Combination of phenotype assessments and CYP2C9-VKORC1 polymorphisms in the determination of warfarin dose requirements in heavily medicated patients.

The relative contribution of phenotypic measures and CYP2C9-vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms to warfarin dose requirements at day 14 was determined in 132 hospitalized, heavily medicated patients. Phenotypic measures were (1) the urinary losartan metabolic ratio before the first dose of warfarin, (2) the S:R-warfarin ratio at day 1, and (3) a dose-adjusted international normalized ratio (INR) at day 4. CYP2C9 and VKORC1 genotypes were determined by gene chip analysis. In multivariate analyses, the dose-adjusted INR at day 4 explained 31% of variability observed in warfarin doses at day 14, whereas genotypic measures (CYP2C9-VKORC1) contributed 6.5%. When S:R-warfarin ratio was used, genotypes contributed more significantly (23.5%). Finally, urinary losartan metabolic ratio was of low predictive value. The best models obtained explained 51% of intersubject variability in warfarin dose requirements. Thus, combination of a phenotypic measure to CYP2C9-VKORC1 genotypes represents a useful strategy to predict warfarin doses in patients receiving multiple drugs (11+/-4 drugs/day).

Glutamine: the emperor or his clothes?

In this introduction to the Proceedings of the Symposium on Glutamine, we consider various lines of evidence that might potentially lead to an answer to the question posed in the title. We begin with a short summary of the multiple functions of glutamine, which are extensive and, superficially at least, equally as impressive as those of glutamate. However, each of these amino acids may serve an equivalent role in some of these functions due to their ready metabolic interconversion. We raise the question whether glutamine is of primordial or rudimentary significance or whether it is a product of somebody else's existence. Thus, there is a short account of the prebiotic events of evolution that led to the appearance of glutamine and life on Earth. In doing this, it then appears that glutamine is a rather schizophrenic molecule, stable and thermodynamically reliable in biochemical environments, but labile in chemical ones. We then turn to the involvement of glutamine in mammalian N (nitrogen) commerce, with initial emphasis on the nitrogen cycle on Earth, then N transport and N excretion, before assessing its contribution to carbon/energy or C/E commerce. We hypothesize that, in addition to its utilization in immune cell function and in normal intestinal tissues, glutamine is a particularly key anapleurotic and energy-yielding substrate in conditions of hypoxia, anoxia and dysoxia. It also serves as a quantitatively important gluconeogenic metabolite under normal postabsorptive conditions. We postulate that in certain conditions, this carbon-energy econometric function might be by-passed with ornithine. In conclusion, the answer to the question above depends on the context, and this point will receive elaboration in many of the individual contributions that collaborate to form these Proceedings.

Ringer's ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats.

OBJECTIVE: Pyruvate has been shown to be protective in numerous in vitro and in vivo models of oxidant-mediated cellular or organ system injury. Unfortunately, the usefulness of pyruvate as a therapeutic agent is abrogated by its very poor stability in solution. In an effort to take advantage of the ability of pyruvate to scavenge reactive oxygen species while avoiding the problems associated with the instability of pyruvate in solution, we sought to determine whether a simple derivative, ethyl pyruvate, would be protective in an animal model of reactive oxygen species-mediated tissue injury, namely mesenteric ischemia and reperfusion in rats. DESIGN: Prospective, randomized trial. SETTING: Animal research center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Under general anesthesia, rats were subjected to 60 mins of mesenteric ischemia followed by 60 mins of reperfusion. Controls (n = 6) received intravenous lactated Ringer's solution according this dosing schedule: 1.5 mL/kg bolus before ischemia, 3.0 mL/kg bolus before resuscitation, and 1.5 mL.kg-1.hr-1 by continuous infusion. Two experimental groups received similar volumes of either pyruvate (n = 6 each) or ethyl pyruvate (n = 9) solution made up exactly like lactated Ringer's solution except for the substitution of either pyruvate or ethyl pyruvate for lactate, respectively. MEASUREMENTS AND MAIN RESULTS: To obtain tissues for assessing mucosal permeability and histology, five 10-cm long segments of small intestine were obtained at the following time points: baseline, after 30 and 60 mins of ischemia, and after 30 and 60 mins of reperfusion. Mucosal permeability to fluorescein isothiocyanate dextran (molecular weight 4000 Da) was assessed ex vivo by using an everted gut sac method. Compared with controls, treatment of rats with either pyruvate solution or ethyl pyruvate solution significantly ameliorated the development of intestinal mucosal hyperpermeability during the reperfusion. Treatment with ethyl pyruvate solution also significantly decreased the extent of histologic mucosal damage after mesenteric reperfusion. CONCLUSIONS: Treatment with Ringer's ethyl pyruvate solution ameliorated structural and functional damage to the intestinal mucosa in a rat model of mesenteric ischemia/reperfusion. Ethyl pyruvate solution warrants further evaluation as a novel therapeutic agent for preventing oxidant-mediated injury in various disease states.

Metabolism 2000: the emperor needs new clothes.

Metabolism is one of the corner stones of nutritional science. As biology enters the post-genomic era and with functional genomics beginning to takeoff, we anticipate that the study of metabolism will play an increasingly important role in helping to link advances made via the reductionist paradigm, that has been so successful in molecular and cellular biology, with those emerging from observational studies in animals and human subjects. A reconstructive metabolically-focused approach offers a timely paradigm for enhancing the elegance of nutritional science. Here we give particular attention to the use of tracers as phenotyping tools and discuss the application of our metaprobe concepts with respect to some novel features of metabolism, including 'underground metabolism', 'metabolic hijacking', 'catalytic promiscuity' and 'moonlighting proteins'. The opportunities for enhancing the study of metabolism by new and emerging technologies, and the importance of the interdisciplinary research enterprise are also touched upon. We conclude that: (1) the metaprobe concepts and approach, discussed herein, potentially yield a quantitative physiological (metabolic) phenotype against which to elaborate partial or focused genotypes; (2) physiological (metabolic) phenotypes which have a whole-body or kinetically-discernible inter-organ tissue-directed metabolic signature are an ideal target for this directed tracer-based definition of the 'functional' genotype; (3) metabolism, probed with tracer tool kits suitable for measuring rates of turnover, change and conversion, becomes in the current sociology of the 'Net', like AOL, Yahoo. Alta Vista, Lycos or Ask Jeeves, the portal for an exploration of the metabolic characteristics of the 'Genomics Internet'.

Twenty-four-hour oral tracer studies with L-[1-13C]lysine at a low (15 mg.kg (-1).d (-1) and intermediate (29 mg.kg (-1).d(-1)) lysine intake in healthy adults.

BACKGROUND: We proposed previously that the mean lysine requirement value is approximately 30 mg * kg(-)(1) * d(-)(1) rather than the proposed 1985 FAO/WHO/UNU estimate of the upper range of the requirement, which is 12 mg * kg(-)(1) * d(-)(1). OBJECTIVE: Our objective was to explore the 24-h pattern and rate of whole-body lysine [l-(13)C]oxidation and status of whole-body lysine balance in healthy, young adults given an L-amino acid diet supplying either a low lysine intake (14-15 mg * kg(-)(1) * d(-)(1)) or an intermediate lysine intake (29 mg * kg(-)(1) * d(-)(1)) for 6 d before a continuous tracer study with L-[1-(13)C]lysine. DESIGN: Five subjects received the low lysine intake, 6 subjects received the intermediate intake, and all were studied by using a standard 24-h oral tracer protocol that was described earlier for studies at a generous lysine intake. RESULTS: The rate of lysine oxidation was not significantly different between the 12-h fasted and 12-h fed states. The daily oxidation rate (f1.gif" BORDER="0"> +/- SD) was 27. 9 +/- 8.8 and 27.3 +/- 17.6 mg lysine * kg(-)(1) * d(-)(1) for the low- and intermediate-intake groups, respectively (NS). Daily lysine balance was -12.4 +/- 92 and 1.8 +/- 17.7 mg * kg(-)(1) * d(-)(1), respectively (P < 0.025), for the low and intermediate intakes. The balance was significantly less than zero (P < 0.001) for the low intake. CONCLUSION: The FAO/WHO/UNU lysine requirement value is not sufficient to maintain lysine homeostasis in healthy adults. From the results of this and tracer studies done by others, the mean lysine requirement of healthy adults was determined to be 30 mg * kg(-)(1) * d(-)(1).

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet.

The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an L-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, L-[1-(13)C]cysteine was given as a primed, constant i.v. infusion (3 micromol x kg(-1) x h(-1)) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean +/- SD; day(-1)) of whole blood GSH was 0.65 +/- 0.13 for the adequate diet and 0.49 +/- 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 +/- 243 and 1,216 +/- 162 microM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 +/- 216 and 579 +/- 135 micromol x liter(-1) x day(-1), respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

Glutamate: an amino acid of particular distinction.

In this introductory paper to the symposium, we consider why L-glutamate (GLU) is such an abundant biomolecule. We begin with a brief discussion of the prebiotic dawn of events and some evolutionary features of GLU in the biological and metabolic world. The properties of GLU are then examined with reference to its overall structural motif and to the reactivity of the molecule at the tautomeric 2 carbon and at the 4- and 5-C positions. This chemical viewpoint reveals that the GLU molecule offers a number of features/properties not shared by its homologs (amino adipic and aspartic acids). These properties make GLU a favorable choice for facilitating its involvement in multiple metabolic processes that play major roles in the nitrogen economy of the host, as well as serving as a nutrient, an energy-yielding substrate, a structural determinant and an excitatory molecule.

L-2-[(13)C]oxothiazolidine-4-carboxylic acid: a probe for precursor mobilization for glutathione synthesis.

L-2-oxothiazolidine-4-carboxylic acid (OTZ), a 5-oxoproline analog, is metabolized by 5-oxoprolinase and converted to cysteine, the rate-limiting amino acid for GSH synthesis, with the release of CO(2). [(13)C]OTZ (1.5 mg/kg) was used in 12 healthy men and women (ages 23-73 yr) to indirectly assess precursor mobilization for GSH synthesis when stores were reduced by 2 g acetaminophen. Expired breath samples were analyzed for (13)CO(2), and results were analyzed using noncompartmental and two-compartment open minimal models. Results show an increase in (13)C excretion (higher OTZ hydrolysis) when GSH stores were reduced and 5-oxoprolinase substrate utilization patterns, consequently, were altered (P < 0. 01). A metabolic rate index (MRI) of the OTZ probe was found to be significantly higher after reduction of GSH content by acetaminophen (P < 0.05). The difference in adaptive capacity (difference between control and postacetaminophen metabolic rate indexes) was two times as large in the young than the old subjects (P < 0.01). These data support the use of [(13)C]OTZ as a probe to identify individuals who may be at risk for low GSH stores or who have an impaired capacity to synthesize GSH.

Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects.

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-(13)C]lysine and L-[6,6-(2)H(2)]lysine, respectively. [(15)N(2)]urea or (15)NH(4)Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with (15)NH(4)Cl. [(15)N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [(13)C]- and [(2)H(2)]lysine enrichments during the 12-h fed period were observed between the two (15)N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after (15)NH(4)Cl and [(15)N(2)]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg. kg(-1). day(-1), depending on the [(15)N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.

Plasma L-5-oxoproline carbon and nitrogen kinetics in healthy young adults.

L-5-oxoproline (OP), an intermediate of the gamma-glutamyl cycle of glutathione synthesis and degradation, may serve as a probe for the state of glutathione kinetics. We explored the whole-body carbon and nitrogen kinetics of OP in five male healthy subjects (75.2 kg; 181 cm; 26 y) after a 5-d adaptation to an adequate L-amino acid-based diet (160 mg N x kg(-1) x d(-1); 188 kJ x kg(-1) x d(-1)), using a crossover design. On day 6 of the diet period, we carried out an 8-h tracer protocol (3 h fast; 5 h fed; 2/3 of daily nitrogen intake) with intravenous infusion of L-[1-(13)C]oxoproline and L-[3, 3-(2)H]cysteine or, in randomized order, on the second occasion, L-[(15)N]oxoproline and L-[3,3-(2)H]cysteine. Plasma OP was isolated by cation exchange and after addition of internal standards (DL-[(2)H(3)]-5-oxoproline; L-[(15)N, U-(13)C(5)]-5-oxoproline; DL-[(2)H(3)]-glutamic acid) derivatized to form TBDMS esters and measured by gas chromatography/mass spectrometry. Plasma OP concentration did not differ between fed and fasted state (fast: 59. 4 +/- 8.3; fed 59.2 +/- 8.9 nmol/mL). (13)C- and (15)N OP flux during the fasted and fed state were 19 +/- 3.6, 21.2 +/- 3.2, and 22.6 +/- 3.9, 25.8 +/- 4.3 micromol x kg(-1) x 30 min(-1), respectively. OP oxidation was 15.6 +/- 3.6 and 17.9 +/- 3.5 micromol x kg(-1) x 30 min(-1), in fasting and feeding, respectively, (P < 0.05). More than 80% of the plasma flux was oxidized. These findings are compared with the published literature on GSH turnover in plasma of human subjects and underscore the need to define more completely the dynamic aspects of glutathione metabolism and of the intermediates of the gamma-glutamyl cycle.

1999 Jonathan E. Rhoads lecture. Isotopic metaprobes, nutrition, and the roads ahead.

The 1999 Jonathan E. Rhoads lecture, delivered by Vernon R. Young at the annual meeting of American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.), San Diego, February 2, 1999, with the printed version coauthored with Alfred M. Ajami, is concerned with the application of isotopic probes and how, in particular, they may be used as diagnostic tools to enhance the role of nutrition in the comprehensive medical management of the patient. Following a brief review of the early uses of stable isotopes in metabolic research we consider the present and possible future application of stable isotope probes. The concept of a "gateway" enzyme in a discrete biochemical pathway and how the flow of substrate through this step might be assessed by giving a "metaprobe" is developed. The specific and desirable structural requirements of the metaprobe are considered. A number of examples are given that further exploit the concepts of "underground" metabolism and of metabolic "hijackers." It is our view that we are on the verge of a new era where, for the many pragmatic and exciting reasons discussed, stable isotope probes will find and increasing use in the practice of clinical medicine and in the preventive and public health areas.

Twenty-four-hour intravenous and oral tracer studies with L-[1-13C]-2-aminoadipic acid and L-[1-13C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults.

BACKGROUND: This is a continuation of investigations of the relations between amino acid kinetics and amino acid dietary requirements in healthy adults. OBJECTIVE: The aim was to investigate the 24-h pattern and rate of the metabolism of an L-[1-13C]-2-aminoadipic acid ([13C]AAA) tracer and of whole-body L-[1-13C]lysine ([13C]lysine) oxidation and balance in healthy, young adults receiving a generous intake of lysine. DESIGN: Thirteen healthy adults were given an adequate, L-amino acid-based diet supplying 77 mg lysine x kg(-1) x d(-1) for 6 d before the tracer studies. Two subjects received [13C]AAA intravenously and 2 received it orally; 3 subjects received [13C]lysine intravenously and 6 received it orally. We measured 13CO2 output, plasma [13C]AAA and [13C]lysine enrichment, and urinary [13C]AAA. RESULTS: [13C]AAA oxidation was estimated to be higher after the orally administered than after the intravenously administer tracer; plasma [13C]AAA was similar to urinary [13C]AAA. Whole-body lysine oxidation showed a rhythm that was induced by meal feeding. The intravenous [13C]lysine tracer gave mean estimates of lysine balances (lysine intake minus oxidation) that apparently were too low (-15.7 mg x kg(-1) x d(-1)) or too high (16.6 mg x kg(-1) x d(-1), P < 0.05 from zero balance) on the basis of urinary [13C]AAA or plasma [13C]lysine estimates of oxidation, respectively. For the orally administered tracer and plasma [13C]lysine enrichment, the mean balance was slightly positive (8.7 mg x kg(-1) x d(-1), P < 0.05 from zero). CONCLUSIONS: Use of urinary [13C]AAA as an index of the enrichment of the precursor pool did not appear to significantly improve the estimate of the fasting and feeding components of daily lysine balance. For estimates of daily, whole-body lysine oxidation, we propose use of plasma [13C]lysine with a 24-h, orally administered tracer protocol.

An initial assessment, using 24-h [13C]leucine kinetics, of the lysine requirement of healthy adult Indian subjects.

The international 1985 FAO/WHO/UNU upper dietary requirement for lysine of 12 mg.kg-1.d-1 may be inadequate for healthy Indian adults. To test this, we used a modified indicator amino acid oxidation technique to assess the adequacy of lysine intake of 12 and 28 mg.kg-1.d-1. Seven healthy, male, Indian subjects were studied during each of two randomly assigned 6-d periods while receiving an otherwise adequate diet based on an L-amino acid mixture. Beginning at 1800 on day 6 of the diet, a 24-h infusion protocol in which a [13C]leucine tracer was administered intravenously was used to assess leucine oxidation and daily leucine balance at each test lysine intake. Mean 24-h leucine oxidation was 54.7 compared with 46.9 mg.kg-1.d-1 (P < 0.05) and mean 24-h leucine balances were -4.1 and 3.5 mg.kg-1.d-1 (P < 0.05) for lysine intakes of 12 and 28 mg, respectively. Leucine balances were significantly negative (0.025 < P < 0.05) with the 12-mg lysine intake and not significantly different (P > 0.10) from zero or equilibrium with the 28-mg intake. These findings indicate that the international requirement for lysine appears to be inadequate to maintain body amino acid homeostasis and function in apparently healthy subjects characteristic of the south Asia region. They further indicate that our previously proposed, tentative lysine requirement of 30 mg.kg-1.d-1 is probably adequate for this population.

Effect of cystine intake on methionine kinetics and oxidation determined with oral tracers of methionine and cysteine in healthy adults.

There is evidence based on nitrogen balance that dietary cystine spares, from approximately 16% to 89%, the total methionine requirement. In a previous study we did not detect, by tracer techniques, a sparing effect of cystine when the diet provided methionine at a limiting intake (requirement level: 13 mg.kg-1.d-1). One reason could be that we used an intravenous infusion of the tracer, which may not, therefore, have labeled the carbon dioxide derived from the splanchnic oxidation of dietary methionine. The aim of this study was to compare methionine metabolism and oxidation in eight healthy adults given for 6 d each of three different diets: 13 mg (87.0 mumol) methionine.kg-1.d-1 and no cystine (diet A); 5 mg (33.5 mumol) methionine.kg-1.d-1 and no cystine (diet B); and 5 mg (33.5 mumol) methionine.kg-1.d-1 and 6.5 mg (52.4 mumol) cystine.kg-1.d-1 (diet C). On day 7, tracers ([1-13C, methyl-2H3]methionine and [2H2]cysteine) were administered orally at 30-min intervals for 8 h. Blood and breath samples were obtained for analysis during 3-h fasting and consecutive 5-h feeding periods. During fasting, methionine oxidation and methionine methyl (Qm) and carboxyl (Qc) fluxes and plasma concentrations were not affected by the amount of sulfur amino acids in the three diets. In the fed state methionine oxidation was significantly lower during diets B (3.0 +/- 0.5 mumol.kg-1.h-1) and C (2.8 +/- 0.6 mumol.kg-1.h-1) than during diet A (4.1 +/- 0.9 mumol.kg-1.h-1); there were no significant differences between diets B and C. Qm and Qc decreased with decreased methionine intake but no effect was observed by adding cystine. Cysteine flux (Qcys) was not affected by diet composition but it was lower during feeding than during fasting. In conclusion, replacing approximately 60% of the total requirement for methionine with cystine over a short diet period did not result in a detectable sparing of methionine oxidation.

Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling.

The rates of whole body nitric oxide (NO) synthesis, plasma arginine flux, and de novo arginine synthesis and their relationships to urea production, were examined in a total of seven healthy adults receiving an L-amino acid diet for 6 days. NO synthesis was estimated by the rate of conversion of the [15N] guanidino nitrogen of arginine to plasma [15N] ureido citrulline and compared with that based on urinary nitrite (NO2-)/nitrate (NO3-) excretion. Six subjects received on dietary day 7, a 24-hr (12-hr fed/12-hr fasted) primed, constant, intravenous infusion of L-[guanidino-15N2]arginine and [13C]urea. A similar investigation was repeated with three of these subjects, plus an additional subject, in which they received L-[ureido-13C]citrulline, to determine plasma citrulline fluxes. The estimated rates (mean +/- SD) of NO synthesis over a period of 24 hr averaged 0.96 +/- 0.1 mumol .kg-1.hr-1 and 0.95 +/- 0.1 mumol.kg-1.hr-1, for the [15N]citrulline and the nitrite/nitrate methods, respectively. About 15% of the plasma arginine turnover was associated with urea formation and 1.2% with NO formation. De novo arginine synthesis averaged 9.2 +/- 1.4 mumol. kg-1.hr-1, indicating that approximately 11% of the plasma arginine flux originates via conversion of plasma citrulline to arginine. Thus, the fraction of the plasma arginine flux associated with NO and also urea synthesis in healthy humans is small, although the plasma arginine compartment serves as a significant precursor pool (54%) for whole body NO formation. This tracer model should be useful for exploring these metabolic relationships in vivo, under specific pathophysiologic states where the L-arginine-NO pathway might be altered.

Diurnal pattern of the interrelationships among leucine oxidation, urea production, and hydrolysis in humans.

We investigated in six healthy adult men, who received an adequate intake of protein (1 g.kg-1.day-1), the relationship among urea production, excretion, and hydrolysis. At the end of a 6-day diet-adjustment period, subjects were studied using a 24-h continuous intravenous [1-13C]leucine and [15N,15N]urea tracer protocol (A. E. El-Khoury, N. K. Fukagawa, M. Sánchez, R. H. Tsay, R. E. Gleason, T. E. Chapman, and V. R. Young. Am. J. Clin. Nutr. 59: 1000-1011, 1994) to determine rates of irreversible protein nitrogen loss and urea kinetics. By combining leucine and urea kinetic data, we found a significant degree of urea hydrolysis over the 24-h period but no evidence to support the thesis that there is a net retention or "salvage" of the urea nitrogen liberated. Our measurements revealed little or no urea hydrolysis during the fed 12-h period of the 24-h tracer protocol but substantial hydrolysis during the 12-h fasting phase. Furthermore, a mass balance model and calculations (APPENDIX) indicated that nitrogen salvage, if any, is quantitatively indistinguishable from insensible nitrogen losses and aggregate estimation errors, accounting for no more than 5% of the nitrogen intake. We conclude that urea hydrolysis, via the intestinal microflora, although representing a component of the overall cycles of nitrogen flow within the body, does not contribute via a net retention of amino nitrogen to the maintenance of body nitrogen homeostasis in healthy adults consuming an adequate diet.

Arginine: new and exciting developments for an "old" amino acid.

Arginine (2-amino-5-guanidino pentanoic acid) was shown in 1895 by Hedin to be present in the proteins of horn. Metabolic nitrogen balance studies, conducted in 1957 by Rose in human adults and in 1959 by Snyderman and coworkers in young infants revealed that a dietary source of this amino acid was not an obligatory requirement for growth and maintenance of nitrogen homeostasis in healthy individuals. Hence, it was initially classified as a non-essential (dispensable) amino acid and, perhaps, for reasons of this classification arginine did not receive the earlier attention it now deserves, in relation to an understanding of the nutritional biochemistry and physiology of its metabolism in humans subjects. However, there is currently a considerable interest in the cellular and tissue functions, as well as clinical, therapeutic significance, of arginine. In this paper we review the multiple functions of arginine, including its role in the L-arginine-nitric oxide pathway, cellular regeneration, immune function, protein synthesis and protein breakdown. We then consider some in vivo aspects of the physiology of arginine metabolism, which varies greatly among eukaryotes, with particular reference to humans. Against this background, studies of arginine in the nutrition of humans under various pathophysiological conditions are reviewed briefly. Finally, a new, updated concept for the metabolic basis for the "conditional essentiality" of arginine is proposed.

Plasma methionine and cysteine kinetics in response to an intravenous glutathione infusion in adult humans.

Glutathione (GSH), a tripeptide (gamma-glutamyl-cysteinyl-glycine), is thought to be both a storage and a transport form of cysteine (Cys). In a previous study (T. Hiramatsu, N.K. Fukagawa, J.S. Marchini, J. Cortiella, Y.-M. Yu, T.E. Chapman, and V.R. Young. Am. J. Clin. Nutr. 60: 525-533, 1994), the direct tracer-derived estimate of Cys flux was considerably higher than that predicted from estimates of protein turnover. To further examine the components of plasma Cys flux, seven normal-weight healthy adult men and women (26 +/- 2 yr) received stable isotope tracer infusions of L-[methyl-2H3;1-13C]methionine, L-[3,3-2H2]cysteine, and L-[methyl-2H3]leucine for 460 min. After a 3-h baseline period, GSH was administered at approximately 32 mumol.kg-1.h-1 until the end of the study. Expired breath and blood samples were obtained at timed intervals and analyzed for isotope enrichment using mass spectrometry. Leucine, alpha-ketoisocaproate, and methionine (carboxyl carbon, methyl moiety, remethylation, and transsulfuration) turnover were reduced during GSH administration (P < 0.01). In the final hour of GSH administration, Cys flux increased by 61% from 55.1 +/- 1.7 to 88.7 +/- 5.2 mumol.kg-1.h-1 (P < 0.01), which was essentially equivalent to the rate of exogenous GSH infusion. These data suggest that GSH breakdown accounts for approximately 50% of tracer-derived Cys flux basally and for all of the increase in measured Cys turnover during exogenous GSH infusion.

Urea cycle intermediate kinetics and nitrate excretion at normal and "therapeutic" intakes of arginine in humans.

We investigated the effects of a high dietary supplement of arginine on plasma arginine, ornithine, and leucine kinetics and on urea production and excretion in five healthy young adult men. Subjects received either 56 or 561 mg arginine.kg-1.day-1 for 6 days via a complete L-amino acid diet, and on day 7 a tracer protocol (first 3 h fasted; next 5 h fed) was conducted, involving primed constant intragastric infusions of L-[15N2-guanidino,5,5-2H2]arginine, L-[5-13C]ornithine, L-[5,5,5-2H3]leucine, and [15N2]urea, with a prime of H13CO3. Plasma arginine and ornithine fluxes increased significantly (P < 0.05) with arginine supplementation, as did the rate of conversion of plasma labeled arginine to ornithine (P < 0.05) and rate of ornithine oxidation (P < 0.001). However, absolute changes in ornithine kinetics were less than those for arginine or those based on changes expected from the change in arginine intake, implying a complex compartmentation in both whole body arginine and ornithine metabolism. The plasma NO3 concentration, daily output of total NO3, and conversion of [15N]arginine to NO3 did not differ between the diets. Urea production and excretion were reduced significantly with arginine supplementation, suggesting an anabolic effect on the whole body nitrogen economy, possibly via the raised plasma insulin levels (P = 0.013) during the prandial phase.