Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man

1. Using the forearm balance method, together with systemic infusions of L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine, we examined the effects of infused branched-chain amino acids on whole-body and skeletal muscle amino acid kinetics in 10 postabsorptive normal subjects; 10 control subjects received only saline. 2. Infusion of branched-chain amino acids caused a four-fold rise in arterial branched-chain amino acid levels and a two-fold rise in branched-chain keto acids; significant declines were observed in circulating levels of most other amino acids, including phenylalanine, which fell by 34%. Plasma insulin levels were unchanged from basal levels (8 +/- 1 mu-units/ml). 3. Whole-body phenylalanine flux, an index of proteolysis, was significantly suppressed by branched-chain amino acid infusion (P less than 0.002), and forearm phenylalanine production was also inhibited (P less than 0.03). With branched-chain amino acid infusion total leucine flux rose, with marked increments in both oxidative and non-oxidative leucine disposal (P less than 0.001). Proteolysis, as measured by endogenous leucine production, showed a modest 12% decrease, although this was not significant when compared with saline controls. The net forearm balance of leucine and other branched-chain amino acids changed from a basal net output to a marked net uptake (P less than 0.001) during branched-chain amino acid infusion, with significant stimulation of local leucine disposal. Despite the rise in whole-body non-oxidative leucine disposal, and in forearm leucine uptake and disposal, forearm phenylalanine disposal, an index of muscle protein synthesis, was not stimulated by infusion of branched-chain amino acids. 4. The results suggest that in normal man branched-chain amino acid infusion suppresses skeletal muscle proteolysis independently of any rise of plasma insulin. Muscle branched-chain amino acid uptake rose dramatically in the absence of any apparent increase in muscle protein synthesis, as measured by phenylalanine disposal, or in branched-chain keto acid release. Thus, an increase in muscle branched-chain amino acid concentrations and/or local branched-chain amino acid oxidation must account for the increased disposal of branched-chain amino acids.     

Muscle ammonia and amino acid metabolism during dynamic exercise in man

Summary. The effect of dynamic exercise on muscle and blood ammonia (NH₃) and amino acid contents has been investigated. Eight healthy men cycled at 50% and 97% of maximal oxygen uptake for 10 min and 5·2 min (to fatigue), respectively. Biopsies (quadriceps femoris muscle), arterial and femoral venous blood samples were obtained at rest and during exercise. Muscle NH₃ at rest and after submaximal exercise was (x?±SE) 0·5±0·1 mmol/kg dry muscle (d.m.) and increased to 4·1 ±0·5 mmol/kg d.m. at fatigue (P<0·001). The total adenine nucleotide (TAN) pool (TAN=ATP+ADP+AMP) did not change after submaximal exercise but decreased significantly at fatigue (P<0·01). The decrease in TAN was similar to the increase in NH₃. Muscle lactate was 3±1 mmol/kg d.m. at rest and increased to 104±5 mmol/kg d.m. at fatigue. Whole blood and plasma NH₃ did not change significantly during submaximal but both increased significantly during maximal exercise (P<0·001). During maximal exercise the leg released 7,120 μmol/min of lactate, whereas only 89 μmol/min of NH₃ were released. NH₃ accumulation in muscle could buffer only 3% of the hydrogen ions released from lactate, and NH₃ release could account for only 1% of the net hydrogen ion transport out of the cell. Muscle glutamine was constant throughout the study, whereas glutamate decreased and alanine increased during exercise (P<0·001). No significant changes in either arterial whole blood glutamine or glutamate were observed. Arterial plasma glutamine and glutamate concentrations, however, increased and decreased (P<0·001), respectively, during exercise. It is concluded that (1) muscle and blood NH₃ levels increase only during strenuous exercise and (2) NH₃ accumulation is of minor importance for regulating acid-base balance in body fluids during exercise.     

Splanchnic and peripheral glucose and amino acid metabolism in diabetes mellitus

Splanchnic and leg exchange of glucose, lactate, pyruvate, and individual plasma amino acids was studied in diabetics 24 hr after withdrawal of insulin and in healthy controls. Measurements were made in the basal postabsorptive state and during the administration of glucose at a rate of 2 mg/kg per min for 45 min.In the basal state, net splanchnic glucose production did not differ significantly between diabetics and controls. However, splanchnic uptake of alanine and other glycogenic amino acids was 1(1/2)-2 times greater in the diabetics, while lactate and pyruvate uptake was increased by 65-115%. Splanchnic uptake of these glucose precursors could account for 32% of hepatic glucose output in the diabetics, as compared to 20% in the controls. This increase in precursor uptake was a consequence of a two- to threefold increment in fractional extraction of these substrates inasmuch as arterial levels of alanine, glycine, and threonine were reduced in the diabetics, while the levels of the remaining substrates were similar in the two groups. Peripheral output of alanine and other glycogenic amino acids as reflected in arterio-femoral venous differences was similar in both groups. An elevation in arterial valine, leucine, and isoleucine was observed in the diabetics, but could not be accounted for on the basis of alterations in splanchnic or peripheral exchange of these amino acids.Administration of glucose (2 mg/kg per min) for 45 min resulted in an 80% reduction in splanchnic glucose output in controls, but failed to inhibit hepatic glucose release in the diabetics despite a twofold greater increment in arterial glucose levels. In both groups no consistent changes in arterial glucagon were observed during the infusion.It is concluded that in nonketotic diabetics (a) total splanchnic output of glucose is comparable to controls, but the relative contribution of gluconeogenesis may be increased by more than 50%; (b) accelerated splanchnic uptake of glucose precursors is a consequence of increased hepatic extraction of available substrates rather than a result of augmented substrate supply; and (c) the failure of glucose infusion to inhibit hepatic glucose output suggests that the exquisite sensitivity of the liver to the infusion of glucose in normal man is a consequence of glucose-induced insulin secretion.     

Effect of cholestyramine on bile acid metabolism in normal man

The effect of cholestyramine administration on the enterohepatic circulation of bile acids was studied in eight normal volunteers. In six subjects the metabolism of sodium taurocholate-(14)C was determined after its intravenous injection before and during the 6th wk of cholestyramine administration, 16 g/day. In two subjects, the metabolism of cholic acid-(14)C was observed before and during the 2nd wk of cholestyramine, 16 g/day. Bile acid sequestration resulted in a more rapid disappearance of the injected primary bile acid and its metabolic products. The composition of fasting bile acids was promptly altered by cholestyramine to predominantly glycine-conjugated trihydroxy bile acid. In four subjects, unconjugated bile acid-(14)C was administered during cholestyramine administration; the relative proportion of glycine-conjugated bile acid-(14)C before enterohepatic circulation was similar to the relative proportion of unlabeled glycine-conjugated bile acid present in duodenal contents after an overnight fast, indicating that a hepatic mechanism was responsible for the elevated ratios of glycine- to taurine-conjugated bile acid (G: T ratios) observed. The relative proportions of both dihydroxy bile acids, chenodeoxycholic and deoxycholic, were significantly reduced. Steatorrhea did not occur, and the total bile acid pool size determined after an overnight fast was unaltered by cholestyramine. These findings suggest that in normal man bile acid sequestered from the enterohepatic circulation by cholestyramine is replaced by an increase in hepatic synthesis primarily via the pathway leading to production of glycocholic acid.     

Studies of amino acid and protein metabolism in normal man with L-[U-14C]tyrosine.

1. Six men were infused intravenously for 10 h with a tracer amount of L-[U-14C]tyrosine while on a standardized food intake. 2. Measurements of plasma L-[14C]-tyrosine specific radioactivity and the excretion rate of 14CO2 were made at frequent intervals and showed plateau labelling of plasma and expired carbon dioxide within 6-8 h. The tyrosine flux was calculated from the specific radioactivity in plasma at plateau value. 3. The excretion rate of 14CO2, corrected for retention of label within the bicarbonate pool, showed that oxidation accounted for 20% of the tyrosine flux. Urinary excretion of label was negligible. 4. Rates of protein synthesis were calculated from the flux of tyrosine after subtracting the proportion oxidized. Although the mean rate of synthesis was consistent with other measurements of protein turnover, the individual values ranged from 284 to 387 g/day. The variation was not reduced by relating turnover to body weight, lean body mass or energy expenditure. 5. Estimating the rates of protein breakdown from the tyrosine flux involved some assumptions about pathways of phenylalanine metabolism. The use of a labelled essential amino acid would therefore give more accurate values for short-term measurements of whole body protein turnover.     

Free amino acids in muscle tissue and plasma during exercise in man

Summary. Blood and muscle samples (percutaneous biopsy of m. quadriceps femoris) were taken before exercise and after 10 and 20 min of exercise in four healthy subjects working on a bicycle ergometer with a load of 70% of VO₂ max. Free amino acids were determined in plasma and muscle and the intracellular concentration of each amino acid was calculated by the chloride method. The plasma concentration of alanine, arginine and glutamine increased during exercise. In muscle there was an increase in glutamine and alanine concentration and a decrease in glutamate concentration at 10 min of exercise; after 20 min of exercise the increase in alanine and glutamine concentrations was less marked, but the decrease in glutamate concentration was of a similar magnitude. The results demonstrate that glutamate is a quantatively important ammonia acceptor during heavy exercise.     

Effect of ketone infusions on amino acid and nitrogen metabolism in man.

To evaluate the role of hyperketonemia in the hypoalaninemia and decreased protein catabolism of prolonged starvation, Na dl-beta-hydroxybutyrate was administered as a primed continuous 3-6-h infusion in nonobese subjects and in obese subjects in the postabsorptive state and after 3 days and 3-5 1/2 wk of starvation. An additional obese group received 12-h ketone infusions on 2 consecutive days after 5-10 wk of fasting. The ketone infusion in nonobese and obese subjects studied in the postabsorptive state resulted in total blood ketone acid levels of 1.1-1.2 mM, a 5-15 mg/100 ml decrease in plasma glucose, and unchanged levels of insulin, glucagon, lactate, and pyruvate. Plasma alanine fell by 21% (P smaller than 0.001) in 3 h. In contrast, other amino acids were stable or varied by less than 10%. Infusions lasting 6 h reduced plasma alanine by 37%, reaching levels comparable to those observed in prolonged starvation. Equimolar infusions of NaC1 and/or administration of NaHCO3 failed to alter plasma alanine levels. During prolonged fasting, plasma alanine, which had fallen by 40% below prefast levels, fell an additional 30% in response to the ketone infusion. In association with repeated prolonged (12 h) infusions in subjects fasted 5-10 wk, urinary nitrogen excretion fell by 30%, returning to base line after cessation of theinfusions and paralleling the changes in plasma alanine. Ketone infusins resulted in two- to fourfold greater increments in blood ketone acids in fasted as compared to postabsorptive subjects. It is concluded that increased blood ketone acid levels induced by infusions of Na DL-beta-hydroxybutyrate result in hypoalaninemia and in nitrogen conservation in starvation. These data suggest that hyperketonemia may be a contributory factor in the decreased availability or circulating alanine and reduction in protein catabolism characteristic of prolonged fastings9     

Overall renal and tubular function during infusion of amino acids in normal man

1. Amino acids have been used to test renal reserve filtration capacity. Previous studies suggest that amino acids increase glomerular filtration rate (GFR) by reducing distal tubular flow and tubuloglomerular feedback activity. 2. Glomerular function and the renal tubular handling of sodium during infusion of amino acids was studied in 12 normal volunteers. 3. Clearance of sodium (CNa) was unchanged. Effective renal plasma flow increased slightly, but significantly, by 9% (P less than 0.05). GFR was increased by 13% (P less than 0.001). Clearance of lithium (CLi) (used as an index of proximal tubular outflow) increased by 38% (P less than 0.001). Calculated absolute proximal reabsorption (GFR-CLi) remained unchanged. Fractional proximal reabsorption [1-(CLi/GFR)] was decreased by 10% (P less than 0.001). Calculated absolute distal sodium reabsorption [(CLi-CNa) x PNa, where PNa is plasma sodium concentration] increased by 40% (P less than 0.001). Plasma renin concentration did not change significantly. 4. The results suggest that amino acids increase GFR by a primary effect on renal haemodynamics or, less likely, by reducing the signal to the tubuloglomerular feedback mechanism. The increase in proximal tubular outflow was compensated for in the distal tubules, so that the sodium excretion rate remained unchanged.     

Nitrogen metabolism and renal amino acid excretion during total parenteral feeding of hypermetabolic patients with various carbohydrate regimes

During an investigative study about the utilization of parenterally administered amino-acids (AA) in severe catabolic states nitrogen balances, urea production rates and catabolic nitrogen as well as urinary losses of amino-acids were determined in six adult polytraumatized patients treated in an intensive care unit. In addition to 19 g nitrogen (0.3 g N/kg/day) 3,000 kcal (12.5 MJ) were given simultaneously as carbohydrates. Either a mixture of laevulose 20%, glucose 10%, xylitol 10% (LGX) or glucose 50% were infused alternatively for four days in a randomized cross-over scheme. Another group of three patients received the LGX-regime alone for six days altogether. Higher urinary N-losses, increased urea production rates corresponding to a rise in the s.c. catabolic nitrogen were obtained on days where the LGX-mixture was infused; there was also an increased excretion of alpha-amino-N due to a distinct aminoaciduria. Not all amino-acids did react in the same manner. When comparing excretion to supply higher losses (greater than 20%) were observed for: THR greater than TRY greater than VAL greater than ILE greater than PHE. Methionine and alanine were also excreted in higher amounts, whereas under glucose an elevated excretion was especially noted with glutamine and alpha-aminobutyric acid. The increased urea production rate associated with a reduced incorporation of amino-acids as well as the accentuated renal AA-losses under LGX may be ascribed to a slight increase in protein catabolism connected with some impairment of AA-utilization due to a temporary metabolic hepatic 'block'. Under glucose an intensified endogenous mobilization as well as the higher exogenous supply of insulin might be responsible for a somewhat better AA-utilization. Amino-acid clearances under the carbohydrate mixture in the second group were - with the exception of lysine and leucine - raised distinctly similar to values seen in aminoaciduria of other origin. This leads to the assumption that the AA-overflow might result from a partial insufficiency or overload of renal transport ('carrier') systems being responsible for amino-acid reabsorption. The question of an adequate supply - concerning essential amino acids in particular - taking into consideration the relatively high losses under one of the nutritional regimes investigated is shortly touched upon. Finally the possibility of adapting AA-mixtures to the respective carbohydrate solutions in order to improve nitrogen balances and utilization is discussed.     

Influence of amino acid administration on whole-body leucine kinetics and resting metabolic rate in postabsorptive normal subjects

1. We have investigated the effect of an amino acid mixture (Vamin 14; 57.4 +/- 10.2 mumol h-1 kg-1) on whole-body leucine kinetics, calculated by a steady-state reciprocal pool model, and resting metabolic rate in eight postabsorptive normal subjects. 2. Vamin 14 infusion increased whole-body leucine flux (P less than 0.001), leucine employed for protein synthesis (P less than 0.001), leucine oxidation (P less than 0.001), metabolic clearance rate of alpha-ketoisocaproic acid (P less than 0.05) and levels of all three branched-chain amino acids (P less than 0.001) compared with the basal situation. In contrast, whole-body proteolysis was reduced (P less than 0.05). 3. Resting metabolic rate was increased during Vamin 14 infusion (P less than 0.05) and was positively correlated with whole-body protein synthesis (n = 16, r = 0.6342, P less than 0.01; y = 0.605x + 173.7), as was the change in metabolic rate with the change in protein synthesis (n = 8, r = 0.772, P less than 0.05; y = 0.493x - 10.85). 4. Overall, Vamin 14 infusion was associated with increased blood glucose (P less than 0.001), although the observed increase in plasma glucagon (t = 2.012) and plasma insulin (t = 1.683) failed to reach statistical significance. 5. These data lend a measure of support to the hypothesis that the apparent increase in whole-body protein synthesis in insulin-dependent diabetic (type I) subjects during insulin withdrawal may be substrate related.     

Effects of brief starvation on muscle amino acid metabolism in nonobese man.

A reduction in the release of substrate amino acids from skeletal muscle largely explains the decrease in gluconeogenesis characterizing prolonged starvation. Brief starvation is associated with an increase in gluconeogenesis, suggesting increased release of amino acids from muscle. In the present studies, accelerated amino acid release from skeletal muscle induced by brief starvation was sought to account for the accompanying augmentation of gluconeogenesis. To do this amino acid balance across forearm muscles was quantified in 15 postabsorptive (overnight fasted) subjects and in 7 subjects fasted for 60 h. Fasting significantly reduced basal insulin (11.3-7.5 muU/ml) and increased glucagon (116-134 pg/ml). Muscle release of the principal glycogenic amino acids increased. Alanine release increased 59.4%. The increase in release for all amino acids averaged 69.4% and was statistically significant for threonine, serine, glycine, alanine, alpha-aminobutyrate, methionine, tyrosine, and lysine. Thus, with brief starvation, muscle release of glycogenic amino acids increases strikingly. This contrasts with the reduction of amino acid release characterizing prolonged starvation. The adaptation of peripheral tissue metabolism to brief starvation is best explained by the decrease in insulin.     

The effects of nutrition and trauma on whole-body protein metabolism in man

Whole-body protein metabolism was determined by a primed constant-rate infusion of L-[ 1-14C ]leucine in patients before and after elective surgery, the nutritional intake being carefully controlled and the surgical stress in individuals being assessed. Pre-operatively, whole-body protein flux (P less than 0.05) and synthesis (P less than 0.05), along with amino acid oxidation (P less than 0.01), increased with nutritional intake whereas protein breakdown remained unaltered. Whole-body protein balance also correlated with intake (P = 0.001). Postoperatively, whole-body protein metabolism was determined with patients either fasted (group 1) or fed (group 2) and the change in metabolism in each individual from a pre-operative study, carried out in the fed state, was calculated. Whole-body protein breakdown increased in both groups (group 1, + 0.91 +/- 0.74 g day-1 kg-1; mean +/- SD, n = 7: group 2, + 0.47, + 0.63 and + 1.01 g day-1 kg-1, n = 3), the change being significant in those fasted after surgery (P less than 0.05). However, the pattern of change in whole-body protein synthesis was entirely different in each group, rising in those fed throughout (+ 0.32, + 0.41 and + 0.66 g day-1 kg-1, n = 3) but falling in those fasted after surgery (-0.38, -0.80 and -1.33 g day-1 kg-1, n = 3). The changes in metabolism appeared more marked in those undergoing greatest surgical stress. Some of the factors involved in the calculations are discussed and their effects on the overall conclusions are considered. A concept of whole-body protein metabolism in the metabolic response to trauma is advocated whereby protein breakdown is largely obligatory to the response, whereas synthesis responds to substrate availability.     

Evidence that histidine is an essential amino acid in normal and chronically uremic man.

The requirement for dietary histidine was investigated in four normal and three chronically uremic men. Subjects lived in a metabolic unit where they were fed three isonitrogenous diets in the following order: a 40-g protein diet (28 plus or minus SD 8 days), a semi-synthetic amino acid diet deficient in histidine (35 plus or minus 2 days), and an amino acid diet which contained histidine (31 plus or minus 5 days). With ingestion of the histidine-deficient diet, nitrogen balance gradually became negative, and serum albumin decreased in six subjects. Plasma histidine fell by 82 plus or minus 6 per cent; muscle histidine decreased by 62 plus or minus 19 per cent; the hematocrit fell by 25 plus or minus 9 per cent; and serum iron rose. Subjects felt unwell, and in five cases a skin lesion consisting of fine scales, dry skin, and mild erythema developed. After administration of the histidine-repletion diet, nitrogen balance became positive in six subjects; serum albumin increased in five cases; plasma and muscle histidine rose; serum iron fell abruptly; a reticulocytosis ensued; and the hematocrit rose. The clinical symptoms and skin lesions disappeared. These observations indicate that histidine is an essential amino acid in normal and chronically uremic man. The absence of dietary histidine is associated with failure of normal erythropoiesis.     

Amino acid metabolism in exercising man

Arterial concentration and net exchange across the leg and splanchnic bed of 19 amino acids were determined in healthy, postabsorptive subjects in the resting state and after 10 and 40 min of exercise on a bicycle ergometer at work intensities of 400, 800, and 1200 kg-m/min. Arterio-portal venous differences were measured in five subjects undergoing elective cholecystectomy.In the resting state significant net release from the leg was noted for 13 amino acids, and significant splanchnic uptake was observed for 10 amino acids. Peripheral release and splanchnic uptake of alanine exceeded that of all other amino acids, accounting for 35-40% of total net amino acid exchange. Alanine and other amino acids were released in small amounts (relative to net splanchnic uptake) by the extrahepatic splanchnic tissues drained by the portal vein.During exercise arterial ananine rose 20-25% with mild exertion and 60-96% at the heavier work loads. Both at rest and during exercise a direct correlation was observed between arterial alanine and arterial pyruvate levels. Net amino acid release across the exercising leg was consistently observed at all levels of work intensity only for alanine. Estimated leg alanine output increased above resting levels in proportion to the work load. Splanchnic alanine uptake during exercise exceeded that of all other amino acids and increased by 15-20% during mild and moderate exercise, primarily as a consequence of augmented fractional extraction of alanine. For all other amino acids, there was no change in arterial concentration during mild exercise. At heavier work loads, increases of 8-35% were noted for isoleucine, leucine, methionine, tyrosine, and phenylalanine, which were attributable to altered splanchnic exchange rather than augmented peripheral release.The data suggest that (a) synthesis of alanine in muscle, presumably by transamination of glucose-derived pyruvate, is increased in exercise probably as a consequence of increased availability of pyruvate and amino groups; (b) circulating alanine serves an important carrier function in the transport of amino groups from peripheral muscle to the liver, particularly during exercise; (c) a glucose-alanine cycle exists whereby alanine, synthesized in muscle, is taken up by the liver and its glucose-derived carbon skeleton is reconverted to glucose.