Burn injury differentially alters whole-blood and organ glutathione synthesis rates: An experimental model.

Previous studies from our laboratories revealed a reduced rate of whole-blood (WB) glutathione (GSH) synthesis in severely burned patients. To determine whether WB GSH metabolism is an indicator of the status of GSH metabolism in one or more of the major organs, we used a burn rabbit model to determine GSH concentrations and rates of synthesis in WB, liver, lungs, kidney, and skeletal muscle. L-[1-(13)C]-cysteine was infused intravenously for 6 h in rabbits at 3 days post-burn and in sham burn controls. WB and organ (13)C-enrichment of cysteine and GSH was determined by gas chromatography/mass spectrometry. Plasma cysteine metabolic flux was increased significantly (P < 0.01) following burn injury. WB, liver, and lung GSH concentrations (P = 0.054, P < 0.05, and P < 0.05, respectively) and fractional rates of GSH synthesis (P < 0.05, P < 0.01, and P < 0.05, respectively) were reduced at 3 days post-burn. Kidney was unaffected. There also appears to be an increased rate of GSH transport out of the liver after burn injury. Hence, there is a differential impact of burn injury on tissue and organ GSH status, with WB qualitatively reflecting the changes in lung and liver. It will be important to determine whether these changes are due to alterations in the intrinsic capacity for GSH synthesis and/or availability of amino acid precursors of GSH.

Adaptation to a long term (4 weeks) arginine- and precursor (glutamate, proline and aspartate)-free diet.

BACKGROUND & AIMS: It is not known whether arginine homeostasis is negatively affected by a "long term" dietary restriction of arginine and its major precursors in healthy adults. To assess the effects of a 4-week arginine- and precursor-free dietary intake on the regulatory mechanisms of arginine homeostasis in healthy subjects. METHODS: Ten healthy adults received a complete amino acid diet for 1 week (control diet) and following a break period, six subjects received a 4-week arginine, proline, glutamate and aspartate-free diet (APF diet). The other four subjects continued for 4 weeks with the complete diet. On days 4 and 7 of the first week and days 25 and 28 of the 4-week period, the subjects received 24-h infusions of arginine, citrulline, leucine and urea tracers. RESULTS: During the 4-week APF, plasma arginine fluxes for the fed state, were significantly reduced. There were no significant differences for citrulline, leucine or urea fluxes. Arginine de novo synthesis was not affected by the APF intake. However, arginine oxidation was significantly decreased. CONCLUSIONS: In healthy adults, homeostasis of arginine under a long term arginine- and precursor-free intake is achieved by decreasing catabolic rates, while de novo arginine synthesis is maintained.

Negative chemical ionization gas chromatography/mass spectrometry to quantify urinary 3-methylhistidine: application to burn injury.

A rapid method for measuring 3-methylhistidine (3MH) in rat and human urine with higher sensitivity and precision than any previously reported method is described using internal standard [1-(13)C]3MH (M+1) and negative chemical ionization (NCI) gas chromatography/mass spectrometry (GC/MS). Internal standard [1-(13)C]3MH (M+1) was added to rat and human urine samples, hydrolyzed, and absorbed onto cation exchange columns. The column eluent was dried and derivatized for GC/MS analysis. Quantification of 3MH levels was accomplished by monitoring the m/z 204 fragment. The m/z 204 fragment was chosen due to the fragment's abundance and stability as determined by analysis of [methyl-(2)H(3), (18)O(2)]3MH (M+7) and [methyl-(13)C]3MH (M+1) fragmentation patterns under NCI conditions. This method shows excellent linearity (0.9989) over the range studied (0-0.5 mol), high recovery (95.9%), and low coefficient of variation (4.7%). The described method is sensitive enough to detect 6.8 pmol amount of urinary 3MH with a precision of 9.1%. The in vivo utility of this method to quantify urinary 3MH was tested in a burn injury rat model and on urine specimens from pediatric burn patients. Data obtained from the urine of burn-injured rats and pediatric burn patients match previously reported trends and validate the in vivo utility of this method.

The daily phenylalanine requirement of healthy Indian adults.

BACKGROUND: The daily requirement for phenylalanine is not known with certainty. Earlier 24-h tracer studies have suggested that the requirement is between 30 and 40 mg . kg(-1) . d(-1). OBJECTIVE: The objective was to assess the phenylalanine requirement in healthy well-nourished Indians with the use of 8 test phenylalanine intakes (19, 23, 27, 31, 35, 38, 43, and 47 mg . kg(-1) . d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and 24-h indicator amino acid balance (24-h IAAB) methods. DESIGN: Thirty-two healthy, well-nourished Indian men were studied during each of 2 randomly assigned 6-d diet periods in which phenylalanine intakes of 19, 23, 27, 31, 35, 38, 43, and 47 mg . kg(-1) . d(-1) were supplied; the diet was devoid of tyrosine. A 24-h [13C]leucine tracer infusion was used to measure 24-h IAAO and 24-h IAAB on day 7. The breakpoint in the relation between these values and the phenylalanine intake was determined. RESULTS: Two-phase linear regression of daily leucine oxidation and balance against phenylalanine intake estimated a breakpoint in the response curve at phenylalanine intakes of 37 and 38 mg . kg(-1) . d(-1) (95% CI for both: 31, >47 mg . kg(-1) . d(-1)), respectively. CONCLUSION: On the basis of the 24-h IAAO and 24-h IAAB methods, a mean phenylalanine requirement of 38 mg . kg(-1) . d(-1) is proposed for healthy well-nourished Indian adults in the absence of tyrosine intake. This finding is similar to that in Western adults.

The daily valine requirement of healthy adult Indians determined by the 24-h indicator amino acid balance approach.

BACKGROUND: The 1985 FAO/WHO/UNU requirement for valine was set at 10 mg x kg(-1) x d(-1) on the basis of nitrogen balance studies carried out in Western subjects. It is likely that the requirement is higher, however, because the requirement of another branched-chain amino acid, leucine, was found to be about 3 times as high (40 mg x kg(-1) x d(-1)) as the 1985 FAO/WHO/UNU value (14 mg x kg(-1) x d(-1)). OBJECTIVE: We assessed the valine requirement in healthy, well-nourished Indians by using 7 test valine intakes (5, 10, 15, 20, 25, 30, and 35 mg x kg(-1) x d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) method, with phenylalanine as the indicator amino acid, while maintaining leucine intake at 40 mg x kg(-1) x d(-1). DESIGN: Eighteen healthy, well-nourished Indian men were studied during each of 3 randomly assigned 7-d diet periods supplying valine intakes that were equally placed on either side of a putative mean valine requirement of 20 mg x kg(-1) x d(-1). Twenty-four-hour IAAO and 24-h IAAB were measured on day 7 by use of a 24-h [13C]phenylalanine tracer infusion. The breakpoint in the relation between these values and the valine intake was determined. RESULTS: Two-phase linear regression of daily phenylalanine oxidation or balance against valine intake estimated a breakpoint in the response curve at a valine intake of 17 mg x kg(-1) x d(-1) (95% Fieller's CI: 11, > 35 and 11, 28 mg x kg(-1) x d(-1), respectively). CONCLUSION: From the 24-h IAAO/IAAB approach, a mean valine requirement of 17 mg x kg(-1) x d(-1) is proposed for healthy, well-nourished Indian adults.

Effect of cystine on the methionine requirement of healthy Indian men determined by using the 24-h indicator amino acid balance approach.

BACKGROUND: The 1985 FAO/WHO/UNU requirement for methionine in healthy adults consuming a cystine-free diet is 13 mg.kg(-1).d(-1). It is unclear whether this daily requirement is influenced by dietary cystine. OBJECTIVE: We assessed the effect of 2 intakes of cystine (5 and 12 mg.kg(-1).d(-1)) on methionine requirements in well-nourished Indian men by using 7 test methionine intakes (3, 6, 9, 13, 18, 21 and 24 mg.kg(-1).d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) methods. We combined these data with those from an experiment with zero cystine intake and in which the exact same method was used. DESIGN: Two studies were performed in which a diet containing either 5 or 12 mg cystine.kg(-1).d(-1) was fed to 21 well-nourished Indian men over three 7-d periods. The 24-h IAAO and 24-h IAAB values were measured on day 7 with the use of a 24-h intravenous [13C]leucine tracer infusion. The breakpoints in the relation between these values and methionine intake in each study were assessed by two-phase linear regression. RESULTS: Breakpoints in the response curve were obtained at methionine intakes of 20 (95% Fiellers CI: 17, 26) and 10 (95% Fiellers CI: 8, 16) mg.kg(-1).d(-1) with cystine intakes of 5 and 12 mg.kg(-1).d(-1) intakes, respectively, which suggested a sparing effect of cystine. Although the 5- and 12-mg cystine breakpoints differed from one another, they did not differ significantly from that estimated previously with 0 mg cystine. CONCLUSION: Cystine may spare the methionine requirement in healthy men, although the amount of sparing is difficult to quantify.

Daily requirement for total sulfur amino acids of chronically undernourished Indian men.

BACKGROUND: Earlier studies of the requirement for total sulfur amino acids (SAAs; methionine in the absence of cystine) in healthy, well-nourished Indians indicated a value of 15 mg.kg(-1).d(-1), but it is unknown whether this estimate is applicable to chronically undernourished subjects. OBJECTIVE: We assessed the total SAA requirement in otherwise clinically healthy, young, chronically undernourished adult Indians by using 7 test methionine intakes (3, 6, 9, 13, 18, 21, and 24 mg.kg(-1).d(-1)), without cystine, and by using both the 24-h indicator amino acid oxidation (24-h IAAO) and the 24-h indicator amino acid balance (24-h IAAB) methods. DESIGN: Twenty-one men were studied during each of 3 randomly assigned 7-d diet periods supplying methionine intakes (diet devoid of cystine) above and below the putative total 1985 FAO/WHO/UNU SAA requirement of 13 mg.kg(-1).d(-1). Twenty-four-hour IAAO and IAAB were measured on day 7 by use of a 24-h [(13)C]leucine tracer infusion. The breakpoint in the relation between these values and methionine intake was determined. RESULTS: Two-phase linear regression of daily leucine oxidation or the daily leucine balance against methionine intake estimated a breakpoint in the response curve at a methionine intake of 16 mg.kg(-1).d(-1) (95% Fiellers CI: 13, 22 mg.kg(-1).d(-1)). CONCLUSIONS: On the basis of the 24-h IAAO-IAAB approach, a mean total SAA requirement of 16 mg.kg(-1).d(-1) is proposed for undernourished Indian adults. This is not significantly different from that determined in Western and Indian well-nourished adults.

Short-term enteral glutamine does not enhance protein accretion in burned children: a stable isotope study.

OBJECTIVE: Glutamine is a nonessential amino acid that, in recent years, has been found to play important roles in several metabolic and immunologic processes. It has been theorized that, in a stressed state, it may become "conditionally essential" because the patient's ability to manufacture glutamine may not be adequate to meet their needs under this condition. We chose to evaluate the ability of 48 hours of enteral glutamine to enhance immediate nitrogen accretion in stressed pediatric burn patients. METHODS: Nine children with serious burns who were tolerating tube feedings were enrolled in a human studies committee-approved protocol in which they received 48 hours of enteral feedings with glutamine replacing 20% of essential and nonessential amino acids and 48 hours of isonitrogenous, isocaloric standard enteral feedings. This interval was chosen to help ensure that the study periods were comparable from a metabolic perspective. At the end of each period, protein kinetics were determined by a primed constant infusion of L-[1-(13)C] leucine tracer. The order of the studies was randomized. Seven children completed both phases of the study. Results were compared by paired t test and are presented as mean +/- standard error of the mean. RESULTS: During the glutamine feeding period, the leucine flux and leucine oxidation rate were significantly lower than those in the conventional feeding period. This reflects a reduction in total leucine intake from 80 +/- 11 to 62 +/- 10 micromol/kg per hour. However, there was no significant difference in the net balance of leucine accretion into proteins between these 2 dietary periods, which indicated that enriched glutamine feeding for 48 hours did not result in an immediate whole body protein gain in this group of pediatric patients. In addition, plasma glutamine concentration showed a moderate increase after 48 hours of supplementation but did not reach significance. CONCLUSION: Rapid protein accretion does not occur with short-term enteral glutamine supplementation. Several days of glutamine supplementation may be required to restore plasma glutamine levels and stimulate protein synthesis.

Introduction to the 3rd Amino Acid Assessment Workshop.

A series of Amino Acid Assessment Workshops (AAAWs) are being organized and conducted to bring together experts in amino acid nutrition, metabolism, cell and molecular biology, toxicology, and regulation/policy with the eventual goal to establish a paradigm for the characterization of risks associated with ingestion of specific intakes of amino acids by humans. In this brief introductory article, I present the rationale behind these AAAWs, which basically emerges from the fact that there is little systematic information about the adverse effects and the pathophysiological mechanisms of excessive intakes of single amino acids or of mixtures of amino acids in human subjects. This 3rd AAAW extends, as well as builds upon, the information collected at the 1st and 2nd AAAWs. The previous two workshops focused attention largely on the metabolism, mechanism of action, and functions of amino acids. This 3rd AAAW will focus particular attention on intakes needed to meet physiological requirements and above, host and diet factors that affect these needs and responses, as well as variation in responses to and levels of intake of amino acids among individuals. In this context, the overall objective is to establish the science and knowledge base required for use in determining and/or predicting the upper level of the safe range of intake of specific amino acids under various host, agent (diet), and environmental conditions.

The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinaemia.

N and S metabolisms are closely interwoven throughout both the plant and animal kingdoms. The essentiality of S relates to its participation in the structure of S-containing amino acids (SAA), to its inclusion in many sulfonated molecules, and to a myriad of metabolic and catalytic reactions of vital importance. Methionine (Met) is the indispensable SAA supplied by food proteins and its plasma homeostasis is achieved via a number of highly efficient regulatory mechanisms. In all conditions characterised by a negative body protein balance such as in dietary restriction or cytokine-induced hypercatabolic losses, N and S endogenous pools manifest parallel tissue depletion rates. Adaptive conservation of N and S body stores is reached by a functional restraint of the trans-sulfuration cascade, through the depression of cystathionine beta-synthase activity. As a result, upstream accumulation of homocysteine favours its re-methylation conversion to Met which helps maintain metabolic pathways of survival value. In addition to the measurement of vitamin indices, that of plasma transthyretin, a sensitive marker of protein nutritional status, is proposed to identify the fluctuations of the total body N component accountable for the alterations of homocysteine concentrations in body fluids.

Intestinal parasites increase the dietary lysine requirement in chronically undernourished Indian men.

BACKGROUND: We previously used the 24-h indicator amino acid balance method to show that the lysine requirement in undernourished Indian men from low socioeconomic and unsanitary environments is approximately 50% higher than the mean requirement of 30 mg lysine. kg(-1). d(-1) in well-nourished men. OBJECTIVE: It is possible that this higher lysine requirement in persons with chronic undernutrition is due to environmental influences, including the presence of intestinal parasites. We assessed this possibility by using 24-h indicator amino acid balance (with leucine) at both the "normal" requirement for lysine intake and the higher requirement, before and after successful treatment to eradicate intestinal parasites in affected, undernourished men. DESIGN: Fourteen chronically undernourished men were studied before and after treatment for intestinal parasites, during each of two 7-d (6-d dietary adaptation plus 1-d tracer experiment) diet periods supplying either 30 (n = 7) or 45 (n = 7) mg lysine. kg(-1). d(-1) from an L-amino acid diet. Twenty-four-hour indicator amino acid balance was estimated on day 6 by [(13)C]leucine tracer infusion. RESULTS: Before the parasite treatment, subjects were in neutral 24-h leucine balance at both lysine intakes. After the eradication of intestinal parasites, there was a significant (P < 0.001) improvement in 24-h leucine balances, which were positive at both lysine intakes. CONCLUSIONS: On the basis of the 24-h indicator amino acid balance approach, it appears that intestinal infestation with parasites increases the requirement for lysine and that this may be one factor responsible for the higher lysine requirement observed in persons with chronic undernutrition.

Metabolic fate of extrahepatic arginine in liver after burn injury.

Increased nitrogen loss in the form of urea is a hallmark of the metabolic aberrations that occur after burn injury. As the immediate precursor for urea production is arginine, we have conducted an investigation on the metabolic fate of arginine in the liver to shed light on the metabolic characteristics of this increased nitrogen loss. Livers from 25% total surface burn (n = 8) and sham burn rats (n = 8) were perfused in a recycling fashion with a medium containing amino acids and stable isotope labeled l-[(15) N(2)-guanidino, 5,5-(2)H(2)]arginine for 120 minutes. The rates of glucose and urea production and oxygen consumption were measured. The rate of unidirectional arginine transport and the intrahepatic metabolic fate of arginine in relation to urea cycle activity were quantified by tracing the disappearance rate of the arginine tracer from and the appearance rate of [(15)N(2)]urea in the perfusion medium. Perfused livers from burned rats showed higher rates of total urea production (mean +/- SE, 4.471 +/- 0.274 v 3.235 +/- 0.261 mumol. g dry liver(-1). min(-1); P <.01). This was accompanied by increased hepatic arginine transport (1.269 +/- 0.263 v 0.365 +/- 0.021 mumol. g dry liver(-1). min(-1)) and an increased portion of urea production from the transported extrahepatic arginine (12.9% +/- 2.9% v 3.5% +/- 0.4%, P <.05). The disposal of arginine via nonurea pathways was also increased (0.702 +/- 0.185 v 0.257 +/- 0.025 mumol/g dry weight(-1)/min(-1); P <.05). We propose that increased inward transport and utilization of extrahepatic arginine by the liver contributes to the accelerated urea production after burn injury and accounts, in part, for its conditional essentiality in the nutritional support of burn patients.

Introduction to the 2nd Amino Acid Assessment Workshop.

The proportions of amino acids in diets typical of human populations usually differ from the proportions in which they are required, although adverse effects due to such differences are not common. However, there is little systematic information about the adverse effects and the pathophysiological mechanisms of excessive intakes of single or mixtures of amino acids in human subjects. To promote the safe and effective application of amino acids in clinical nutrition and for health promotion it is necessary to establish a sound scientific basis for evaluating their efficacy and safety under various conditions of use. Hence, a series of Amino Acid Assessment Workshops (AAAW) are being organized to bring together experts in amino acid nutrition, metabolism, cell and molecular biology, toxicology and regulation/policy with the eventual purpose of establishing a paradigm for the characterization of risks associated with the ingestion of specific intakes of amino acids by humans. In this introductory paper I summarize the major issues arising at the 1st AAAW, held in Tokyo June, 2001, and provide an introductory context to the present, 2nd AAAW.

Trace element biology: the knowledge base and its application for the nutrition of individuals and populations.

Impressive strides are being made in the understanding of trace element metabolism and function. This is underscored by the many contributions in these proceedings. However, not so impressive are: i) the precise recognition of mild trace element deficiencies and how to establish their functional consequences, possibly confounded by concurrent trace element inadequacies, are difficult to assess, ii) approaches to the quantitative determination of requirements for trace elements remain unsatisfactory and archaic, in so many ways, iii) our understanding of the extent of the biological basis for the variation in requirements among apparently similar individuals is poor, and iv) much needs to be learned about the quantitative extent to which genetic, epigenetic and dietary factors interact to determine the nutritional phenotype. Some ideas are presented as to how we might embrace, in the context of a reconstructive approach, the exciting new knowledge and related techniques emerging during the postgenome era and develop new paradigms for assessing trace element needs and status, and for establishing effective nutrient intake under different conditions of complex genotype-environment interactions. Metabolites are functional cellular entities and I also urge a vigorous application of metabolomics and of metabolic profiling that is closely linked with genomics, proteomics, trace element kinetics and system analysis, as components of the new integrative paradigm. We need to understand the system and its strategy, not only the molecular details of its component parts and its individual controls. An interdisciplinary research and teaching enterprise will be necessary to best achieve this aim. All of this is related to our common goal to promote, through expanded biological knowledge and its effective application, the enhanced role of trace elements for human well-being.

Daily methionine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique.

BACKGROUND: The 1985 FAO/WHO/UNU upper requirement for the sulfur-containing amino acids in healthy adults, which was set at 13 mg . kg(-)(1) . d(-)(1), is based on nitrogen balance studies in Western subjects. Short-term tracer-based studies also estimated a mean requirement of 13 mg . kg(-)(1) . d(-)(1), but whether this estimate is applicable to healthy populations worldwide is unknown. OBJECTIVE: Using a 24-h indicator amino acid oxidation and balance method with 7 test methionine intakes (3, 6, 9, 13, 18, 21, and 24 mg . kg(-)(1) . d(-)(1)), we assessed methionine requirements in healthy, well-nourished Indians. DESIGN: Twenty-one healthy, well-nourished Indian men were studied during each of 3 randomly assigned 7-d diet periods in which methionine intakes (diet devoid of cysteine) were equally placed on either side of the putative mean methionine requirement of 13 mg . kg(-)(1) . d(-)(1). Twenty-four-hour indicator amino acid oxidation and balance were measured on day 7 by using a 24-h [(13)C]leucine tracer infusion. The breakpoint in the relation between these values and the methionine intake was determined. RESULTS: Two-phase linear regression of daily leucine oxidation against methionine intake estimated a breakpoint in the response curve at a methionine intake of 14 mg . kg(-)(1) . d(-)(1) (95% CI: 11, 23 mg . kg(-)(1) . d(-)(1)). The breakpoint estimated from the leucine balance-methionine intake relation was 15 mg . kg(-)(1) . d(-)(1) (95% CI: 11, 27 mg . kg(-1) . d(-1)). CONCLUSIONS: From the 24-h indicator amino acid oxidation and balance approach, a mean methionine requirement, in the absence of cysteine intake, of 15 mg . kg(-1) . d(-1) is proposed for healthy, well-nourished Indian adults. This requirement is similar to that established in Western adults.