05), while a between the significant correlation was present galactose elimination capacity and the sum of BCAA (p <0.05).
DISCUSSION A number of studies have reported a reduction in BCAA plasma levels in cirrhotic patients [8-111. Different mechanisms have been claimed to be involved in the pathogenesis of this alteration in plasma amino acids. Portal-systemic shunting reduces BCAA both in experimental animals [25-271 and in humans [28], though the pathogenetic mechanism remains unclear. BCAA can also be consumed to participate in the ammonia detossification by muscle tissues via glutamine synthesis [29-311. A reciprocal relationship has been demonstrated between BCAA plasma levels
in well-nourished
and malnourished
Well-nourished patients (14)
Malnourished patients (9)
Controls
48.1 _t 3.0 2.9* 0.7 1z3.9+ 9.3 121.6, 7.gb 46.1 k 6.04 627.3 + 33.9 248.8k 18.1 202.7* 10.7 352.5 k 27. I 35.9+ 2.7 21.4+ 2.4 221.7* 15.8 107.7+ 7.15 34.5+ 2.6b 69.8* 5.9 126.3* 8.9 115.1+ 10.3s 67.3* 4.7” 96.2f 7.8h 189.3$- 12.2 66.6k 3.3 87.4+ 4.4 2.4& 0.2”
59.3+ 8.4 4.2+ 0.7b 132.9i 10.8 115.5+ 5.6h 52.1* 9.5 614.Ok45.6 210.9* 18.7 210.9k 12.8 342.8 + 36.4 40.8k 2.1 17.3+ 2.2 179.6* 6.gdb 122.8k22.8 38.3* 4.0b 49.6i 4Zb 95.81 6.5”b 120.5; 6.5” 88.8k 7.5”s 86.8+ 6.5 180.8k 9.5 67.7* 3.3 83.1+ 5.5 1.59+_ 0.1”s
57.11 li: 4.35 1.93* 0.2 113.4 i 8.2 88.8 + 6.0 41.9 f 6.9 629.7 f 33.6 294.8 k43.4 190.5 f 9.5 367.2 + 22.2 42.6 + 5.6 17.8 i 1.6 222.1 k 10.1 92.6 & 12.3 24.04* 2.1 59.7 + 3.4 121.5 i 6.3 66.2 & 4.9 52.0 + 2.5 71.2 i 6.7 183.7 + 11.8 71.3 + 5.3 83.2 + 4.9 3.46& 0.22
“Well-nourished vs malnourished (significance %ignificant OS controls (at least * < 0.05).
at least p <0.05).
(10)
2.5”
AMINOACID
IMBALANCE
AND
MALNLJTRII‘ION
IN LIVER
and insulin in physiological and pathological conditions [32], and it has been hypothesised that also in cirrhotic patients the high insulin levels might be the cause of the decrease of BCAA [33-351. In this study we have focused our attention on the possible contribution of the nutritional status in the plasma amino acid abnormalities in cirrhotic patients. From our data it would appear that the cirrhotic patients showing greater weight loss, as well as reduced values for the percent ideal body weight, muscle mass (as evaluated by AMC) and fat stores (as evaluated by TSF), ie., the malnourished patients, demonstrated lower levels of branched chain amino acids when compared with the group of patients who were better nourished. Plasma phenylalanine was slightly but significantly increased in the malnourished group, while the ratio BCAA/AAA was reduced. The BCAA valine, leucine, and isoleucine are essential amino acids and their circulating levels have been shown to depend on the dietary protein intake [16-181. At the same time the BCAA plasma concentration is known to be influenced by the muscle amino acid release derived from protein catabolism and by the simultaneous insulin mediated muscle amino acid uptake [36]. In muscle the BCAA are utilised for protein synthesis as well as oxidized as metabolic fuels [14, 151. In malnourished cirrhotic patients all these factors could play a role in determining the low BCAA levels. Chronic protein energy malnutrition has been shown to decrease the plasma levels of most essential amino acids and a chronic low protein intake could have easily occurred in the group of malnourished cirrhotic patients. It should be noted however that the decrease in BCAA was rather selective, other not being essential amino acid concentrations different from that observed in the well-nourished group. As previously stated, BCAA can be specifically used in muscle (and adipose tissue) as energy sources, and their oxidation has been shown to be increased in conditions in which the utilisation of other substrates is impaired [15, 37-391. A more severe energy deficiency in the malnourished cirrhotic patients REFERENCES
[II Felig I’, Marliss E, Ohman J, et al 1970 Plasma amino
acid levels in diabetic keto-acidosis. Diabetes 19: 727729 121 Fdrst I’, Bergstrom J, Chao L, et al 1979 Influence of amino acid metabolism in severe trauma. Acta Chirurgica Scandinavica Supplement 494: 136-141 [31 Askanazi J, Carpentier Y A, Michelson C B, et al 1980 Muscle and plasma amino acids following injury. Influence of inter-current infection. Annals of Surgery 192: 78-85
CIRRHOSIS
could therefore explain a higher BCAA consumption in the peripheral tissues. On the other hand, the progressive reduction in muscle mass, as stated by the lower AMC values in the malnourished group, may directly influence the BCAA levels by decreasing their release from muscle [40]. The significant correlation between plasma BCAA and AMC may indeed support the relationship between the decrease in BCAA and the progressive muscle wastage. A similar correlation has also been observed by Young, et al., in surgical patients [41]. did not insulin Although we measure concentration, it seems unlikely that higher insulin concentrations could have mediated the decrease of BCAA we observed in malnourished cirrhotic patients. Moreover, ammonia plasma concentrations were not different between the two groups nor were the clinical signs of portal-systemic shunting. Clinical signs of malnutrition are frequently associated with chronic liver disease f19] and seem to show a higher prevalence as liver insufficiency progresses [20]. For this reason it can be difficult to separate the effect of malnutrition from that of liver disease. It could be inferred that the differences we observed in the two groups of patients were mainly a reflection of different degrees of hepatocellular damage. The galactose elimination capacity was not significantly different in the two groups of patients we examined; however, we cannot exclude that the degree of liver insufficiency was somewhat more severe in the malnourished group. If the difference in the higher insufficiency could explain liver observed in concentrations phenylalanine malnourished patients, the lower levels of BCAA observed should not be attributed to a more severe hepatic insufficiency. In fact, while AAA are mainly catabolised in the liver [12], the net splanchnic exchange of BCAA has been constantly found close to zero in normal individuals [13] and in cirrhotic patients [42] in the basal state. This makes it difficult to give a real value to the correlation empirically found between GEC and BCAA values. In conclusion, these data suggest malnutrition may contribute to the low BCAA plasma levels present in cirrhotic patients. [4] Kopple J D, Fhigel R, Jones M R 1981 Branched chain amino acids in chronic renal failure. In: Walser M, Williamson J R (eds) Metabolism and clinical implications of branched chain amino and keto-acids. Elsevier North-Holland, New York, pp 555-567 [5] Holt E, Snyderman S, Norton I’, et al 1963 The plasma aminogram in Kwashiorkor. Lancet II: 13431348 [6] Felig I’, Owen D E, Wahren J, et al 1969 Amino acid metabolism during prolonged starvation. Journal of Clinical Investigation 48: 584-594
(:I.INICAL. Adibi S A 1976 Metabolism of branched chain amino acids in altered nutrition. Metabolism 25: 1287-1302 Iber F L, Rosen H, Levenson S M, et al 1957 The plasma amino acids in patients with liver failure. Journal of Laboratory Clinical Medicine 50: 417-425 Iob V, Coon W W, Sloan M 1967 Free amino acids in liver, plasma, and muscle of patients with cirrhosis of the liver. Journal of Surgical Research 7: 41-43 [l(‘l Rosen H, Yoshimura N, Hodgman J, et al 1977 Plasma amino acid patterns in hepatic encephalopathy in differing aetiology. Gastroenterology 72: 48%487 [III Cascino A, Cangiano C, Calcaterra V, et al 1978 Plasma amino acids imbalance in patients with liver disease. American Journal of Diagnostic Diseases 23: 591-598 [W Miller L L 1962 The role of the liver and the nonhepatic tissues in the regulation of free amino acid levels in the blood. In: Holden J T (ed) Amino acid pools. Elsevier, Amsterdam, pp 708-721 [1 31 Felig P 1975 Amino acid metabolism in man. Annual Review of Biochemistry 44: 933-955 A L 1972 Oxidation of leucine Ll.41 Odessey R, Goldberg by non-skeletal muscle. American Journal of Physiology 223: 137G-1383 Ll51 Goldberg A L, Odessey R 1972 Oxidation of amino acids by diaphragms from fed and fasted rats. American Journal of Physiology 223: 1384-1391 [lhl Grimble R F, Whitehead R G 1970 Fasting serum amino acid pattern in Kwashiorkor and after administration of different levels of protein. Lancet I: 918-921 and plasma I171 Harper A E 1974 Amino acid requirements amino acids. In: Brown H (ed) Protein nutrition. Charles C Thomas, Springfield IL, pp 130-179 General. [I81 Harper A E 1977 Amino acid requirements: In: Green H L, Hollyday M A, Munro H N (eds) Clinical nutrition update: amino acids. American Medical Association, Chicago, pp 58-65 [I91 Morgan A G, Kelleher J, Walker B E, et al 1976 Nutrition in cryptogenic cirrhosis and chronic aggressive hepatitis. Gut 17: 113-l 18 C L, Anderson S, Wessner R E, et al 1984 [201 Mendenhall Protein calorie malnutrition associated with alcoholic hepatitis. American Journal of Medicine 76: 211-222 B G, Summerskill W M J, Dawson A Pll Parson-Smith M, et al 1957 The electroencephalograph in liver disease. Lancet 2: 867-871 [=I Jeliffe D B 1966 The assessment of the nutritional status of the community with special reference to field surveys in developing regions of the world. WHO Monograph 53, Geneva, World Health Organization of the hepatic [231 Tygstrup N 1966 Determination elimination capacity (Lm) of galactose by single injection. Scandinavian Journal of Clinical Laboratory Investigations 18: Suppl. 32: 118-125 [241 Riggio 0, Merli M, Cantafora A, et al 1984 Total and individual free fatty acid concentrations in liver cirrhosis. Metabolism 33: 646651 P51 Soeters P B, Weir G, Eberol A M, et al. 1977 Insulin, glucagon, portal systemic shunting and hepatic failure in the dog. Journal of Surgical Research 23: 183-188 [261 Funovics J M, Cummings M G, Shuman L, et al 1975 An improved non-suture method for portacaval anastomosis in the rat. Surgery 77: 661-664 v71 James J H, Hodgman J M, Funovics J M, et al 1976 Brain tryptophan, plasma free tryptophan and Submitted
1985.
.fbr
pubiicatiox:
9 Aug.
1985.
Accepred:30 Aug.
NU’I‘RlTION
:! I)3
distribution of plasma neutral amino acids. Metabolism 25: 471476 [281 lwasoky Y, Soto H, Ohkubo A, et al 1980 Effect of spontaneous portal-systemic shunting on plasma insulin and amino acid concentrations. Gastroenterology 78: 677 u91 Hayashi M, Ohnishi H, Kawada Y, et al 1981 Augmented utilization of branched-chain amino acids by skeletal muscle in decompensated liver cirrhosis in special relation to ammonia detoxication. Gastroenterologica Japonica 16: 64-70 [301 Riggio 0, James J H, Peters J C, et al 1984 Influence of ammonia on leucine utilization in muscle and adipose tissue in vitro. In: Kleinberger G, Ferenci P (eds) Advances in hepatic encephalopathy and urea cycle diseases. Karger, Basel, pp 519-526 [311 Leweling H, Holm E, Staedt U, et al 1984 Intra- and extracellular amino acid concentrations in ammoniuminfused rats. Evidence that hyperammoniemia reduces BCAA levels. In: Kleinberger G, Ferenci P (eds ) Advances in hepatic encephalopathy and urea cycle diseases. Karger, Basel, pp 552-554 M, Berchtold P, [321 Berger M, Zimmermann-Telschow et al (1978 Blood amino acid levels in patients with insulin excess (functioning insulinoma) and insulin deficiency (diabetic ketosis). Metabolism 27: 793-806 [331 Limberg B, Kommerell B 1981 Why decreased serum branched-chain amino acids in cirrhosis (letter). Gastroenterology 80: 21 l-212 [341 Soeters P B, Fisher J E 1976 Insulin, glucagon, amino acid imbalance and hepatic encephalopathv. The L.ancet II: 880-882 [351 Munro H N, Fenstrom J D, Wurtman R .I 1975 Insulin plasma amino acid imbalance and hepatic coma. Lancet I: 722-724 [361 Pozefsky T, Felig P, Tobin J B, et al 1969 Amino acid balance across tissues of the prearm in fast absorptive man. Effects of insulin at two dose levels. Journal of Clinical Investigation 48: 22752282 1371 Buse MG, Herlong H F, Weigand D A 1976 The effect of diabetes, insulin and the redox potential on lcucine metabolism by isolated rat hemidiaphragm. Endocrinology 98: 1166-l 175 [381 Adibi S A, Stank0 R T, Morse E L 1982 Modulation of leucine oxidation and turnover by graded amounts of carbohydrate intake in obese subjects. Metabolism 31: 578-588 I391 Hogg S A, Morse E L, Adibi S A 1982 Effect of exercise on rates of oxidation, turnover and plasma clearance of leucine in human subjects. American Journal of Physiology 242 (Endocrinol. Metab. 5): E407-E410 [401 Zoli M, Bianchi G P, Marzocchi A, et al L984 Splanchnic, peripheral and renal exchange of amino acids in cirrhotic patients with portal hypertension. In: Kleimberger G, Ferenci P, Riederer P, Thaler H, Vienna (eds) Advances in Hepatic Encephalopathy and Urea Cycle Diseases, Karger, Basel, pp 538-544 1411 Young G A, Graham L H 1981 Evaluation of protein energy malnutrition in surgical patients from plasma valine and other amino acids, proteins, and anthropometric measurements. The American Journal of Clinical Nutrition 34: 166-172 L, Wahren J 1982 ~421 Eriksson L S, Hagenfeldt Intravenous infusion of z-0x0-isocaproate: influence on amino acid and nitrogen metabolism in patients with liver cirrhosis. Clinical Science 62: 285-292
Aminoacid Cirrhosis
NUTRITION
Imbalance and Malnutrition
(1985) 4: :’ t!I “i’i
in Liver
M. Merli*, 0. Riggio *, S. Iapichinot, P. Miazzoj-, and L. Capocacciat
*Institute of Medical Sciences, University of L’Aquila, Italy, and t2nd Gastroenterological Unit, 3rd Department of
Internal Medicine, University of Rome ‘La Sapienza’, Italy (Reprint requests to L.C.)
The reason branched chain aminoacids are decreased and aromatic aminoacids increased in chronic liver failure is unclear. Branched chain aminoacids are mainly catabolised in muscles, and it is known that protein energy malnutrition may decrease the concentration of these aminoacids in plasma. In this study we have evaluated the nutritional status of a group of cirrhotics and compared it with their plasma aminoacid imbalance. Fourteen patients were considered as well-nourished and nine as malnourished. Plasma levels of branched chain aminoacids were significantly decreased and the phenylalanine increased in the malnourished group. Arm muscle circumference was significantly correlated with branched chain aminoacids. In conclusion our data suggest that malnutrition may contribute to the low levels of these aminoacids in patients with liver cirrhosis.
ABSTRACT
INTRODUCTION
PATIENTS
AND METHODS
Plasma amino acid concentrations are known to be altered in different pathological situations [l-4] and in altered nutritional status [5-71. Several reports have shown that chronic liver failure is associated with typical changes in plasma amino acid pattern [8111: branched chain amino acids (BCAA) are reduced, while aromatic amino acids @AA) and methionine are increased. However, the pathogenesis of this abnormal amino acid profile has not been clarified. Aromatic amino acids and methionine are catabolised mainly in the liver [12, 131; therefore it has been suggested that their elevation might depend on the progressive liver dysfunction and portalsystemic shunting. On the other hand, branched chain amino acids escape hepatic metabolism and are muscle [13-l 51; catabolised mainly in skeletal therefore the reason for their decreased levels is still unclear. Interestingly, protein-energy malnutrition produces a similar decrease in branched chain amino acids [l&18]. Clinical signs of malnutrition are frequent in patients with chronic liver disease [19, 301, and the nutritional status could therefore contribute to the plasma amino acid imbalance in these patients. In order to evaluate the possible influence of the nutritional status on the plasma amino acid profile in patients with liver cirrhosis, we have studied different nutritional parameters, liver function tests, and plasma amino acid concentrations in well-nourished and malnourished cirrhotic patients.
Twenty-three patients with clinical and histological diagnosis of liver cirrhosis were investigated. They were 12 males and 11 females ranging in age from 45 to 74 years (mean =59+ 10 years). A history of alcohol abuse was present in six patients, while seven had post-necrotic and ten cryptogenic cirrhosis. Different liver function tests (bilirubin, GPT, prothrombine time, ammonia) and the galactose elimination capacity were performed in all subjects. Plasma proteins such as albumin, prealbumin and transferrin were determined as indices of protein synthesis. These proteins were not included in the nutritional assessment, as we have previously demonstrated that they are mainly influenced by the degree of liver damage. Oesophageal varices were present in 15 patients, and in three cases previous hematemesis had occurred. Mild ascites was present in eight subjects at the time of the study, as revealed by ultrasonography. Hepatic encephalopathy grade I according to the classification of Parson-Smith, et al. 1211 was present in four cases. None of the patients had overt diabetes or renal insufficiency as determined by serum creatinine and serum urea nitrogen. All subjects were in hospital at the time of the study and consumed a controlled diet provided 30 kcal/kg and 1 g/kg of proteins for 3 days prior to the study. The patients showing clinical signs of hepatic encephalopathy grade I were submitted to moderate protein restriction, 0.8 g/kg protein, and lactulose therapy. Patients were categorised as malnourished or well-nourished, according to their
“50
AMINOACID IMBALANCE AND MALNU’I‘RII‘ION IN LIVER CIRRHOSIS
nutritional status. For this purpose weight, height, weight loss, arm muscle circumference and triceps skinfold were regularly recorded. Malnourished patients were identified as those showing two or more anthropometric parameters below the standard values according to Jeliffe [22]. Patients showing no, or no more than one, altered nutritional parameter were considered well nourished. The mean values obtained for the nutritional parameters in the well-nourished (14 patients) and malnourished (9 patients) group are shown in Table 1. Serum bilirubin, glutamic pyruvic transaminase, prothrombine activity and albumin were determined by standard laboratory techniques. Plasma ammonia was determined enzymatically. The galactose elimination capacity (GEC) was assessed according to Tygstrup [23] after intravenous galactose injection. The results were expressed in milligrams per kilogram per min (N.V. 2 7). Prealbumin and transferrin were determined by radial immunodiffusion techniques using commercial kits (Behering, Scoppito, L’Aquila, Italy). Anthropometric measurements were performed as previously reported [24]. Arm muscle circumference (cm) was measured with a steel tape on the left relaxed arm at the mid point between the tip of the acromion and oleocranon process. Triceps skinfold (mm) was measured to the nearest mm with a Harpenden Skinfold Caliper (British Indicators, Ltd, St Albans, Herts, England) at the same site. The percentage weight loss was calculated from the observed weight subtracted from the usual weight as given by the patient. Blood samples for plasma amino acid determination were always taken in the morning in the post-
Table 1
absorbtive state. Plasma amino acids were measured by means of a Carlo Erba 3A29 Automatic Amino Analyzer using lithium citrate buffers. 1 ml of heparinized plasma was deproteinized by the addition of 0.5 ml of sulfosalicilic acid 5”,, (w/v) containing norleucine as internal standard. Analyses were performed on 0.2ml samples. Control values for plasma amino acids were assessed in 10 subjects similar in age (62k 14 years), with normal liver function, who were hospitalised mild for cardiovascular problems.
Statistical
analysis
Student’s t test for unpaired data was used for the comparison between malnourished and wellnourished patients. Correlation coefficients were determined by standard procedures. All data are presented as the mean k SEM.
RESULTS The well-nourished and the malnourished patients constitute two groups comparable in age (585 11 years vs. 605 10 years respectively). The ratio males vs. females was 6:8 in the well-nourished and 4:5 in malnourished group. The anthropometric the measurements for the two groups are shown in Table 1. Clinical signs, such as the presence of ascites or oesophageal varices or hepatic encephalopathy, were equally distributed in the two groups. Liver function tests in the two groups of patients are reported in Table 2. Malnourished patients tended to show a more severe liver insufficiency as documented by
Nutritional parameters in well-nourished and malnourished cirrhotic patients AMC (cm)
TSF (mm)
2.OkO.7
25.6iO.7
18.2f2.3
10.4L2.2”
21.5k0.5”
9.2+ 1.01”
IBW (%) Well-nourished patients (14)
WL (SC)
110.9k5.7 84.7k2.2”
Malnourished patients (9) “p < 0.05.
Table 2
Liver function tests in well-nourished and malnourished cirrhotic patients Bilirubin mg/dl
Well-nourished
patients (14)
Malnourished patients (9) “p < 0.05 at least.
GPT (mu/ml)
Prothrombin Activity (%)
Ammonia (fimolil)
GEC (mg/k/min)
1.11*0.1
42.9 k 10.4
71.8k3.5
78.6k7.4
4.41*
2.25 kO.6”
36.2*
62.9k4.6a
82.7k5.2
3.77 * 0.3
8.9
1.1
CLINICAL
serum bilirubin and prothrombin activity. However, the galactose elimination capacity taken as a global index of liver cell mass did not show a statistically significant difference between the two groups. All the plasma proteins tended to be more reduced in the malnourished vs. the well-nourished patients, ie., 2.9f0.2 vs. 3.8kO.l g/d1 @<0.05), albumin prealbumin 9.87 + 1.6 vs. 12.lf1.5mg/dl and transferrin 214 + 19 vs. 266+ 17 mg/dl respectively. Plasma amino acid concentrations in well-nourished and malnourished cirrhotic patients and in control subjects are reported in Table 3. Methionine, phenylalanine, tyrosine and serine were significantly increased in the cirrhotic patients when compared with controls, while the BCAA were significantly decreased only in the malnourished group. Plasma levels of valine, leucine and isoleucine were significantly reduced in malnourished patients when well-nourished patients. compared with Phenylalanine was significantly increased in the malnourished group, while tyrosine and methionine differences. did not show significant The BCAAIAAA ratio (which was significantly lower in both groups vs. controls) was consequently much more reduced in the malnourished group. muscle Arm circumference was the only anthropometric parameter which correlated
Table 3 Plasma amino acids (mmol/ml) cirrhotic patients and in controls
Taurine Aspartate Threonine Serine Glutamate Glutamine Proline Glycine Alanine Citrulline y-amino-butyrate Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine ZBCAAjZAAA
NUl‘RIl‘ION
251
significantly with leucine (p < O.Ol), valine (p < 0.05) and the sum of BCAA (p < 0.05). No correlation was found between AMC and tyrosine or phenylalanine plasma levels. The sum of AAA (tyrosine and phenylalanine) demonstrated a weak correlation with the galactose elimination capacity (t. = 0.42) (0.1 >p > 0.05), while a between the significant correlation was present galactose elimination capacity and the sum of BCAA (p <0.05).
DISCUSSION A number of studies have reported a reduction in BCAA plasma levels in cirrhotic patients [8-111. Different mechanisms have been claimed to be involved in the pathogenesis of this alteration in plasma amino acids. Portal-systemic shunting reduces BCAA both in experimental animals [25-271 and in humans [28], though the pathogenetic mechanism remains unclear. BCAA can also be consumed to participate in the ammonia detossification by muscle tissues via glutamine synthesis [29-311. A reciprocal relationship has been demonstrated between BCAA plasma levels
in well-nourished
and malnourished
Well-nourished patients (14)
Malnourished patients (9)
Controls
48.1 _t 3.0 2.9* 0.7 1z3.9+ 9.3 121.6, 7.gb 46.1 k 6.04 627.3 + 33.9 248.8k 18.1 202.7* 10.7 352.5 k 27. I 35.9+ 2.7 21.4+ 2.4 221.7* 15.8 107.7+ 7.15 34.5+ 2.6b 69.8* 5.9 126.3* 8.9 115.1+ 10.3s 67.3* 4.7” 96.2f 7.8h 189.3$- 12.2 66.6k 3.3 87.4+ 4.4 2.4& 0.2”
59.3+ 8.4 4.2+ 0.7b 132.9i 10.8 115.5+ 5.6h 52.1* 9.5 614.Ok45.6 210.9* 18.7 210.9k 12.8 342.8 + 36.4 40.8k 2.1 17.3+ 2.2 179.6* 6.gdb 122.8k22.8 38.3* 4.0b 49.6i 4Zb 95.81 6.5”b 120.5; 6.5” 88.8k 7.5”s 86.8+ 6.5 180.8k 9.5 67.7* 3.3 83.1+ 5.5 1.59+_ 0.1”s
57.11 li: 4.35 1.93* 0.2 113.4 i 8.2 88.8 + 6.0 41.9 f 6.9 629.7 f 33.6 294.8 k43.4 190.5 f 9.5 367.2 + 22.2 42.6 + 5.6 17.8 i 1.6 222.1 k 10.1 92.6 & 12.3 24.04* 2.1 59.7 + 3.4 121.5 i 6.3 66.2 & 4.9 52.0 + 2.5 71.2 i 6.7 183.7 + 11.8 71.3 + 5.3 83.2 + 4.9 3.46& 0.22
“Well-nourished vs malnourished (significance %ignificant OS controls (at least * < 0.05).
at least p <0.05).
(10)
2.5”
AMINOACID
IMBALANCE
AND
MALNLJTRII‘ION
IN LIVER
and insulin in physiological and pathological conditions [32], and it has been hypothesised that also in cirrhotic patients the high insulin levels might be the cause of the decrease of BCAA [33-351. In this study we have focused our attention on the possible contribution of the nutritional status in the plasma amino acid abnormalities in cirrhotic patients. From our data it would appear that the cirrhotic patients showing greater weight loss, as well as reduced values for the percent ideal body weight, muscle mass (as evaluated by AMC) and fat stores (as evaluated by TSF), ie., the malnourished patients, demonstrated lower levels of branched chain amino acids when compared with the group of patients who were better nourished. Plasma phenylalanine was slightly but significantly increased in the malnourished group, while the ratio BCAA/AAA was reduced. The BCAA valine, leucine, and isoleucine are essential amino acids and their circulating levels have been shown to depend on the dietary protein intake [16-181. At the same time the BCAA plasma concentration is known to be influenced by the muscle amino acid release derived from protein catabolism and by the simultaneous insulin mediated muscle amino acid uptake [36]. In muscle the BCAA are utilised for protein synthesis as well as oxidized as metabolic fuels [14, 151. In malnourished cirrhotic patients all these factors could play a role in determining the low BCAA levels. Chronic protein energy malnutrition has been shown to decrease the plasma levels of most essential amino acids and a chronic low protein intake could have easily occurred in the group of malnourished cirrhotic patients. It should be noted however that the decrease in BCAA was rather selective, other not being essential amino acid concentrations different from that observed in the well-nourished group. As previously stated, BCAA can be specifically used in muscle (and adipose tissue) as energy sources, and their oxidation has been shown to be increased in conditions in which the utilisation of other substrates is impaired [15, 37-391. A more severe energy deficiency in the malnourished cirrhotic patients REFERENCES
[II Felig I’, Marliss E, Ohman J, et al 1970 Plasma amino
acid levels in diabetic keto-acidosis. Diabetes 19: 727729 121 Fdrst I’, Bergstrom J, Chao L, et al 1979 Influence of amino acid metabolism in severe trauma. Acta Chirurgica Scandinavica Supplement 494: 136-141 [31 Askanazi J, Carpentier Y A, Michelson C B, et al 1980 Muscle and plasma amino acids following injury. Influence of inter-current infection. Annals of Surgery 192: 78-85
CIRRHOSIS
could therefore explain a higher BCAA consumption in the peripheral tissues. On the other hand, the progressive reduction in muscle mass, as stated by the lower AMC values in the malnourished group, may directly influence the BCAA levels by decreasing their release from muscle [40]. The significant correlation between plasma BCAA and AMC may indeed support the relationship between the decrease in BCAA and the progressive muscle wastage. A similar correlation has also been observed by Young, et al., in surgical patients [41]. did not insulin Although we measure concentration, it seems unlikely that higher insulin concentrations could have mediated the decrease of BCAA we observed in malnourished cirrhotic patients. Moreover, ammonia plasma concentrations were not different between the two groups nor were the clinical signs of portal-systemic shunting. Clinical signs of malnutrition are frequently associated with chronic liver disease f19] and seem to show a higher prevalence as liver insufficiency progresses [20]. For this reason it can be difficult to separate the effect of malnutrition from that of liver disease. It could be inferred that the differences we observed in the two groups of patients were mainly a reflection of different degrees of hepatocellular damage. The galactose elimination capacity was not significantly different in the two groups of patients we examined; however, we cannot exclude that the degree of liver insufficiency was somewhat more severe in the malnourished group. If the difference in the higher insufficiency could explain liver observed in concentrations phenylalanine malnourished patients, the lower levels of BCAA observed should not be attributed to a more severe hepatic insufficiency. In fact, while AAA are mainly catabolised in the liver [12], the net splanchnic exchange of BCAA has been constantly found close to zero in normal individuals [13] and in cirrhotic patients [42] in the basal state. This makes it difficult to give a real value to the correlation empirically found between GEC and BCAA values. In conclusion, these data suggest malnutrition may contribute to the low BCAA plasma levels present in cirrhotic patients. [4] Kopple J D, Fhigel R, Jones M R 1981 Branched chain amino acids in chronic renal failure. In: Walser M, Williamson J R (eds) Metabolism and clinical implications of branched chain amino and keto-acids. Elsevier North-Holland, New York, pp 555-567 [5] Holt E, Snyderman S, Norton I’, et al 1963 The plasma aminogram in Kwashiorkor. Lancet II: 13431348 [6] Felig I’, Owen D E, Wahren J, et al 1969 Amino acid metabolism during prolonged starvation. Journal of Clinical Investigation 48: 584-594
(:I.INICAL. Adibi S A 1976 Metabolism of branched chain amino acids in altered nutrition. Metabolism 25: 1287-1302 Iber F L, Rosen H, Levenson S M, et al 1957 The plasma amino acids in patients with liver failure. Journal of Laboratory Clinical Medicine 50: 417-425 Iob V, Coon W W, Sloan M 1967 Free amino acids in liver, plasma, and muscle of patients with cirrhosis of the liver. Journal of Surgical Research 7: 41-43 [l(‘l Rosen H, Yoshimura N, Hodgman J, et al 1977 Plasma amino acid patterns in hepatic encephalopathy in differing aetiology. Gastroenterology 72: 48%487 [III Cascino A, Cangiano C, Calcaterra V, et al 1978 Plasma amino acids imbalance in patients with liver disease. American Journal of Diagnostic Diseases 23: 591-598 [W Miller L L 1962 The role of the liver and the nonhepatic tissues in the regulation of free amino acid levels in the blood. In: Holden J T (ed) Amino acid pools. Elsevier, Amsterdam, pp 708-721 [1 31 Felig P 1975 Amino acid metabolism in man. Annual Review of Biochemistry 44: 933-955 A L 1972 Oxidation of leucine Ll.41 Odessey R, Goldberg by non-skeletal muscle. American Journal of Physiology 223: 137G-1383 Ll51 Goldberg A L, Odessey R 1972 Oxidation of amino acids by diaphragms from fed and fasted rats. American Journal of Physiology 223: 1384-1391 [lhl Grimble R F, Whitehead R G 1970 Fasting serum amino acid pattern in Kwashiorkor and after administration of different levels of protein. Lancet I: 918-921 and plasma I171 Harper A E 1974 Amino acid requirements amino acids. In: Brown H (ed) Protein nutrition. Charles C Thomas, Springfield IL, pp 130-179 General. [I81 Harper A E 1977 Amino acid requirements: In: Green H L, Hollyday M A, Munro H N (eds) Clinical nutrition update: amino acids. American Medical Association, Chicago, pp 58-65 [I91 Morgan A G, Kelleher J, Walker B E, et al 1976 Nutrition in cryptogenic cirrhosis and chronic aggressive hepatitis. Gut 17: 113-l 18 C L, Anderson S, Wessner R E, et al 1984 [201 Mendenhall Protein calorie malnutrition associated with alcoholic hepatitis. American Journal of Medicine 76: 211-222 B G, Summerskill W M J, Dawson A Pll Parson-Smith M, et al 1957 The electroencephalograph in liver disease. Lancet 2: 867-871 [=I Jeliffe D B 1966 The assessment of the nutritional status of the community with special reference to field surveys in developing regions of the world. WHO Monograph 53, Geneva, World Health Organization of the hepatic [231 Tygstrup N 1966 Determination elimination capacity (Lm) of galactose by single injection. Scandinavian Journal of Clinical Laboratory Investigations 18: Suppl. 32: 118-125 [241 Riggio 0, Merli M, Cantafora A, et al 1984 Total and individual free fatty acid concentrations in liver cirrhosis. Metabolism 33: 646651 P51 Soeters P B, Weir G, Eberol A M, et al. 1977 Insulin, glucagon, portal systemic shunting and hepatic failure in the dog. Journal of Surgical Research 23: 183-188 [261 Funovics J M, Cummings M G, Shuman L, et al 1975 An improved non-suture method for portacaval anastomosis in the rat. Surgery 77: 661-664 v71 James J H, Hodgman J M, Funovics J M, et al 1976 Brain tryptophan, plasma free tryptophan and Submitted
1985.
.fbr
pubiicatiox:
9 Aug.
1985.
Accepred:30 Aug.
NU’I‘RlTION
:! I)3
distribution of plasma neutral amino acids. Metabolism 25: 471476 [281 lwasoky Y, Soto H, Ohkubo A, et al 1980 Effect of spontaneous portal-systemic shunting on plasma insulin and amino acid concentrations. Gastroenterology 78: 677 u91 Hayashi M, Ohnishi H, Kawada Y, et al 1981 Augmented utilization of branched-chain amino acids by skeletal muscle in decompensated liver cirrhosis in special relation to ammonia detoxication. Gastroenterologica Japonica 16: 64-70 [301 Riggio 0, James J H, Peters J C, et al 1984 Influence of ammonia on leucine utilization in muscle and adipose tissue in vitro. In: Kleinberger G, Ferenci P (eds) Advances in hepatic encephalopathy and urea cycle diseases. Karger, Basel, pp 519-526 [311 Leweling H, Holm E, Staedt U, et al 1984 Intra- and extracellular amino acid concentrations in ammoniuminfused rats. Evidence that hyperammoniemia reduces BCAA levels. In: Kleinberger G, Ferenci P (eds ) Advances in hepatic encephalopathy and urea cycle diseases. Karger, Basel, pp 552-554 M, Berchtold P, [321 Berger M, Zimmermann-Telschow et al (1978 Blood amino acid levels in patients with insulin excess (functioning insulinoma) and insulin deficiency (diabetic ketosis). Metabolism 27: 793-806 [331 Limberg B, Kommerell B 1981 Why decreased serum branched-chain amino acids in cirrhosis (letter). Gastroenterology 80: 21 l-212 [341 Soeters P B, Fisher J E 1976 Insulin, glucagon, amino acid imbalance and hepatic encephalopathv. The L.ancet II: 880-882 [351 Munro H N, Fenstrom J D, Wurtman R .I 1975 Insulin plasma amino acid imbalance and hepatic coma. Lancet I: 722-724 [361 Pozefsky T, Felig P, Tobin J B, et al 1969 Amino acid balance across tissues of the prearm in fast absorptive man. Effects of insulin at two dose levels. Journal of Clinical Investigation 48: 22752282 1371 Buse MG, Herlong H F, Weigand D A 1976 The effect of diabetes, insulin and the redox potential on lcucine metabolism by isolated rat hemidiaphragm. Endocrinology 98: 1166-l 175 [381 Adibi S A, Stank0 R T, Morse E L 1982 Modulation of leucine oxidation and turnover by graded amounts of carbohydrate intake in obese subjects. Metabolism 31: 578-588 I391 Hogg S A, Morse E L, Adibi S A 1982 Effect of exercise on rates of oxidation, turnover and plasma clearance of leucine in human subjects. American Journal of Physiology 242 (Endocrinol. Metab. 5): E407-E410 [401 Zoli M, Bianchi G P, Marzocchi A, et al L984 Splanchnic, peripheral and renal exchange of amino acids in cirrhotic patients with portal hypertension. In: Kleimberger G, Ferenci P, Riederer P, Thaler H, Vienna (eds) Advances in Hepatic Encephalopathy and Urea Cycle Diseases, Karger, Basel, pp 538-544 1411 Young G A, Graham L H 1981 Evaluation of protein energy malnutrition in surgical patients from plasma valine and other amino acids, proteins, and anthropometric measurements. The American Journal of Clinical Nutrition 34: 166-172 L, Wahren J 1982 ~421 Eriksson L S, Hagenfeldt Intravenous infusion of z-0x0-isocaproate: influence on amino acid and nitrogen metabolism in patients with liver cirrhosis. Clinical Science 62: 285-292