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Background: Patients with chronic heart failure frequently experience profound wasting during the course of the disease, a condition termed cardiac cachexia. Although protein is the primary structural and functional component of most tissues, few studies have examined the effect of heart failure on protein metabolism. Moreover, no study has assessed the relationship of protein turnover to hormonal alterations thought to promote cachexia. Thus, our goal was to determine if whole-body protein metabolism is altered in heart failure patients and to assess the relationship of protein kinetics to circulating levels of anabolic and catabolic hormones., Methods: We measured whole-body protein metabolism using 13C-leucine, body composition, and circulating anabolic and catabolic hormone levels in 10 patients with chronic heart failure and 11 elderly controls., Results: No differences in leucine rate of appearance, oxidation, or nonoxidative disposal were noted between heart failure patients and controls. However, in a subgroup of patients characterized by increased resting energy expenditure for their metabolic body size (n = 4; > or = 20% above that predicted from fat-free mass), leucine rate of appearance (mean +/- SE; 146 +/- 6 micromol/min), an index of protein breakdown, tended to be higher compared with patients with normal resting energy expenditure (n = 5; 120 +/- 8 micromol/min) and controls (127 +/- 4 micromol/min; p = .06). Alterations in anabolic/catabolic hormone balance did not explain increased protein breakdown in this subgroup, and no correlations were found between hormone levels and protein breakdown in the heart failure group as a whole. In contrast, increased circulating interleukin-6 soluble receptor (r = 0.829; p < .01) and reduced insulin-like growth factor-I (r =-.751; p < .05) levels were related to greater rates of leucine oxidation in heart failure patients., Conclusion: Our results demonstrate that, although increased protein turnover is not a generalized feature of heart failure, there is a subgroup of patients characterized by resting hypermetabolism and increased protein breakdown. Moreover, hormonal alterations related to the heart failure syndrome were related to increased protein oxidation.

Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day.

Carbon (C) in the 1-position of leucine is released as CO(2) with the decarboxylation of alpha-ketoisocaproate (KIC). Carbon in the 2-position of leucine undergoes several additional metabolic steps before entering the tricarboxylic acid (TCA) cycle in the 1-position of acetyl-CoA, where it can be released as CO(2) or be incorporated into other compounds. This study examined the metabolic fate of C in the 2-position of leucine. We infused 11 healthy subjects with [1-(13)C]leucine and [1,2-(13)C(2)]leucine for 3.5--4 h to measure leucine kinetics and the oxidation of the tracers from enrichments of (13)C in blood and expired CO(2). The fraction of leucine infused that was oxidized (f(ox)) was used to define the degree of recovery of the (13)C label(s) for each tracer. As expected, leucine appearance (means +/- SE) did not differ between tracers ((13)C(1): 92.1 +/- 3.1 vs. (13)C(2): 89.2 +/- 3.2 micromol x kg(-1) x h(-1)) when calculated using plasma leucine enrichments as an index of intracellular enrichment. A small (3%) but significant (P = 0.048) difference between tracers was found when KIC was used to calculate leucine appearance ((13)C(1): 118.0 +/- 4.1 vs. (13)C(2): 114.4 +/- 4.5 micromol x kg(-1) x h(-1)). The value of f(ox) was 14 +/- 1% for [1,2-(13)C(2)]leucine and was lower than the f(ox) for [1-(13)C]leucine (19 +/- 1%). From the f(ox) data, we calculated that the recovery of the 2-(13)C label in breath CO(2) was 58 +/- 6% relative to the 1-(13)C label. These findings show that, although a majority of the 2-(13)C label of leucine is recovered in breath CO(2), a significant percentage (approximately 42%) is retained in the body, presumably by transfer to other compounds, via TCA exchange reactions.

To examine the whole body and splanchnic tissue substrate handling during endotoxemia, an intravenous bolus of endotoxin was given to six healthy volunteers during primed, continuous infusions of [1-13C]leucine, [ring-2H5]phenylalanine, and [6,6-2H2]glucose. Whole body protein breakdown, based on whole body Leu and Phe appearance rates (Ra), increased in response to endotoxin given at time 0 (RaLeu 77 +/- 2 mol.kg-1 x h-1, t = 0 h; 88 +/- 6, t = 4 h; P < 0.05) (RaPhe 39 +/- 2 mol.kg-1 x h-1, t = 0; 46 +/- 3, t = 4 h; P < 0.05). Splanchnic amino acid balance (Bal) increased (BalLeu 7 +/- 4 mol.kg-1 x h-1, t = 0; 21 +/- 5, t = 2 h; P < 0.05) (BalPhe 3 +/- 2 mol.kg-1 x h-1, t = 0; 16 +/- 4, t = 2 h; P < 0.05) and can be accounted for by increased splanchnic uptake (Rd) of Phe and Leu (RdLeu 21 +/- 3 mol.kg-1 x h -1, t = 0; 37 +/- 7, t = 120 min; P < 0.05) (RdPhe 10 +/- 3 mol.kg-1 x h-1, t = 0; 24 +/- 5, t = 120 min; P < 0.05). Splanchnic conversion of Leu to ketoisocaproate increased with endotoxin administration (0.7 +/- 0.6 mol.kg-1 x h-1, t = 0; 8 +/- 3, t = 360 min; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

To explore the effects of altered protein intake on the uptake and utilization of dietary amino acids in human subjects, six healthy male Massachusetts Institute of Technology students were studied during three dietary periods each of 9 days, with tracer infusion studies conducted on days 8 and 9. During one period the diet provided a generous protein intake (1.5 g.kg-1.day-1) and during the other two a low-protein intake (0.1 g.kg-1.day-1). Tracer infusions (fed state) were given while subjects received either a liquid formula, supplying the prior protein intake or (on day 9) a generous intake. Combinations of [2H3]Leu and [13C]Leu and of [15N]Lys and [2H4]Lys were given via continuous intravenous or intragastric infusion for 4 h to estimate leucine kinetics and the first-pass splanchnic uptake of tracer. Dietary-induced changes in leucine flux and oxidation were similar irrespective of the tracer and/or route of administration and sampled pool (leucine or KIC) used for calculation. Estimates of absolute changes in the rates of leucine disappearance via nonoxidative metabolism (protein synthesis) and of appearance via protein breakdown depended on the route of [13C]Leu administration. However, it appears that both altered rates of protein synthesis and breakdown contribute to the adaptations required in the body nitrogen economy when protein intakes are restricted and subsequently resupplied. From the intragastric [13C]Leu protocol, the first-pass splanchnic uptake of tracer was the same (approximately 30%) for generous, low-protein, and refed conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the fate of enterally delivered essential amino acids, leucine and phenylalanine, 14 healthy adults were infused in the postabsorptive state with [1-13C]leucine, [5,5,5-2H3]leucine, and [phenyl-2H5]phenylalanine for 7 h in a crossover design by intravenous and nasogastric tube routes. The amount of enterally delivered tracer that was sequestered by the splanchnic bed on the first pass was 21 +/- 1, 17 +/- 3, and 29 +/- 2 for the [13C]leucine, [2H]leucine, and [2H]phenylalanine tracers, respectively. Less than 2% of the nasogastric [1-13C]leucine tracer was oxidized on the first pass. We estimate that 40% of the nasogastric leucine tracer that was sequestered on the first pass was converted to alpha-ketoisocaproate and released, and 50% was incorporated into newly synthesized proteins. Assuming that less phenylalanine is incorporated into protein than leucine because of the lower abundance of phenylalanine in protein compared with leucine, we estimate that 80% of the extracted nasogastric phenylalanine tracer was converted to tyrosine. The study design also indicated a significant effect of duration of tracer infusion on the results, presumably due to recycling of tracer from rapidly turning over protein.

Leucine has been reported to be an important regulator of protein metabolism. We investigated the effect of intravenous infusion of L-leucine versus saline on amino acid metabolism in eight healthy human subjects. Plasma concentrations of amino acids were measured and protein turnover was estimated using L-(1-13C)lysine and L-(3,3,3,-2H3)leucine as tracers. Glucose kinetics were measured using D-(6,6-2H2)glucose as a tracer. Leucine infusion increased the plasma leucine concentration from 103 +/- 8 to 377 +/- 35 mumol/L (P less than .01). Plasma concentrations of essential amino acids, including threonine, methionine, isoleucine, valine, tyrosine, and phenylalanine were significantly decreased by leucine infusion. Leucine infusion did not change lysine flux significantly (108 +/- 4 during saline v 101 +/- 4 mumol/kg/h-1 during leucine infusion), but decreased lysine oxidation (13.2 +/- 0.9 v 10.7 +/- 1 mumol/kg/h, P less than .05) and endogenous leucine flux (from 128 +/- 4 to 113 +/- 7 mumol/kg/h, P less than .05) when plasma (2H3) ketoisocaproate (KIC) was used for calculation. During leucine infusion, the (2H3) KIC to (2H3) leucine plasma enrichment ratio increased from 0.76 +/- 0.02 to 0.88 +/- 0.01 (P less than .001), while estimation of leucine flux using plasma (2H3) leucine showed no change in endogenous leucine flux. Leucine infusion decreased hepatic glucose production and metabolic clearance of glucose, but did not change plasma concentrations of glucose, insulin, C-peptide, glucagon, epinephrine, norepinephrine, or free fatty acids. We conclude that leucine spares glucose and lysine catabolism and decreases plasma concentrations of essential amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

The complexity of amino acid and protein metabolism has limited the development of comprehensive, accurate whole body kinetic models. For leucine, simplified approaches are in use to measure in vivo leucine fluxes, but their domain of validity is uncertain. We propose here a comprehensive compartmental model of the kinetics of leucine and alpha-ketoisocaproate (KIC) in humans. Data from a multiple-tracer administration were generated with a two-stage (I and II) experiment. Six normal subjects were studied. In experiment I, labeled leucine and KIC were simultaneously injected into plasma. Four plasma leucine and KIC tracer concentration curves and label in the expired CO2 were measured. In experiment II, labeled bicarbonate was injected into plasma, and labeled CO2 in the expired air was measured. Radioactive (L-[1-14C]leucine, [4,5-3H]KIC, [14C]bicarbonate) and stable isotope (L-[1-13C]leucine, [5,5,5-2H3]KIC, [13C]bicarbonate) tracers were employed. The input format was a bolus (impulse) dose in the radioactive case and a constant infusion in the stable isotope case. A number of physiologically based, linear time-invariant compartmental models were proposed and tested against the data. The model finally chosen for leucine-KIC kinetics has 10 compartments: 4 for leucine, 3 for KIC, and 3 for bicarbonate. The model is a priori uniquely identifiable, and its parameters were estimated with precision from the five curves of experiment I. The separate assessment of bicarbonate kinetics (experiment II) was shown to be unnecessary. The model defines masses and fluxes of leucine in the organism, in particular its intracellular appearance from protein breakdown, its oxidation, and its incorporation into proteins. An important feature of the model is its ability to estimate leucine oxidation by resolving the bicarbonate model in each individual subject. Finally, the model allows the assessment of the domain of validity of the simpler commonly used models.

The metabolism of amino acids is far more complicated than a 1- to 2-pool model, yet, such simple models have been extensively used with many different isotopically labeled tracers to study protein metabolism. A tracer of leucine and measurement of leucine kinetics has been a favorite choice for following protein metabolism. However, administering a leucine tracer and following it in blood will not adequately reflect the complex, multi-pool nature of the leucine system. Using the tracer enrichment of the ketoacid metabolite of leucine, alpha-ketoisocaproate (KIC), to reflect intracellular events of leucine was an important improvement. Whether this approach is adequate to follow accurately leucine metabolism in vivo or not has not been tested. From data obtained using simultaneous administration of leucine and KIC tracers, we developed a 10-pool model of the in vivo leucine-KIC and bicarbonate kinetic system. Data from this model were compared with conventional measurements of leucine kinetics. The results from the 10-pool model agreed best with the simplified approach using a leucine tracer and measurement of KIC enrichment.

In amino acid tracer kinetic studies of the fed state, ingested amino acid may be taken up during its initial transit through splanchnic tissues and thus not enter the plasma compartment where tracer is infused. To investigate this possibility, adult human subjects received simultaneous intravenous (iv) and intragastric (ig) leucine tracer infusions, first during a postabsorptive (PA) 4-h primed continuous ig infusion of L-[1-13C]-leucine and L-[5,5,5-2H3]leucine iv, followed on a separate day by a fed infusion, in which an ig infusion of a liquid formula was started 2 h before the tracer infusion and continued throughout the tracer study. Subjects were accustomed to a constant experimental diet supplying 1.5 g protein.kg-1.day-1 and 41-45 kcal.kg-1.day-1 for 7 and 12 days before the PA and fed studies, respectively. For the PA study, plasma enrichment for the ig tracer was 3.34 +/- 0.27 (SE) mol + excess and for the iv tracer it was 4.18 +/- 0.10 (P less than 0.02). Enrichments of alpha-keto-isocaproic acid (KIC) were 3.24 +/- 0.16 (ig) and 3.02 +/- 0.14 (iv), respectively [not significant (NS)]. For the fed study, plasma leucine enrichment for the ig tracer was 2.15 +/- 0.14 and for the iv tracer was 2.84 +/- 0.09 (P less than 0.02). KIC enrichments were 2.02 +/- 0.08 (ig) and 2.24 +/- 0.08 (iv), respectively (NS). In the PA study, the ratio of the plasma leucine enrichments for the ig and iv tracers was 0.80 +/- 0.06 and in the fed experiment, 0.76 +/- 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of meal consumption on plasma leucine and alanine kinetics were studied using a simultaneous, primed, continuous infusion of L-[1-13C]leucine and L-[3,3,3-2H3]alanine in four healthy, young, adult male subjects. The study included an evaluation of the effect of sampling site on plasma amino acid kinetics, with blood being drawn simultaneously from an antecubital and dorsal heated hand vein. In comparison with the postabsorptive state, the ingestion of small hourly meals resulted in a 35% increase in plasma leucine flux and a 77% increase in leucine oxidation. Calculated entry of leucine into the plasma compartment from endogenous sources decreased by 65%. Plasma alanine flux more than doubled, indicating a significant enhancement in de novo alanine synthesis. 13C enrichment of leucine in venous and arterialized plasma did not differ significantly, but alanine flux calculated from isotopic measurement in venous plasma was substantially greater than that based on analysis of arterialized blood plasma.

In vivo effects of insulin on plasma leucine and alanine kinetics were determined in healthy postabsorptive young men (n = 5) employing 360-min primed, constant infusions of L-[1-13C]leucine and L-[15N]alanine during separate single rate euglycemic insulin infusions. Serum insulin concentrations of 16.4 +/- 0.8, 29.1 +/- 2.7, 75.3 +/- 5.0, and 2,407 +/- 56 microU/ml were achieved. Changes in plasma 3-methyl-histidine (3-MeHis) were obtained as an independent qualitative indicator of insulin-mediated reduction in proteolysis. Hepatic glucose output was evaluated at the lowest insulin level using D-[6,6-2H2]glucose. The data demonstrate a dose-response effect of insulin to reduce leucine flux, from basal values of 77 +/- 1 to 70 +/- 2, 64 +/- 3, 57 +/- 3, and 52 +/- 4 mumol(kg X h)-1 at the 16, 29, 75, and 2,407 microU/ml insulin levels, respectively (P less than 0.01). A parallel, progressive reduction in 3-MeHis from 5.8 +/- 0.3 to 4.3 +/- 0.3 microM was revealed. Leucine oxidation estimated from the 13C-enrichment of expired CO2 and plasma leucine (12 +/- 1 mumol[kg X h]-1) and from the 13C-enrichment of CO2 and plasma alpha-ketoisocaproate (19 +/- 2 mumol[kg X h]-1) increased at the 16 microU/ml insulin level to 16 +/- 1 and 24 +/- 2 mumol(kg X h)-1, respectively (P less than 0.05 for each), but did not increase at higher insulin levels. Alanine flux (206 +/- 13 mumol(kg X h)-1) did not increase during the clamp, but alanine de novo synthesis increased in all studies from basal rates of 150 +/- 13 to 168 +/- 23, 185 +/- 21, 213 +/- 29, and 187 +/- 15 mumol(kg X h)-1 at 16, 29, 75, and 2,407 microU/ml insulin levels, respectively (P less than 0.05). These data indicate the presence of insulin-dependent suppression of leucine entry into the plasma compartment in man secondary to a reduction in proteolysis and the stimulation of alanine synthesis during euglycemic hyperinsulinemia.

We examined whole body leucine metabolism in healthy young and elderly adults while in the postabsorptive state and during an intravenous glucose administration at two rates. Leucine flux, incorporation into whole body protein, and oxidation were estimated from a continuous intravenous infusion of L-[1-13C]leucine and determination of 13C enrichment in plasma leucine and expired air. Per unit of body weight, leucine flux and rates of incorporation into protein were similar in young and old men. Old women showed lower rates compared with young women. Rates were similar for both age groups when expressed per unit of total body water and/or of muscle protein mass. Intravenous glucose infusion (4 mg . kg-1 . min-1) reduced plasma leucine levels and flux similarly in both age groups. Thus, age-related differences in muscle mass and sensitivity of peripheral tissues to insulin-mediated glucose uptake and metabolism are not paralleled by alterations in whole body leucine kinetics in the postabsorptive state.

Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.

To explore leucine metabolism in relation to leucine intake, five young adult men received an L-amino acid diet that supplied 40, 30, 20, and 10 mg leucine.kg-1.d-1 for 6 d. A stable-isotope-tracer infusion study was then conducted for 5 h while subjects received an intragastric infusion of the test diet. Primed, constant infusions of L-[1-13C]leucine (intragastric) and L-[2H3]leucine (intravenous) were given simultaneously. A final infusion study was conducted in subjects in the postabsorptive state after an additional 2 d with the 10-mg diet. Estimates were made of leucine flux and oxidation, rates of uptake and release of absorbed leucine by the splanchnic region, and leucine balance. The rate of appearance of dietary leucine in the systemic circulation (Leud) decreased (p less than 0.01) between the 40- and 10-mg diets. At the latter intake, splanchnic uptake was approximately 37% of absorbed leucine. The correlation between Leud and plasma leucine concentration was highly positive. A leucine intake of approximately 40 mg. kg-1.d-1 was close to that required to maintain leucine balance under these conditions.

Whole-body leucine and lysine metabolism was explored in young adult men by a primed constant intravenous infusion of a mixture of L-[1-13C]leucine and L-[alpha-15N]lysine over a 4-h period. Subjects were studied after an overnight fast (postabsorptive state) or while consuming hourly meals (fed state) after adaptation to diets providing either a surfeit level of protein (1.5 g.kg body-1.day-1), a level approximating maintenance requirements (marginal intake) (0.6 g.kg body wt-1.day-1), or a grossly inadequate level (0.1 g.kg-1.day-1). The change in protein intake from a marginal to a surfeit level was associated with an increased leucine flux and incorporation of leucine into body protein. In the fed state, oxidation of leucine increased sharply and release of leucine from tissue protein diminished. When dietary protein intake was reduced from the requirement to inadequate level, leucine flux and body protein synthesis and protein breakdown were reduced, together with a smaller reduction in leucine oxidation. The response of the metabolism of [15N]lysine was responsible for maintenance of leucine and other essential amino acid economy, and they appear to be related to the nitrogen and amino acid requirements of the subject. These findings also demonstrate an effect of meals, modulated by their protein content, on the dynamics of whole-body amino acid metabolism.

Severe muscle wasting is a well-recognized characteristic of untreated insulin-deficient diabetes mellitus, a condition in which leucine turnover and oxidation are accelerated. To ascertain whether a similar circumstance exists in type II diabetes when insulin is present but with reduced efficacy, we investigated leucine turnover and oxidation in five obese type II diabetic women by tracer infusion of L-[1-13C,15N]leucine in the postabsorptive state both before and after intensive insulin therapy. With conventional treatment, the type II diabetic women received 61 +/- 33 (SD) U/day of insulin, and their fasting plasma glucose averaged 194 +/- 41 (SD) mg/dl. Leucine carbon flux (QC), nitrogen flux (QN), and oxidation (C) averaged 6.4 +/- 1.2, 15.6 +/- 4.6, and 1.4 +/- 0.3 mmol/h, respectively. These values were not different from the respective values of 6.6 +/- 1.3, 17.0 +/- 8.3, and 1.0 +/- 0.2 mmol/h in matched obese nondiabetic controls, suggesting that leucine metabolism is not altered in insulin-treated type II diabetics. After a week of intensive insulin therapy in which the same diabetic subjects received 94 +/- 36 U/day of insulin, postabsorptive plasma glucose declined to 117 +/- 26 mg/dl. Leucine QC (6.2 +/- 1.0), QN (14.8 +/- 3.7), and C (1.5 +/- 0.5 mmol/h) were unaltered by the increased insulin therapy. Thus, obese type II diabetics had normal leucine kinetics but were hyperglycemic while receiving conventional insulin therapy. Additional intensive insulin therapy in these diabetic subjects improved plasma glucose but did not alter leucine kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

L-Glutamine markedly enhances insulin release evoked by L-leucine in rat pancreatic islets. The metabolic situation found in the islets exposed to both L-glutamine and L-leucine was investigated. L-Leucine slightly decreased the rate of L-glutamine deamidation, inhibited the conversion of glutamate to 2-ketoglutarate by transamination, increased the oxidative deamination of L-glutamate, stimulated the recirculation of 2-ketoglutarate to glutamate and inhibited the further oxidative metabolism of 2-ketoglutarate. L-Glutamine slightly decreased the rate of L-leucine conversion to 2-ketoisocaproate, but inhibited more severely the conversion of 2-ketoisocaproate to acetoacetate and CO2. Several of these findings appeared attributable to activation of glutamate dehydrogenase by L-leucine. When allowance was made for the influence of exogenous amino acids on the oxidation of endogenous fatty acids, a close parallelism was found between the rate of generation of reducing equivalents or O2 uptake and the insulin secretory response to L-leucine and/or L-glutamine. These findings reinforce the view that the process of nutrient-stimulated insulin release coincides with and may be attributable to an increase in catabolic fluxes in the islet cells.

Low plasma levels of branched-chain amino acids, leucine, isoleucine, and valine are postulated to play an etiologic role in hepatic encephalopathy. Supplementation is advocated to reverse encephalopathy and improve nutritional status and survival. We measured in vivo leucine metabolism in normal individuals (n = 5) and in two groups of patients with cirrhosis (n = 8) with a primed continuous infusion of L-[15N, 1-13C] leucine to quantitate the following parameters of leucine metabolism: nitrogen and carbon fluxes, oxidation, contribution to protein synthesis, breakdown of endogenous protein to leucine, deamination and reamination to/from ketoisocaproate. Studies were performed in the fasting and fed states with a conventional enteral diet (Propac) and a branched chain-enriched diet (one third Propac plus two thirds Hepatic-Aid). In vivo leucine metabolism was similar in the fasting and fed states in normal individuals in patients with cirrhosis and with both diets when studied at a protein intake of 0.6 gm/kg ideal body weight/day. When fed these diets, oxidation increased (p less than 0.05) and breakdown decreased (p less than 0.05). The Hepatic-Aid diet increased (p less than 0.05) nitrogen and carbon fluxes significantly more than did the standard diet. Four additional patients with cirrhosis on a diet with more protein were studied (0.75 gm/kg ideal body weight/day). Carbon and nitrogen fluxes, oxidation, synthesis, and deamination were increased (p less than 0.05) when patients with cirrhosis were fed the Propac diet compared with those who fasted. The Hepatic-Aid diet further increased (p less than 0.05) all parameters except synthesis and did not decrease protein breakdown. These data show that patients with cirrhosis metabolize leucine in vivo in a manner identical to that of normal subjects and that leucine-enriched formulas increase oxidation to CO2 without improving protein synthesis.

Previous results of short-term diet studies of leucine kinetics have suggested that the currently accepted requirement value for the amino acid in adults is too low. In the present study the effects of a more prolonged diet period at low leucine intakes on leucine kinetics and nitrogen balance (NB) were explored in healthy young men. They (4 or 5 subjects per group) received an adequate leucine intake (80 mg/kg/d) for 1 or 2 weeks (Period 1) followed by either 7, 14 or 30 mg/kg/d for 3 weeks (Period 2) with a return to 80 mg/kg/d for 1 week (Period 3). Estimates of leucine fluxes (LF), oxidation (LO) and balance (LB) were based on a constant intravenous infusion of L-[1-13C]leucine, at end of Period 1, at 1 and 3 weeks of Period 2 and on days 1 and 3 of Period 3. At all three intakes LF and LO, during the fed state, fell between 1 and 3 weeks of Period 2. LB was negative at 1 week of Period 2 for all groups but had approached equilibrium by 3 weeks. N balance at 3 weeks was similar for all groups but during Period 3 was significantly higher (P less than 0.05) and markedly positive (+18 mgN/kg/d) for the 7 and 14 mg groups, compared with the 30 mg group (+4 mgN/kg/d), indicating that 'depletion' had occurred at the lower leucine intakes during Period 2. Our interpretation is that LB was approached by an adaptation in the 30 mg group whereas it was achieved in the 7 and 14 mg groups by an accommodation, associated with a reduced and low rate of leucine uptake into protein (LF minus LO). Thus, the leucine requirement was judged to be greater than 14 mg/kg/d, a level currently accepted as the upper range of the requirement for healthy adults. The significance of these findings for assessment of nutrient requirements is discussed, with emphasis on the limitation of NB measurements for evaluation of human amino acid requirements.

To elucidate the relative roles of insulin (I) and amino acid (AA) availability on body protein economy and AA kinetics, we compared whole body leucine kinetic responses, using a 360-min constant infusion of L-[1-13C]leucine, during administration of an L-AA solution to six healthy young (21-25 yr) and six healthy old (72-87 yr) men (study 1) to those when the AA solution was given in conjunction with a euglycemic I clamp (study 2). In study 1, serum I increased significantly (P less than 0.02) by 4 +/- 1 and 4 +/- 2 microU/ml in young (Y) and old (O) men, respectively. In study 2, I was raised to 91 +/- 7 (Y) and 88 +/- 7 (O) microU/ml; the glucose infusion to maintain euglycemia in the Y was significantly greater than in the O (8.0 +/- 0.1 vs. 6.8 +/- 1.9 mg.kg-1.min-1). Leucine flux and oxidation increased significantly in both age groups during the administration of AA. Estimates of leucine released from protein breakdown declined (P less than 0.01) by 18 and 20% in study 1 and 2, respectively, in the young and by 12 and 44%, respectively, in the elderly. Rates of leucine incorporation into protein increased (P less than 0.01) similarly in both age groups and in both studies. These findings emphasize the importance of AA availability in the stimulation of protein synthesis and suggest that insulin's major role in vivo is to repress whole body proteolysis. Furthermore, despite evidence of an age-related decline in glucose disposal, the elderly had similar leucine kinetic responses to hyperaminoacidemia.

A study was carried out with 12 young men to examine the relationships between the intake of leucine and indices of leucine kinetics, using L-[1-13C]leucine as a tracer. Six subjects received L-amino acid diets during 7-day periods supplying leucine in the range of 79 to 20 mg.kg-1.day-1 (Group I) and another six subjects (Group II) received leucine intakes ranging from 20 to 4 mg.kg-1.day-1. Estimations were made of leucine kinetics, at the end of each diet period, when subjects were receiving small isonitrogenous, isocaloric meals during the isotope infusion period. Leucine flux declined with reduced leucine intake and leucine oxidation tended not to change at intakes below 20 mg.kg-1.day-1 (slope not statistically different than zero). Plasma valine increased markedly with further restriction in leucine intake below this level. The daily mass balance of leucine, estimated from the difference between intake and oxidation, became negative at an intake of about 20 mg.kg-1.day-1. These findings are discussed in relation to the published mean and upper range of requirement for leucine in healthy adults, currently taken to be 11 mg.kg-1.day-1 and 14 mg.kg-1.day-1, respectively.

The keto analog of leucine, alpha-ketoisocaproate (KIC), is formed intracellularly from leucine and is released, in part, into the systemic circulation. Therefore. KIC can be used to estimate intracellular leucine tracer enrichment in man during labeled-leucine tracer experiments without requiring tissue biopsy samples. This approach was studied in young, healthy, male adults maintained on different dietary protein intakes from generous (1.5 g kg-1d-1) to deficient (0.0 g kg-1d-1) for 5-7 day periods. At the end of each dietary period, the volunteers were given a primed, continuous infusion of L-[1-13C]leucine either after an overnight fast (postabsorptive state) or while being fed hourly aliquots of the same diet. The plasma concentrations of all 3 branched-chain amino and keto acid pairs were measured from early morning blood samples taken from 4 subjects at 4 different levels of protein intake. Leucine concentration showed a weak correlation, and valine concentration showed a strong correlation with protein intake; isoleucine and the 3 keto acids did not. However, each branched-chain amino acid concentration was strongly correlated with its corresponding keto acid concentration. In plasma samples obtained during the L-[1-13C]leucine infusions, the ratio of [1-13C]KIC to [1-13C]leucine enrichment ratio remained relatively constant (77 +/- 1% over the wide range of dietary protein intakes and for both the fed and postabsorptive states. For the tissues from which the plasma KIC originates, the rate of plasma leucine into cells will account for approximately 77% of the intracellular leucine flux with the remaining 23% coming primarily from leucine release via protein breakdown. The constant nature of the plasma KIC to leucine 13C enrichment ratio implies that relative changes in leucine kinetics will appear the same under many dietary circumstances regardless of whether plasma leucine or KIC enrichments are used for the calculations.

Physiological elevations of plasma cortisol levels, as are encountered in stress and severe trauma, were produced in six normal subjects by infusing them with 140 micrograms.kg-1.h-1 of hydrocortisone for 64 h. Amino acid kinetics were measured in the postabsorptive state using three 4-h infusions of L-[1-13C]leucine, L-[phenyl-2H5]-phenylalanine, L-[2-15N]glutamine, and L-[1-13C]alanine tracers 1) before, 2) at 12 h, and 3) at 60 h of cortisol infusion. Before and throughout the study, the subjects ate a normal diet of adequate protein (0.8 g.kg-1.day-1) and energy intake. The cortisol infusion raised plasma cortisol levels significantly from 10 +/- 1 to 32 +/- 4 micrograms/dl, leucine flux from 83 +/- 3 to 97 +/- 3 mumol.kg-1.h-1, and phenylalanine flux from 34 +/- 1 to 39 +/- 1 (SE) mumol.kg-1.h-1 after 12 h of cortisol infusion. These increases were maintained until the cortisol infusion was terminated (64 h). These nearly identical 15% increases in two different essential amino acid appearance rates are reflective of increased whole body protein breakdown. Glutamine flux rose from 325 +/- 28 to 453 +/- 28 mumol.kg-1.h-1 by 12 h of cortisol infusion and remained elevated at the same level at 64 h. The increase in flux was primarily due to a 55% increase in glutamine de novo synthesis. Alanine flux increased from 207 +/- 13 to 285 +/- 23 mumol.kg-1.h-1 with acute hypercortisolemia and increased further to 475 +/- 59 mumol.kg-1.h-1 at 60 h of cortisol infusion, a result primarily of increased alanine de novo synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The adaptive response of whole body leucine metabolism to nutritional supplementation was determined in 6 adolescents with Crohn's disease and growth failure. Five healthy adolescents served as controls for body composition studies. In the first study period, all subjects received a constant diet comparable to usual intakes. In the second period, the patients were given overnight intragastric supplemental feedings that increased dietary protein and energy intakes approximately 40% (to 3.2 g/kg . day and 96 kcal/kg . day, respectively). During each dietary period, O2 consumption, nitrogen balance, whole body potassium (40K), and urinary creatinine measurements were obtained on all adolescents, and the patients received a primed, constant, intravenous infusion of [13C]leucine for 4 h in the fed state. Plasma leucine and expired carbon dioxide 13C-enrichments were determined by mass spectrometric techniques. In the first study, O2 consumption and nitrogen balances were similar between groups; in patients, 40K and urinary creatinine were reduced by 30% and 36%, respectively. With nutritional supplementation, nitrogen balance increased fourfold; O2 consumption and 40K increased by 32% and 10%, respectively. Similarly, whole body leucine flux increased from 166.9 +/- 5.9 to 201.3 +/- 11.2 mumol/kg . h (p less than 0.05) due to a 66% increase in leucine incorporation into body protein (p less than 0.01) and a 41% decrease in leucine oxidation (p less than 0.05). Thus, these studies demonstrate that the mechanisms responsible for lean body mass accretion during nutritional supplementation in adolescents with Crohn's disease and growth failure are increased rates of amino acid incorporation into body protein (via protein synthesis) and decreased rates of amino acid oxidation.

Leucine catabolism is regulated by either of the first two degradative steps: (reversible) transamination to the keto acid or subsequent decarboxylation. A method is described to measure rates of leucine transamination, reamination, and keto acid oxidation. The method is applied directly to humans by infusing the nonradioactive tracer, L-[15N,1-13C]leucine. Leucine transamination was found to be operating several times faster than the keto acid decarboxylation and to be of equal magnitude in adult human males under two different dietary conditions, postabsorptive and fed. These results indicate that decarboxylation, not transamination, is the rate-limiting step in normal human leucine metabolism.

Adaptations of leucine and glucose metabolism to 3 d of fasting were examined in six healthy young men by use of L-[1-13C]leucine and D[6,6-2H2]glucose as tracers. Leucine flux increased 31% and leucine oxidation increased 46% after 3 d of fasting compared with leucine flux and oxidation after an overnight fast. Glucose production rate declined 38% and resting metabolic rate decreased 8% during fasting. Plasma concentrations of testosterone, insulin, and triiodothyronine were reduced by fasting whereas plasma glucagon concentrations were increased. We conclude that there is increased proteolysis and oxidation of leucine on short-term fasting even though glucose production and energy expenditure decreased.