Intravenous Amino Acids as the Sole Nutritional Substrate Utilization and Metabolism in Fasting Normal Human Subjects DAVID E. F. TWEEDLE, Ch.M., GARRY F. FITZPATRICK, M.D., MURRAY F. BRENNAN, M.B.Ch.B., JESUS M. CULEBRAS, M.D., BRUCE M. WOLFE, M.D., MARGARET R. BALL, A.B., FRANCIS D. MOORE, M.D.
From the Department of Surgery of the Harvard Medical School at the Peter Bent Brigham Hospital, Boston, Massachusetts
The fasting normal human volunteer subject provides an ideal experimental setting for the initial investigation of foodstuffs whose use is proposed for the acutely ill surgical patient. In the normal human subject many variables can be controlled; the achievement of an ideal body fuel economy is quite simple; if a favorable utilization of injected foodstuffs cannot be achieved in this setting, it is unlikely, and remains to be proven, that utilization will be satisfactory under the challenges of acute surgical trauma. In this experimental model, employing four normal human volunteer subjects, nutrition has been provided by the intravenous infusion of isotonic amino acids (FreAmineR II) at a 3.4% concentration. No other source of calories or nutrients was provided. In this setting, utilization was very poor; the subjects were in negative nitrogen balance throughout. The nitrogen excretion was significantly greater than the total of infused nitrogen. The changes in protein, fat and carbohydrate intermediates, as well as the alteration in hormone concentrations, suggest the following endocrine governance of fuel economy in this setting: a sharp rise in glucagon with maintenance of insulin concentration; rapid gluconeogenesis at the expense of both injected and endogenous amino acids; a progressive ketosis without any associated improvement in protein economy; fat oxidation to meet caloric need. The changes in plasma amino acid concentrations are of outstanding interest. They demonstrate changes appropriate to the infusion gradient with the exception of three amino acids whose concentrations did not respond to high infusate levels (serine, lysine, and alanine); likewise, by the fact that methionine rose remarkably though present in only low concentrations in the infusion. These data, taken with other information reported in the literature, as well as continuing studies in these laboratories, strongly suggest that the utilization of infused anino acids for protein synthesis is favored by the provision of an additional caloric source such as glucose.
sidered within the framework of their synthetic utilization and metabolic effectiveness, as balanced against costs, either fiscal or clinical. The metabolic settings of trauma, sepsis, or complex illness, and the concomitant administraion of other foodstuffs renders interpretation difficult. Solutions of crystalline amino acids are now available in several different molar mixtures. It is appropriate to document the metabolic fate of one such solution, employing it without any other substrate, and in the absence of trauma or acute illness. Its effect on the plasma amino acid pattern, as well as its general metabolism and endocrine reception, should be investigated. In studies previously reported from this laboratory, we have described a metabolic model that is very useful for such studies. This is the fasting normal human volunteer subject fed intravenously. After preliminary diet stabilization, the subject is given nothing but water by mouth over a period of 8-14 days. He is provided intravenously with the nutrient under study. With water by mouth and normal ward activity, the subjects experience little discomfort or hunger. Appropriate study of the blood and urine provides an accurate demonstration of the metabolic utilization of the feedings, the interaction between substrates, the effect of the exogenous mixture on metabolism of the patient's body stores of protein, fat, and carbohydrate and the influence of the nutrient combination on the patient's hormonal response and plasma amino acid pattern. Initially, using this model, it was shown that glucose by vein demonstrated a progressive and linear improvement in protein economy ("protein sparing") that went far beyond the initial quantum described by Gamble18 and was quantitatively related to the weight of glucose
A s AN INCREASING variety of nitrogen-containk ing compounds and protein precursors become available for intravenous infusion in starving patients, their evaluation becomes critically important. Widespread use of intravenous nutriments must be conSubmitted for publication: July 8, 1976. This work was carried out under contract between the United States Army Medical Research and Development Command, and Harvard Medical School; it was supported by a grant from the National Institutes of Health.
60
AMINO ACIDS IN FASTING MAN
Vol. 186 * No. I
provided.24 Finally, a "nitrogen floor" was reached, eivdently a basal urine nitrogen excretion not readily reduced further. There was a brisk and remarkable decrease in the draft on native lean tissue proteins, largely muscle. Glucose concentrations in the blood were normalized; there was an appropriate increase in insulin concentration with a reduction in glucagon activity, diminished mobilization of free fatty acids, and a reduction in blood urea nitrogen to 3-5 mg/dl. Total urinary nitrogen loss was reduced to 1.5-1.7 gm/M2/day. A remarkable feature of this demonstration was a sharp rise in alanine concentration at the highest levels of glucose administration, interpreted as a result of sharp reduction in glucagon secretion. The normally circulating alanine was not consumed for gluconeogensis when adequate glucose was provided. This model has now been used for a number of other related studies, including the recent report that the protein-sparing effect of an intravenous fat emulsion could be reproduced by providing an equivalent amount of available glycerol intravenously as the sole substrate.7 At no point in these studies have we observed any evidence to suggest that a state of ketosis favors protein economy, although this has been
suggested.6"6'27 The fasting normal human volunteer subject thus provides an ideal setting for the study of the metabolic fate of amino acids given intravenously in the absence of any other nutritional substrate that might be used in the bodily economy of protein, fat, or carbohydrate. It is the purpose of this paper to report such studies carried out in four normal human volunteer subjects, and to address three specific questions: What is the effect of peripherally infused amino acids on the nitrogen economy? What is their effect on specific concentrations of amino acids in the blood? Does this setting provide any evidence to support the concept that progressive ketosis, associated with amino acid infusion, favors protein economy in man? Although these subjects-normal young men were neither starving nor traumatized, basal data of this type should provide important information against which similar observations can be made in trauma, starvation, and complex illness. Normal subjects are very readily maintained in zero nitrogen balance; if an intravenous program is incapable of accomplishing this in the fasting normal, it is unlikely that it will be effective under the conditions of stress. Methods
Subjects Four male subjects, age 20 to 35 years, were interviewed, examined, and screened by electrocardio-
61
graphic, radiographic, hematologic, and biochemical investigations to ensure that they were free of disease. The nature of the experiment was explained to them as well as the fact that, although unlikely, they might experience mild hunger senstations and that they would receive intravenous infusions and undergo blood sampling.* It was explained that they could withdraw from the study if they so wished. One of these subjects (S.B.) was about 25% overweight, for his height. Nutritional Intake
Subjects were given a measured oral intake at their habitual level, for two days prior to the study infusions, to ensure stability and nutritional repletion. During the 8-day period of study, they were infused via a peripheral vein with a 3.4% solution of amino acids (FreAmineR II) delivered throughout the 24 hours at a constant rate (125 ml per hour) by an infusion pump (IMED Corp.). This solution was prepared under sterile laminar flow conditions by diluting 400 ml of commercial solution (8.5% FreAmineR II, McGaw Labs., California) with 600 ml of distilled water, adding extra sodium and potassium chloride. Ten consecutive 1-liter bags of this solution were analyzed by macro-Kjeldahl technique. As shown in Table 1, they were found to contain 4.5 + 0.2 (S.D.) gm of nitrogen per liter, 30.8 gm of amino acids.t The osmolality of 3.4% FreAmineR II with electrolytes added is 408 mO/l. Each subject received 2.0 liters of this solution on the first day of the intravenous portion of the study, and 3.0 liters on subsequent days. Water-soluble vitamins (2 ml Berocca C.R, Roche Labs.) and trace metals (2 mg Zn, 0.4 mg Cu, 0.2 mg Mn and 0.06 mg I) were given daily. Fat-soluble vitamins (5 ml Multivitamin infusionR, U.S.V. Pharm. Corp.) were given weekly. Distilled water was permitted by mouth, ad libitum. Collection and Analysis of Samples Each study day commenced at 8 a.m. All urine was collected in acid and stored at -20° until analyzed. Analyses for urea, creatinine, and uric acid were carried out by the AutoAnalyzer (Technicon, Tarrytown, New York) utilizing a modification of the carbamido * These precautions and the informed consent procedure were reviewed and approved by the Committee on Human Subjects of the Peter Bent Brigham Hospital. t The reason for this discrepancy (i.e., 3.4% FreAmineR II is actually 3.08% amino acids) related to the fact that 8.5% FreAmineR II is made by the solution of 85 gm of FreAmineR II powder, in water. Some ash and phosphate salt are included in this weighing procedure. As noted in the table, the label claim and the analytic data for individual amino acids agree within satisfactory limits.
Ann. Surg. * July 1977
TWEEDLE AND OTHERS
62
diacetyl reaction, the alkaline picrate (Jaffe) reaction and the phosphotungstic acid reactions, respectively. Ammonia was analyzed using an ammonia electrode (Orion Research Inc., Model 95-10). Venous blood samples were obtained at 8 a.m. after an overnight fast. Plasma glucose was determined by the Hoffman reaction and blood urea nitrogen on the AutoAnalyzer. Serum free fatty acids (FFA) were measured by a single extraction method.12 Serum triglycerides (TG), aceto-acetate, and beta-hydroxybutyrate (blood ketones) were analyzed by enzymatic methods.3 Whole blood alanine was determined by enzymatic analysis using alanine dehydrogenase.4 Serum immunoreactive insulin (IRI) and human growth hormone (HGH) were measured by combined radioimmunoassay.'9 Plasma immunoreactive glucagon (IRG) was analyzed by radioimmunoassay using a 30k antibody.'3 Statistical significance of means was analyzed by Student's "t" test; slopes by co-variance analysis. Amino acid analyses were carried out on plasma TABLE 1. Composition of the Infused Solution. (3.4% FreAmineR II)
Amino Acids Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Alanine
Arginine Histidine Proline Serine
Glycine Cysteine Total
Total nitrogen (gm/L) Electrolytes (mEq/L)
Data calculated from FreAmineR II manufacturer's label (mM/L) 18.0 23.5 17.0 12.1 11.6 11.4 2.5 19.1 26.9 7.1 6.2 33.0 16.0 90.6
From the Department of Surgery of the Harvard Medical School at the Peter Bent Brigham Hospital, Boston, Massachusetts
The fasting normal human volunteer subject provides an ideal experimental setting for the initial investigation of foodstuffs whose use is proposed for the acutely ill surgical patient. In the normal human subject many variables can be controlled; the achievement of an ideal body fuel economy is quite simple; if a favorable utilization of injected foodstuffs cannot be achieved in this setting, it is unlikely, and remains to be proven, that utilization will be satisfactory under the challenges of acute surgical trauma. In this experimental model, employing four normal human volunteer subjects, nutrition has been provided by the intravenous infusion of isotonic amino acids (FreAmineR II) at a 3.4% concentration. No other source of calories or nutrients was provided. In this setting, utilization was very poor; the subjects were in negative nitrogen balance throughout. The nitrogen excretion was significantly greater than the total of infused nitrogen. The changes in protein, fat and carbohydrate intermediates, as well as the alteration in hormone concentrations, suggest the following endocrine governance of fuel economy in this setting: a sharp rise in glucagon with maintenance of insulin concentration; rapid gluconeogenesis at the expense of both injected and endogenous amino acids; a progressive ketosis without any associated improvement in protein economy; fat oxidation to meet caloric need. The changes in plasma amino acid concentrations are of outstanding interest. They demonstrate changes appropriate to the infusion gradient with the exception of three amino acids whose concentrations did not respond to high infusate levels (serine, lysine, and alanine); likewise, by the fact that methionine rose remarkably though present in only low concentrations in the infusion. These data, taken with other information reported in the literature, as well as continuing studies in these laboratories, strongly suggest that the utilization of infused anino acids for protein synthesis is favored by the provision of an additional caloric source such as glucose.
sidered within the framework of their synthetic utilization and metabolic effectiveness, as balanced against costs, either fiscal or clinical. The metabolic settings of trauma, sepsis, or complex illness, and the concomitant administraion of other foodstuffs renders interpretation difficult. Solutions of crystalline amino acids are now available in several different molar mixtures. It is appropriate to document the metabolic fate of one such solution, employing it without any other substrate, and in the absence of trauma or acute illness. Its effect on the plasma amino acid pattern, as well as its general metabolism and endocrine reception, should be investigated. In studies previously reported from this laboratory, we have described a metabolic model that is very useful for such studies. This is the fasting normal human volunteer subject fed intravenously. After preliminary diet stabilization, the subject is given nothing but water by mouth over a period of 8-14 days. He is provided intravenously with the nutrient under study. With water by mouth and normal ward activity, the subjects experience little discomfort or hunger. Appropriate study of the blood and urine provides an accurate demonstration of the metabolic utilization of the feedings, the interaction between substrates, the effect of the exogenous mixture on metabolism of the patient's body stores of protein, fat, and carbohydrate and the influence of the nutrient combination on the patient's hormonal response and plasma amino acid pattern. Initially, using this model, it was shown that glucose by vein demonstrated a progressive and linear improvement in protein economy ("protein sparing") that went far beyond the initial quantum described by Gamble18 and was quantitatively related to the weight of glucose
A s AN INCREASING variety of nitrogen-containk ing compounds and protein precursors become available for intravenous infusion in starving patients, their evaluation becomes critically important. Widespread use of intravenous nutriments must be conSubmitted for publication: July 8, 1976. This work was carried out under contract between the United States Army Medical Research and Development Command, and Harvard Medical School; it was supported by a grant from the National Institutes of Health.
60
AMINO ACIDS IN FASTING MAN
Vol. 186 * No. I
provided.24 Finally, a "nitrogen floor" was reached, eivdently a basal urine nitrogen excretion not readily reduced further. There was a brisk and remarkable decrease in the draft on native lean tissue proteins, largely muscle. Glucose concentrations in the blood were normalized; there was an appropriate increase in insulin concentration with a reduction in glucagon activity, diminished mobilization of free fatty acids, and a reduction in blood urea nitrogen to 3-5 mg/dl. Total urinary nitrogen loss was reduced to 1.5-1.7 gm/M2/day. A remarkable feature of this demonstration was a sharp rise in alanine concentration at the highest levels of glucose administration, interpreted as a result of sharp reduction in glucagon secretion. The normally circulating alanine was not consumed for gluconeogensis when adequate glucose was provided. This model has now been used for a number of other related studies, including the recent report that the protein-sparing effect of an intravenous fat emulsion could be reproduced by providing an equivalent amount of available glycerol intravenously as the sole substrate.7 At no point in these studies have we observed any evidence to suggest that a state of ketosis favors protein economy, although this has been
suggested.6"6'27 The fasting normal human volunteer subject thus provides an ideal setting for the study of the metabolic fate of amino acids given intravenously in the absence of any other nutritional substrate that might be used in the bodily economy of protein, fat, or carbohydrate. It is the purpose of this paper to report such studies carried out in four normal human volunteer subjects, and to address three specific questions: What is the effect of peripherally infused amino acids on the nitrogen economy? What is their effect on specific concentrations of amino acids in the blood? Does this setting provide any evidence to support the concept that progressive ketosis, associated with amino acid infusion, favors protein economy in man? Although these subjects-normal young men were neither starving nor traumatized, basal data of this type should provide important information against which similar observations can be made in trauma, starvation, and complex illness. Normal subjects are very readily maintained in zero nitrogen balance; if an intravenous program is incapable of accomplishing this in the fasting normal, it is unlikely that it will be effective under the conditions of stress. Methods
Subjects Four male subjects, age 20 to 35 years, were interviewed, examined, and screened by electrocardio-
61
graphic, radiographic, hematologic, and biochemical investigations to ensure that they were free of disease. The nature of the experiment was explained to them as well as the fact that, although unlikely, they might experience mild hunger senstations and that they would receive intravenous infusions and undergo blood sampling.* It was explained that they could withdraw from the study if they so wished. One of these subjects (S.B.) was about 25% overweight, for his height. Nutritional Intake
Subjects were given a measured oral intake at their habitual level, for two days prior to the study infusions, to ensure stability and nutritional repletion. During the 8-day period of study, they were infused via a peripheral vein with a 3.4% solution of amino acids (FreAmineR II) delivered throughout the 24 hours at a constant rate (125 ml per hour) by an infusion pump (IMED Corp.). This solution was prepared under sterile laminar flow conditions by diluting 400 ml of commercial solution (8.5% FreAmineR II, McGaw Labs., California) with 600 ml of distilled water, adding extra sodium and potassium chloride. Ten consecutive 1-liter bags of this solution were analyzed by macro-Kjeldahl technique. As shown in Table 1, they were found to contain 4.5 + 0.2 (S.D.) gm of nitrogen per liter, 30.8 gm of amino acids.t The osmolality of 3.4% FreAmineR II with electrolytes added is 408 mO/l. Each subject received 2.0 liters of this solution on the first day of the intravenous portion of the study, and 3.0 liters on subsequent days. Water-soluble vitamins (2 ml Berocca C.R, Roche Labs.) and trace metals (2 mg Zn, 0.4 mg Cu, 0.2 mg Mn and 0.06 mg I) were given daily. Fat-soluble vitamins (5 ml Multivitamin infusionR, U.S.V. Pharm. Corp.) were given weekly. Distilled water was permitted by mouth, ad libitum. Collection and Analysis of Samples Each study day commenced at 8 a.m. All urine was collected in acid and stored at -20° until analyzed. Analyses for urea, creatinine, and uric acid were carried out by the AutoAnalyzer (Technicon, Tarrytown, New York) utilizing a modification of the carbamido * These precautions and the informed consent procedure were reviewed and approved by the Committee on Human Subjects of the Peter Bent Brigham Hospital. t The reason for this discrepancy (i.e., 3.4% FreAmineR II is actually 3.08% amino acids) related to the fact that 8.5% FreAmineR II is made by the solution of 85 gm of FreAmineR II powder, in water. Some ash and phosphate salt are included in this weighing procedure. As noted in the table, the label claim and the analytic data for individual amino acids agree within satisfactory limits.
Ann. Surg. * July 1977
TWEEDLE AND OTHERS
62
diacetyl reaction, the alkaline picrate (Jaffe) reaction and the phosphotungstic acid reactions, respectively. Ammonia was analyzed using an ammonia electrode (Orion Research Inc., Model 95-10). Venous blood samples were obtained at 8 a.m. after an overnight fast. Plasma glucose was determined by the Hoffman reaction and blood urea nitrogen on the AutoAnalyzer. Serum free fatty acids (FFA) were measured by a single extraction method.12 Serum triglycerides (TG), aceto-acetate, and beta-hydroxybutyrate (blood ketones) were analyzed by enzymatic methods.3 Whole blood alanine was determined by enzymatic analysis using alanine dehydrogenase.4 Serum immunoreactive insulin (IRI) and human growth hormone (HGH) were measured by combined radioimmunoassay.'9 Plasma immunoreactive glucagon (IRG) was analyzed by radioimmunoassay using a 30k antibody.'3 Statistical significance of means was analyzed by Student's "t" test; slopes by co-variance analysis. Amino acid analyses were carried out on plasma TABLE 1. Composition of the Infused Solution. (3.4% FreAmineR II)
Amino Acids Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Alanine
Arginine Histidine Proline Serine
Glycine Cysteine Total
Total nitrogen (gm/L) Electrolytes (mEq/L)
Data calculated from FreAmineR II manufacturer's label (mM/L) 18.0 23.5 17.0 12.1 11.6 11.4 2.5 19.1 26.9 7.1 6.2 33.0 16.0 90.6
