Leucine dose response in the reduction of urea production from septic proteolysis and in the stimulation of acute-phase proteins.

The administration of branched-chain amino acids (BCAAs) has been proved useful in reducing both urea nitrogen production and muscle proteolysis in trauma patients with sepsis, but the optimum infusion rate to achieve these effects is still in question. In this prospective randomized study, a group of 16 posttrauma patients with sepsis received a branched chain-enriched (BCAA = 49.4%) amino acid mixture (8 patients; 120 observations) or a standard amino acid infusion (BCAAs = 15.5%; 8 patients; 227 observations). Total calories, percent lipid calories, and amino acid nitrogen administration were not different in the two groups. Each patient was studied at 8-hour intervals for the plasma levels of amino acids, six hepatic acute-phase proteins, albumin, and other metabolic parameters, including urinary urea nitrogen and 3-methylhistidine excretion. The total intake of each amino acid and its clearance were calculated and the dose of leucine during each 8-hour period was related to the leucine clearance, plasma acute-phase protein levels, and the urinary production of urea and 3-methylhistidine, as an indicator of proteolysis. The results show a significant (r2 = 0.691; p less than 0.0001) reduction of urea nitrogen production and proteolysis as a function of the increase in leucine dose. The identification of a critical mean rate of leucine infusion has been derived from the analysis of the significant linear correlation between leucine intake and leucine clearance (r2 = 0.594; p less than 0.0001). Significant positive correlations between the leucine intake dose and the platelet count (r2 = 0.402; p less than 0.0001), the plasma fibrinogen level (r2 = 0.218; p less than 0.0001), and the regression-derived sum of six acute-phase proteins plus albumin (r2 = 0.696; p less than 0.0001) were found. The increase in leucine clearance was progressively less marked above a mean daily leucine intake rate of 1.4 mumol/kg/min, which also appeared to be the dose level that maximized the acute-phase protein and coagulation effects and reduced proteolysis and urea nitrogen production, suggesting that this is a critical BCAA infusion rate at which an optimum leucine effect occurs. From these data a BCAA (leucine) dose nomogram has been derived.

Pharmacologic modulation of increased release of gluconeogenic precursors from extra-splanchnic organs in sepsis.

The effect of sterile inflammation and sepsis on the release of lactate and amino acids by peripheral tissues was investigated by removing the splanchnic organs (liver and small intestines) from the circulation and monitoring changes in plasma substrates for 30 min. Functional hepatectomy was performed in rats 5-7 days following the intraperitoneal introduction of a fecal-agar pellet (1.5 ml) [sterile vs. Bacteriodes fragilis (10(8) CFU) + E. coli (10(3) CFU)]. Following functional hepatectomy, dichloroacetate, an activator of the pyruvate dehydrogenase complex, significantly inhibited both lactate and alanine release. L-cycloserine, an inhibitor of alanine aminotransferase, significantly (P less than .05) reduced alanine following hepatectomy. Methionine sulfoximine, an inhibitor of glutamine synthetase, significantly (P less than .005) decreased glutamine accumulation following functional hepatectomy in each of the conditions examined. Treatment with each of these drugs abolished the differences between control and sepsis following hepatectomy. These results demonstrate that alterations in the amino acid profiles during sepsis may be modulated in peripheral organs pharmacologically by utilizing known inhibitors of critical regulatory enzymes.

Effect of dichloroacetate on plasma and hepatic amino acids in sterile inflammation and sepsis.

The effect of sterile inflammation and chronic sepsis on the plasma and hepatic free amino acid concentrations was determined. Relative to control animals, only minor alterations in the plasma amino acid concentrations were observed in sterile inflammation and sepsis. In liver, concentrations of alanine, serine, threonine, asparagine, proline, and glycine were significantly increased to the same extent in sterile inflammation and sepsis, while hepatic glutamine concentrations were significantly decreased. Compared with sterile inflammation, the branched-chain amino acid concentrations were depressed in the liver of septic animals. Following administration of dichloroacetate, hepatic alanine concentrations were significantly reduced more than threefold in each of the conditions examined; in contrast, significant increases in hepatic concentrations of threonine, glycine, glutamine, glutamate, histidine, and proline were observed. Also following administration of dichloroacetate, the branched-chain amino acid concentrations were all significantly elevated in each of the conditions examined, and plasma alanine concentrations were significantly decreased, while those of glutamine and glycine were significantly increased. These results demonstrate that there is a disassociation between the plasma and hepatic concentration of free amino acids in sterile inflammation and sepsis. Furthermore, the results demonstrate that some of the alterations in hepatic amino acid metabolism may be reversed pharmacologically by dichloroacetate.

Determinants of urea nitrogen production in sepsis. Muscle catabolism, total parenteral nutrition, and hepatic clearance of amino acids.

The major determinants of urea production were investigated in 26 patients with multiple trauma (300 studies). The body clearances (CLRs) of ten amino acids (AAs) were estimated as a ratio of muscle-released AAs plus total parenteral nutrition-infused AAs to their extracellular pool. While clinically septic trauma (ST) patients without multiple-organ failure syndrome (MOFS) had a higher level of urea nitrogen production (25.6 +/- 13.4 g of N per day) compared with nonseptic trauma (NST) patients (14 +/- 7.5 g of N per day) and with ST patients with MOFS (4.28 +/- 1.5 g of N per day), in all groups urea N production was found to be a function of muscle protein degradation (catabolism), total parenteral nutrition-administered AAs, and the ratio between leucine CLR and tyrosine CLR (L/T) (r2 = .82, P less than .0001). Since tyrosine is cleared almost exclusively by the liver, the L/T ratio may be regarded as an index of hepatic function. The significant differences between urea N production in ST and NST patients lay in an increased positive dependence on muscle catabolism and increased negative correlation with L/T in the ST group. At any L/T ratio, urea N production was increased in ST patients over NST patients, but in ST patients with MOFS, it fell to or below levels of NST patients. These data show that the ST process is associated with enhancement of ureagenesis, due to increased hepatic CLR of both exogenous and endogenous AAs. In sepsis with MOFS, a marked inhibition of urea synthesis occurs, partially explained by a decreased hepatic CLR of non-branched-chain AAs.

Pharmacological reversal of abnormal glucose regulation, BCAA utilization, and muscle catabolism in sepsis by dichloroacetate.

Sepsis has been shown to decrease skeletal muscle glucose oxidation by inhibiting the pyruvate dehydrogenase activity (PDHa) and to increase proteolysis and use of branched-chain amino acids (BCAA). The effects of dichloroacetate (DCA), which reverses PDHa inhibition, were studied in skeletal muscle from a septic (S) rat model of intra-abdominal abscess (E. coli + B. fragilis) and compared to control (C) and sterile inflammatory abscess (I) animals. In one set of S, I, and C animals, DCA (1 mmol/kg) was injected intraperitoneally at 0, 30, and 60 min. Septic, but not I, rats had a twofold increase in skeletal muscle lactate concentrations over C, but no changes in pyruvate. After DCA, both lactate and pyruvate were reduced (p less than 0.001) to same level in S, I, and C. Skeletal muscle alanine was increased in S compared to I or C, but after DCA was reduced threefold in C, S, and I (p less than 0.001) suggesting that alanine synthesis may be impaired due to decreased pyruvate availability. Like alanine, skeletal muscle BCAA were increased in S compared to C, but not altered in I. Following DCA, BCAA levels in muscle from S were reduced (p less than 0.001) to values seen in C or I. Muscle phenylalanine content was significantly elevated in S (p less than 0.05) compared to C or I, but was reduced (p less than 0.05) after DCA in S but not in C or I. Decreased muscle phenylalanine associated with lowered BCAA suggests DCA may decrease septic muscle protein catabolism and/or enhance protein synthesis. Coupled with an increased PDHa and reduced lactate levels, this suggests that DCA may reverse the excess muscle catabolism and BCAA dependence of sepsis by increasing glucose and lactate oxidation and may be a useful therapeutic modality.

Inhibition of post-traumatic septic proteolysis and ureagenesis and stimulation of hepatic acute-phase protein production by branched-chain amino acid TPN.

Previous studies have shown that severe sepsis after major trauma results in the reprioritization of release of hepatic acute-phase proteins (APP). They suggest competition for leucine for nutritional utilization may be responsible. To test this hypothesis, a branched-chain enriched (46.6%) amino acid mixture (BCAA) was administered on a prospective randomized basis with standard TPN therapy to 16 septic post-trauma patients. After sepsis was diagnosed, a randomized therapy (control-TPN or BCAA-TPN) was given for 12 days, or until death occurred. Total calories and amino acid nitrogen (N) administered were not different in the two groups (t-test) and q 8 h (347 study periods) amino acid clearances, urinary urea nitrogen excretion, muscle proteolysis from 3-methyl-histidine (3-MH) excretion, and standard indices of sepsis severity and hepatic function were measured, as well as platelets (PLAT), leucocytes (WBC), albumin (ALB), and six acute-phase proteins: C-reactive protein (CRP), alpha-1-antitrypsin (A1TRIP), fibrinogen (FIBRIN), alpha-2-macroglobulin (AMACRO), ceruloplasmin (CERUL), and transferrin (TRANS). Using Scheffé analysis of all contrasts the data showed: BCAA resulted in a fall in 24-hour urea N excretion (24.0 to 20.0 gm/24 hr) and in proteolysis (138 to 126 gm/24 hr) (p less than 0.0001). Prestudy CRP levels were all elevated, but compared to control where APP reprioritization occurred, over the initial 10 days of therapy BCAA patients had a more rapid fall in CRP with a more rapid rise in FIBRIN, TRANS, CERUL, ALBUMIN, AMACRO, and A1TRIP (all p less than 0.0001) relative to CRP. Also, the sepsis-reduced clearances of glutamine and glutamate, alanine, and proline were increased (p less than 0.0001) during BCAA even though urea nitrogen production was reduced (p less than 0.0001). The increase in leucine clearance with BCAA-enriched TPN was positively correlated (r2 = 0.601; p less than 0.0001) with the increase in the sum of all APP and ALB and was also associated with an increase both in FIBRIN and in platelets (p less than 0.0001). The BCAA-related increase in FIBRIN (9.1 to 11.9 mg/ml) occurred at the same time as a fall in prothrombin time (p less than 0.0001). BCAA-enriched TPN reduced proteolysis and amino acid catabolism and appeared to increase the levels of the more rapidly appearing anti-inflammatory and nutritional hepatic APP and formed coagulation elements in post-traumatic sepsis.

Increased dependence of leucine in posttraumatic sepsis: leucine/tyrosine clearance ratio as an indicator of hepatic impairment in septic multiple organ failure syndrome.

The body clearance of 10 plasma amino acids (AA) was determined from the rate of compared muscle-released AA and AA administered by infusion of total parenteral nutrition (TPN) compared to their estimated extracellular (ECW) pool in patients with multiple trauma with (n = 10) or without (n = 16) sepsis at 8-hour intervals. In both nonseptic and septic trauma, increasing TPN increased the mean clearance rate of all infused AA. When the individual AA clearance rates were normalized by the total AA infusion rate, regression-covariance analysis revealed that patients with sepsis had relatively impaired clearances of alanine (p less than 0.01) and methionine, proline, phenylalanine, and tyrosine p less than 0.05 for all). In contrast, the clearances of branched-chain AA (BCAA) valine and isoleucine were maintained, and the clearance of leucine was higher (p less than 0.05) in trauma patients with sepsis than in those without. At any AA infusion rate, compared with surviving patients with sepsis (p less than 0.05), patients who developed fatal multiple organ failure syndrome (MOFS) showed increased clearances of all BCAA with further impaired clearance of tyrosine. The clearance ratio of leucine/tyrosine was increased in MOFS at any AA infusion rate (p less than 0.0001), was an indicator of severity, and, if persistent, was a manifestation of a fatal outcome. Because tyrosine metabolism occurs almost entirely in the liver while leucine can be utilized by viscera and muscle, these data suggest early and progressive septic impairment of the pattern of hepatic uptake and oxidation of AA with a greater body dependence on BCAA, especially leucine, as septic MOFS develops.

Reprioritization of hepatic plasma protein release in trauma and sepsis.

We studied the temporal pattern of seven hepatic synthesized plasma proteins in 26 severely injured patients beginning in the immediate posttrauma period. Clinical sepsis developed in ten patients between three and eight days after injury, and 16 patients had nonseptic courses. In the initial five days after injury, except for albumin, all acute-phase protein levels rose. However, if sepsis developed, C-reactive protein, fibrinogen, ceruloplasmin, and alpha 1-antitrypsin levels continued to be elevated after the initial five posttrauma days, while transferrin, albumin, and alpha 2-macroglobulin levels fell. This differential response became more extreme as sepsis progressed. Covariance analysis of the regression of the five true acute-phase hepatic proteins on C-reactive protein showed that, when sepsis occurred after major traumatic injury, the C-reactive protein rise was associated with a significant reprioritization of hepatic acute-phase plasma protein release. This reprioritization response seems to be both a predictor of sepsis as well as a measure of the adequacy of the host response to trauma and sepsis.

Plasma free amino acids in children consuming lupin protein with and without methionine supplementation.

Plasma free amino acids were measured in the fasting state and 3 and 4 hours postprandially in children after 9 days consumption of diets in which lupin provided all of 6.4-6.7% protein kcal, either with supplementation of 2% (grams/gram protein) DL-methionine (L & Met, n = 9) or with an isonitrogenous amount of urea (L & U, n = 10). Fasting concentration of total amino acids (TAA) and of essential amino acids (TEAA) and the TEAA:TAA molar ratio did not differ between diets. Fasting Met concentration (15 +/- 4 mumol/liter) and the Met:TEAA ratio (0.021 +/- 0.005) were markedly depressed after 9 days of L & U. Supplementation with Met caused an expected increase of Met concentration (25 +/- 6 mumol/liter) and the Met:TEAA molar ratio (0.039 +/- 0.007) and a profound decrease of Thr concentration (119 +/- 28 to 77 +/- 22 mumol/liter) and Thr:TEAA (0.165 +/- 0.027 to 0.124 +/- 0.028). Postprandially after L ", Met and Met: TEAA did not change from their low fast ing values. After L rMet, Met and Met:TEAA increased significantly relative to fasting values. Threonine concentration increased but the Thr:TEAA ratio decreased significantly (0.124 +/- 0.028 to 0.111 +/- 0.027). These studies confirm Met as the first-limiting amino acid in lupin protein and suggest that Thr becomes limiting when Met is provided in adequate amounts.

Protein quality and digestibility of sorghum in preschool children: balance studies and plasma free amino acids.

The protein quality and digestibility of two high lysine (2.9-3.0 g/100 g protein) and tow conventional varieties (lysine content 2.1-2.2 g/100 g protein) of whole grain sorghum milled as flour were assessed through balance studies in 13 children 6-30 months of age. Sorghum protein provided 6.4 or 8.0% of dietary energy. Control diets contained 64% kcal protein as casein. Children consumed 100-150 kcal/kg body weight/day. Sorghum consumption was associated with weight loss or poor weight gain. We found no difference by variety in apparent nitrogen absorption or retention. Mean absorption and retention of nitrogen (+/- SD) from 26 six-day sorghum dietary periods were 46 +/- 17% and 14 +/- 10% of intake, respectively (corresponding preceding casein control values: 81 +/- 5% and 38 +/- 3%). Stool weight and energy losses during sorghum periods averaged 2.5 to 3 times control values. Plasma amino acids were determined in eleven children after 16 days of sorghum consumption. Fasting concentration of total amino acids (TAA) was similar to values previously obtained with wheat protein at similar levels of intake. Total concentration of essential amino acids (TEAA) was low as were concentrations of lysine (Lys) and threonine (Thr). Analysis of postprandial changes of the Lys/TEAA and Thr/TEAA molar ratios confirmed that Lys was the first limiting amino acid.

Fasting and postprandial plasma free amino acids of infants and children consuming exclusively potato protein.

Fasting and postprandial plasma free amino acids were studied in nine children on the 9th day of consumption of a diet in which potato protein provided all nitrogen at a marginal level of protein intake (5.03-5.10% protein-calories). The analysis of the potato utilized indicated that 49% of total amino acids (TAA) were free amino acids (AA) and 40% essential amino acids (EAA). Fasting values of TAA and total essential amino acids (TEAA) and the ratios of EAA/TEAA were similar to those obtained previously with milk or casein diets. The low percentage of protein-calories in the diet and relatively poor nitrogen absorption of the potato were reflected in a low (0.236) fasting TEAA/TAA ratio. Met/TEAA and Trp/TEAA ratios were significantly (P less than 0.05) but not markedly lower 3 hours postprandially compared to fasting values, returning to the initial levels 4 hours postprandially. Thr/TEAA ratio was significantly higher (p less than 0.05) at 3 hours but not so at 4 hours. The results suggest that potato protein has an adequate ratio of TEAA/TAA and the balance among individual EAA concentrations should be able to meet the EAA requirements of growing infants and small children if fed and absorbed in sufficient amounts to meet total nitrogen needs.

Nutritive value of brown and black beans for infants and small children.

A precooked, instantized mixture of brown and black beans, with and without 0.3% DL-methione added, served as the only source of protein in the diets of 10 recovered malnourished infants and children 10 to 42 months of age. At 6.4 to 5.7% dietary protein calories stool wet weights were twice as high, apparent N absorption significantly lower (65.6 +/- 5.9 versus 87.5 +/- 2.3% of intake), and apparent N retention much lower (9.8 +/- 6.1 versus 34.5 +/- 10.2% of intake) than during preceding and following isocaloric and isonitrogenous casein-based diets. The addition of methionine resulted in minimal improvement in N retention and a highly suggestive increase in fasting plasma free methionine. Prolonged feeding of the methionine-enriched beans at 8.0 to 10.9% protein calories supported satisfactory growth and serum albumin levels in two of three children, not so in the smallest one, in whom repeated balance studies demonstrated no decrease over time in stool wet weight and on marginal improvement in N absorption and retention. The poor digestibility of the protein in these beans is the first-limiting factor in its utilization by infants and small children.

Fasting plasma free amino acids of infants and children consuming cow milk proteins.

We have analyzed the fasting plasma aminograms of 165 infants and small children. Mean age was 17 months (range, 3-42 months). The majority had been consuming a diet in which all protein was supplied by calcium caseinate, the remainder a modified cow milk formula. Protein provided less than 6.4% of energy for 8 children, 6.4%-7% of energy for 81 children, 8% of energy for 42 children and greater than 8% of energy for 34. Only the values for the concentrations of total essential amino acids, valine, and taurine at less than 6.4% of energy as protein were significantly lower than corresponding values at higher intakes of protein. Analysis of the remainder of the data revealed no significant differences in either absolute concentrations or molar fractions among differing levels or sources of protein intake. Interindividual variation in concentrations of amino acids was greater than that resulting from changes in the level of intake of high quality protein when 6.4% or more of energy was provided as protein.

Postprandial plasma lysine as an indicator of dietary lysine adequacy in infants.

Postprandial plasma free amino acids were studied as an index of the level of dietary lysine. Fasting blood was obtained from 11 children who were then fed a meal in which wheat provided 100% of 0.4 g protein and 30% to 50% of 25 kcal/kg body weight. Blood samples were obtained 3 and 4 hours postprandially. Fasting blood samples were obtained from these same children after 9, 18 and 27 (n=6) days of consuming the same diet (2 g protein, 125 kcal/kg/day). Postprandial blood samples were taken from four additional children after consuming a meal containing 25 kcal and 0.75 g protein (0.5 g from wheat, 0.25 g casein)/kg. This meal was expected to provide adequate lysine while still maintaining lysine in lowest concentration among the essential amino acids relative to needs for protein synthesis. After consuming the unsupplemented wheat diet postprandial values showed a significant rise of total amino acids at 3 hours with a return to fasting levels at 4 hours. Total essential amino acids did not change at 3 or 4 hours. Lysine concentration was significantly lower at 3 and 4 hours. The ratio of lysine to total essential amino acids decreased at 3 and, more so, at 4 hours. In 3 of 4 children studied the postprandial decrease of plasma lysine and its molar fraction was not manifest after consuming wheat + casein. The fasting lysine concentration during continued wheat consumption showed no change at 9 days and then a progressive fall at 18 and 27 days. The results with wheat + casein lend support to the concept that a postprandial fall in the concentration of lysine and its molar fraction indicates not merely that lysine is present in lowest concentration but that it is present in inadequate concentration relative to needs for protein synthesis.

Plasma amino acids of infants consuming soybean proteins with and without added methionine.

Fasting plasma free amino acids were determined in 54 convalescent malnourished infants: seven infants while consuming a diet based on isolated soybean protein, containing 4.0% to 5.3% of dietary metabolizable energy (calories) as protein (A), 20 at 6.4% to 6.7% protein calories (B), 23 at 6.4% to 6.7% protein calories with added DL-methionine (C), and four with 8.0% to 12.3% protein calories (D). There were no differences in total amino acid concentration (TAA) among the four groups; the molar fraction of essential amino acids (EAA:TAA) was lower for group A; there were no differences among the four groups in Lys:EAA or 1/2 cystine:EAA ratios or in Met concentration. Met:EAA was higher in C than B, with considerable overlap of individual values. In 10 of 13 infants who were represented in both B and C, Met concentration and Met:EAA ratio were higher in group C. Fasting plasma AA levels are not consistently reliable for field or clinical assessment of dietary Met adequacy. Fasting and postprandial (3- and 4-hour) plasma AA were determined in 29 infants: in 12 the preceding diet and the test meal were both Met-deficient with less than 6.7% protein calories (E), in five the preceding diet was milk-based but the test meal was Met-deficient at less than 6.7% (F), in five the preceding diet and test meal were based on isolated soybean protein at less than 6.7% with DL-Met added (G), and in seven the test meal was soy-based with greater than 9.0% protein calories (H). Plasma Met concentration and Met:EAA fell significantly at 3 and 4 hours in groups E and F, but not in groups G and H, suggesting that a postprandial fall in Met:EAA ratio can be used to identify dietary Met deficiency in field situations.

Plasma free amino acids of children consuming a diet with uneven distribution of protein relative to energy.

Adequate N retention and growth have been demonstrated in children consuming diets in which the total daily protein is fed in a single meal, energy intake being spread over a 14 hour period. To elucidate how adequate protein nutriture continues when protein is not consumed in parallel to energy, plasma free amino acids were measured in 11 children fed a casein-modified cow's milk based diet providing all protein (6.4% of calories) in one feeding, while energy was evenly distributed among five feedings. Children were fed at 0800 hours, 1200 hours, 1500 hours (protein-containing meal), 1900 hours, and 2200 hours. Fasting plasma aminograms were determined in all 11 children. During a long-term growth study with the diet, six children had additional determinations 3 and 4 hours after the first feeding on the same day on which the fasting sample was obtained; samples prior to and 3 and 4 hours after the 1500 hour feeding were obtained on a different day. In a seventh child, all six samples were obtained on a single day. Fasting values for total amino acids (TAA), total essential amino acids (TEAA) and TEAA/TAA, Val, Ile and Pro were elevated in most cases, a pattern seen in children fed high protein diets. The 1100 hour and 1200 hour samples showed a marked decrease in TAA, TEAA and TEAA/TAA. A further drop at 1500 hour produced a pattern characteristic of protein deficiency. Following the protein containing meal, TAA increased 50-100%, TEAA 100-200%, with disproportionate increases in Leu, Ile, Val and Pro. The persistence of this pattern in some cases up to 17 hours suggested that EAA were available for protein synthesis when additional energy was subsequently consumed. An aminogram characteristic of protein deficiency was seen only 18-20 hours after the last protein intake.

Free catecholamine excretion in the urine in normal infants and in those with marasmus or kwashiorkor.

Free catecholamine (epinephrine + norepinephrine) excretions of normal male infants 2.1-3.2, 4.5-10.6, and 12.5-18.5 months of age, respectively, and of infants and children with marasmus or marasmic kwashiorkor were measured on three consecutive days after admission and after partial rehabilitation. In normal infants, particularly the older ones, the first day's excretion was higher than that of the next two days, probably in response to the stress of the procedures. There was an increase with age, but on a surface area basis, the differences were not significant, the three age groups excreting 15.1 plus or minus 10.2, 23.8 plus or minus 20.9, and 24.7 plus or minus 14.3 mug/m-2/day, respectively. Excretions of marasmic infants on admission were not significantly different from those of the control children. Higher mean values were due to elevated excretions of infants with severe infection. After partial rehabilitation, excretions were similar to those of control subjects. In children with marasmic kwashiorkor excretions were no different from those of control infants, except in severely infected children. Admission values revealed more day-to-day variation than recovery values or than those of normal and marasmic infants, in whom subject to subject variation was more marked.