2nd Amino Acid Workshop Introduction to the 2nd Amino Acid Assessment Workshop1 Vernon R. Young2 Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA 02139 ABSTRACT 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. J. Nutr. 133: 2015S–2020S, 2003. KEY WORDS:  amino acids  excess  risks  safety  metabolism  mechanism

The proportions of amino acids in diets consumed by human populations usually differ from the proportions in which they are required to efficiently support growth and maintenance, although adverse effects due to such differences are not common. However, insufficient or excessive amounts of individual amino acids included in, or added to, diets result in adverse effects (1–3), as clearly demonstrated in experimental animals (2). A particular problem is to assess the human relevance and applicability of these experimental data, especially in view of the sparse and disordered information that is available from human studies, in part because it is possible that the effects of specific amino acid intake levels may differ as a function of species as well as the anatomical location analyzed. A second challenge is to outline an effective way to close this large gap in current knowledge about the metabolic and functional consequences of abnormal or unusual, in particular high, intakes in human subjects. Thus, a series of workshops has been planned and initiated under the auspices of the International Council on Amino Acid Science (ICAAS; [email protected]), with its secretariat in Tokyo, Japan, to address these major issues. This second amino acid assessment workshop (2nd AAAW) will build on the deliberations of the 1st AAAW, held in Tokyo, June 2001. The objective of the latter was to review comprehensively the roles of dietary amino acids in cellular and organ function, as well as

the consequences associated with abnormal or unusual, in particular high, intakes in human subjects. The emphasis of the discussions was intended to be, as far as possible, on mechanisms and quantitative aspects, with the goal of eventually developing a working framework for assessment of the consequences of abnormal amino acid intakes. In addition, an aim was to begin to develop the details of a research program that would generate a sufficient knowledge base for purposes of making sound and effective recommendations and policies concerning amino acid intakes by human subjects. In this introductory paper I will first review, in summary form, the major topics covered and issues arising at the 1st AAAW, because the proceedings were not published. I will outline the proposed preliminary strategy that emerged at a final workshop session as a possible basis for identifying the type of information required and the basic and applied research needed to generate this information. This will serve to introduce the major objectives of the present workshop. These, in part, are to consider how the explosion of knowledge in biology, before and during this postgenome sequencing era and the associated technology, could help to fully identify the mechanisms involved and better predict the metabolic responses and their functional sequelae to widely altered intakes of amino acid and their safety in humans.

A rationale for an AAAW series

1 Presented at the conference ‘‘The Second Workshop on the Assessment of Adequate Intake of Dietary Amino Acids’’ held October 31–November 1, 2002, in Honolulu, Hawaii. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Vernon R. Young, Yuzo Hayashi, Luc Cynober and Motoni Kadowaki. Conference proceedings were published in a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Dennis M. Bier, Luc Cynober, Yuzo Hayashi and Motoni Kadowaki. 2 To whom correspondence should be addressed. E-mail: [email protected].

In the broader sense, the 1st AAAW and its successors have the overall purpose to establish a continuing scientific dialog among experts in amino acid nutrition, metabolism, cell and molecular biology, toxicology and regulation/policy. The eventual goal is to construct a scientific framework that would be used for making a precise prediction of the consequence(s)

0022-3166/03 $3.00 Ó 2003 American Society for Nutritional Sciences.

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of a particular amino acid intake(s) in people under various conditions. This purpose is made necessary because there is no formal, established risk assessment paradigm for intakes of amino acids that are in significant excess of physiological requirements. In contrast, there are established procedures for nonnutrients based on hazard identification, hazard characterization, exposure assessment and risk characterization (4–8). This approach involves a dose response assessment to define levels of intake without appreciable adverse effects. However, the necessary information from systematic research is not available for (essentially) all micro- and macronutrients, especially in humans. It is also quite clear from the recent Food and Nutrition Board/Institute of Medicine (FNB/IOM) initiative, to set new dietary reference intakes, especially with regard to upper levels (9), that a similar application of the approach used for nonnutrients would be unworkable for nutrients, including amino acids. Therefore, the broad objective is to assemble, during the course of a continuing AAAW series, the pieces of the scientific/biological jigsaw puzzle that would help lead to the establishment of a suitable model for application with amino acids and other nutrients; with the appropriate consultation and deliberation, the model might then be applied internationally for assessing safe intakes of amino acids by specific populations. The 1st AAAW Amino acids/mixtures are used in clinical nutrition and for health promotion. Hence, in turn, to promote their safe and effective application, it is necessary to establish a sound scientific basis for evaluating their efficacy and ‘‘safety’’. In this context a series of questions immediately emerge: i) what are their roles, i.e., what is the nature of their metabolism and function(s); ii) what are the adverse effects of excessive intakes and what mechanisms are involved; iii) how might these be predicted or anticipated; iv) what is the effect of genetic and other factors on the response to amino acid intakes; and v) what are the critical research issues? With these kinds of questions in mind, the 1st AAAW was organized and held in Tokyo, June 2001. It included experts from various areas of amino acid biology, and the objective and purpose was i) to review the roles and metabolism of dietary amino acids in relation to cell and organ function, and ii) to identify the possible consequences associated with abnormal or unusual, in particular high, intakes in human subjects. The focus of the discussions was intended to be, as far as possible, on mechanisms and quantification. It was planned that a working framework for assessment of the consequences of abnormal amino acid intakes might be developed, together with a number of the details of a research program required to generate a sufficient knowledge base for purposes of making sound and effective recommendations and policies. The program was arranged with due regard for the context of dietary amino acid adequacy or excess in humans. Specifically it was recognized that i) amino acids are physiologically significant components/derivatives of food proteins; ii) their metabolism is closely interlinked; iii) dietary factors modify the response to specific amino acid intakes; iv) host characteristics also determine responses and these need to be specified and understood; and v) there are adaptations/accommodations to intakes of amino acids depending on the time frame under consideration. The 1st AAAW consisted of four sessions, lasting a total of one and a half days. Session I dealt with amino acid function,

interorgan metabolism requirements and plasma levels. Session II was devoted to inborn errors, excessive intakes of single amino acids, high intakes and the nature of the adverse effects. In Session III emphasis was given to the issues of safety assessment, concepts and the framework for risk assessment. Session IV attempted to bring together the major ideas emerging from Sessions I–III to begin to outline a proposed framework for evaluating amino acid adequacy and safety. It was emphasized during the course of these sessions that i) amino acids serve many different and many multiple functions (Table 1), and ii) their metabolism is highly interconnected. Many examples of this latter characteristic of these nutrient molecules might be given. However, for illustrative purposes the recent observations made by Chen et al. (11) are instructive. Thus, it was shown that arginine supplementation failed to reduce the atherosclerotic burden in apoE(2/2)mice. In fact it obliterated the protective effect of iNOS deficiency in iNOS(2/2)/ApoE(2/2) mice. A possible explanation for these findings has been offered in a thoughtful editorial by Loscalzo (12). He proposes that the arginine supplementation created a methylation demand or stress, associated with a stimulation of creatine synthesis and, in the process, an increase in the rate of L-homocysteine generation with raised plasma homocysteine concentrations (Fig. 1). In this context, it has also been shown that creatine supplementation reduces L-arginine:glycine amidinotransferase expression, attenuating methylation stress and reducing plasma homocysteine in rats (13). This is a contemporary example of amino acid interrelationships which in this case concerns the effect of a generous intake of a specific amino acid (arginine) on the function of a specific system, the atherothrombotic vasculature, that might be due possibly to another amino acid, homocysteine. Clearly such intimate metabolic connections that occur among amino acids must be considered in any comprehensive assessment of amino acid adequacy and safety. Furthermore, the individual amino acids are involved in the functioning of various physiological and anatomic systems, through their participation in multiple metabolic/cellular processes as has been summarized by Reeds (14) (Table 2). These systems might serve as at least an initial focus of inquiry into the consequences of altered and excess intakes of specific amino acids or mixtures of amino acids. The flow of amino acids within and among organs, the differing metabolic activities within organs and their intakes TABLE 1 Some functions of amino acids1 Function Substrates for protein synthesis Regulators of protein turnover Regulators of enzyme activity

Precursor of signal transducer Neurotransmitter Ion fluxes Precursor of N compounds Transporter of N Translational regulator Transcriptional regulator 1

From Young et al (10).

Example Those for which there is a codon Leucine, glutamine Arginine and N-acetyl glutamate synthesis Phe and phenylalanine dehydroxylase activation Arginine and nitric oxide Tryptophan, glutamate Taurine, glutamate, oxoproline Nucleic acid, creatine Glutamine, alanine Leucine [4E-BP1 and P70(s6k) via MTOR-dependent pathway] Leucine limitation induces CHOP expression

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FIGURE 1 Principal catabolic fates of L-arginine. Dashed arrows indicate inhibition of enzyme activity. Abbreviations used: NO, nitric oxide; NOSs, nitric oxide synthases; DDAH, dimethylargine dimethylamino hydrolase; SAH hydrolase, S-adenosylhomocysteine hydrolase. Taken from Loscalzo (12), with permission.

FIGURE 2 Overview of the fate of amino acids. A schematic outline of the regulatory system, including a series of feedback loops, responsible for determining plasma amino acid concentrations. From Harper (1), with permission.

from dietary sources determines, in part, the concentration and pattern of amino acids in the circulation. The involvement of these various processes and other factors in determining the plasma amino acid profile at any moment in time is depicted in Figure 2 (1). Because of the relatively easy access to the plasma compartment in human subjects it was the view of the participants of the 1st AAAW that a more in-depth investigation of the nature of the regulation of plasma amino acids concentrations and the resulting profiles would be valuable as a partial basis for developing suitable biomarkers

of amino acid adequacy and excess. This has also been pointed out by Cynober (15) in his recent review of plasma amino acid levels, based on a paper that he presented at the 1st AAAW. In the second session of the 1st AAAW, particular attention was given to the effects of excessive intakes of single amino acids, a condition that might occur with ready access to amino acid supplements and sources of individual amino acids at retail outlets. In the context of animal studies and with reference to the extrapolation of observations in nonhuman models to human concerns it is also relevant to note, as shown in a series of studies in rats by Benevenga and Harper (16), that there is a metabolic adaptation to high intakes that takes time to be fully expressed. This point illustrated, for example, in Figure 3, which shows that addition of high methionine to a casein-based diet in growing rats results in immediate loss of body weight, whereas the addition of glycine to the high methionine-casein diet prevented the weight loss and with the continued intake of glycine there was eventually some additional growth response. However, when glycine was added to the diet only after rats had received the high methionine-casein for two weeks an immediate effect on improving growth was observed with glycine supplementation. Hence, the time dimension is of importance in the evaluation and prediction of the consequences of a given amino acid intake in rodents. This phenomenon needs to be assessed for its significance in relation to the assessment of human amino acid intakes. Similarly, as summarized in Table 3, the level of dietary protein can modulate the effects of the addition of a high level of an amino acid (for example, tyrosine) to a diet, with the effect being of greater consequence when a diet is relatively low or marginal in protein content (17). Thus, the impact of dietary factors precludes an easy or simple predication of the consequences of a given intake of a single amino acid. Again, this must be recognized in any framework that is developed to evaluate the adequacy of and tolerance to specific levels of amino acid intake in human subjects. The concepts and framework for safety assessment were discussed in the third session of the 1st AAAW. These included brief consideration of three components required for regulatory decisions; hazard identification, risk characterization and risk reduction. With reference to nutrients, and to amino acids in particular, somewhat different approaches are needed in the assessment of amino acid adequacy and safety in comparison to those followed to evaluate xenobiotics. As reviewed in Tokyo

TABLE 2 Involvement of amino acids in physiological and metabolic function1 System Intestine

Skeletal muscle Nervous system

Immune system

Cardiovascular

1

Function Energy generation Proliferation

Product

Glu, As, Glutamine Nucleic acids Glutamine, Gly, Asp Protection Glutathione Cys, Glu, Gly Nitric oxide Arg Mucins Thr, Cys, Ser, Pro Energy generation Creatine Gly, Arg, Met Peroxidative Taurine (?) Cys protection Transmitter Adrenergic Phe synthesis Serotinergic Try Glutaminergic Glu Glycinergic Gly Nitric oxide Arg Peroxidative Taurine (?) Cys protection Lymphoctye (?) Glutamine, Arg, proliferation Asp Peroxidative Glutathione Cys, Glu, Gly protection Blood pressure Nitric oxide Arg regulation Perioxidative Red cell Cys, Glu, Gly protection(?) Glutathione

From Reeds (14).

ATP

Precursor

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FIGURE 3 Adaptation to a high methionine diet. Effect of a methionine addition to a casein diet and the alleviating effect of supplemental glycine on body weight changes in rats. From Benevenga & Harper (16), with permission.

by Andrew Renwick and summarized in Table 4, there are important metabolic differences between micronutrients or, for that matter, macronutrients such as amino acids and xenobiotics. These differences dictate the need for development of new specific approaches for assessing nutrient intake levels that exceed those necessary to meet their physiological functions and that are outside normal short-term variations in intake. From these three sessions, five issues emerged and a number of research suggestions were made. These included i) the desirability of exploring in much greater detail the utility of plasma amino acid levels as a means of identifying the status of amino acid metabolism. Here it was recognized that the pharmacokinetic characteristics of plasma amino acids needed examination, with analysis of responses to altered amino acid intakes in rodents and suitable parallel studies to assess

TABLE 3 Effect of content of dietary protein on response to an excess of tyrosine1 Tyrosine concentration Dietary protein (%) 6 6 1 5% tyrosine 24 24 1 5% tyrosine 1

Weight Oxidation of a gain Plasma Muscle tyrosine load (g/wk) Mortality (mmol/100ml) (mmol/g) via CO2 (%) 21 ÿ4

0/5 3/5

10.6 228.0

0.41 4.83

57 66

56 53

0/5 0/5

17.5 68.0

0.46 1.07

81 87

Combined from data of Ip and Harper (17).

equivalence in humans; ii) the proposal to further explore use of experimental animal models to identify cellular/organ responses that go beyond the well-known function of specific amino acids; iii) a recognition that there is a need to establish dose-response relationships for the parent amino acid and/or the metabolites linked with the toxicological endpoints; and iv) the value of defining the mechanism responsible for the adverse effect. This requires identification of the active compound, whether it is the parent amino acid or a metabolite. An interesting scientific example, in this context, was recently presented in a case report of an adverse event associated with a methionine-loading test in which a substantial overdose of methionine was given, possibly as much as 96 g (18). Additionally, determination of the initial molecular or cellular targets and process involved, perhaps facilitated by the use of trangensic models, will need to be defined and identified. An example of the power of genetic techniques/models is the recent demonstration (19) that b-cell development was impaired in F/A-21/1 transgenic mice over-expressing the enzyme arginase I under the control of the rat intestinal fatty acid binding promoter and enhancer element (20). In this model, circulating and tissue arginine concentrations are reduced to ;35% of controls and the disturbance in b-cell maturation, specifically the transition of progenitor-b to precursor-b cells, disappeared upon arginine supplementation. Finally, it was emphasized that there is a need to establish appropriate default measures and the development of surrogate markers. It was further proposed that a vigorous strategic research effort to carry out comparative studies in experimental animals (rats) and humans, in which plasma metabolites, metabolomics, proteomics and transcriptomics are exploited would lead to important advances in this area and help fill the gap in knowledge concerning metabolism and functional responses to altered amino acid intakes in humans. The 2nd AAAW These various issues noted above provided a partial rationale for the program and series of papers for the present workshop. Here, it is the intent to focus on how relatively recent advances in biology, made before and during this postgenome era (and the associated technology), might help to better understand the mechanisms involved and to improve the ability to predict the responses to altered intakes of amino acids and their safety. Thus, among other things, there is a need to i) determine how to define and identify molecular signatures of a pathological response to amino acid intake; ii) perhaps identify a distinct set of genes that differentiate adequacy from excess for specific amino acids; iii) explore the power of a microarray approach to establish a molecular profile of amino acid adequacy/excess, and iv) decide what organs/tissues on which to focus a major effort. Should it be the brain, the liver and/or the gut? This issue of tissue or organ focus is underscored simply, for example, by the recent observation that excess methionine induced glycine N-methyltransferase activity and abundance in the liver, with the kidney being less responsive and the pancreas being unresponsive (21). Finally, regulation, and in consequence disregulation, can occur at various loci above the genome, including at the level of the proteome, metabolome and the system in which these operate; each should be considered in reference to the issues above. Indeed, it also may now be possible to explore new lines of investigation for this purpose as we move from use of
simple/ single indicators (markers) of adequacy/deficiency/excess to develop and explore new, more comprehensive indices of functional significance: i.e., a more global approach (involving

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TABLE 4 Differences between xenobiotic chemicals and micronutrients1 Property Absorption

Distribution

Elimination

Overall kinetic processes 1

Xenobiotic chemical

Micronutrient

Passive diffusion of un-ionized molecules; limited absorption of polar molecules; few examples where absorption depends on active transport; simple first-order processes. Transported in blood as a simple solution, or reversibly bound to plasma proteins such as albumin and a1-acid glycoprotein or within lipoproteins; tissue uptake is usually by simple passive diffusion; entry to some tissues may be restricted by endothelial:tissue barriers, e.g., the blood:brain barrier; extent of distribution is usually related to the lipophilicity of the molecule. Elimination is by renal excretion for water soluble compounds and by hepatic specific metabolism for lipophilic compounds; the processes show low substrate specificity and are generally first order at the concentrations that would be present in humans.

Usually facilitated or active transport processes; saturable absorption may be used to limit the extent of uptake (zero-order uptake); absorption may be related to physiological need, e.g., calcium and vitamin D. Usually transported in solution or sometimes bound to plasma proteins; potentially toxic micronutrients, e.g., iron, are tightly bound to plasma proteins, the amounts specific of which are controlled by the amount of the micronutrients in the body.

Essentially first-order processes such as passive diffusion and metabolism by enzymes with low substrate specificity.

Elimination is usually via metabolic processes and enzymes, although a number of related compounds may share the same elimination process; renal excretion may involve an active re-uptake transporter in the renal tubule that minimizes the loss of the micronutrient at low concentrations but allows its excretion when the plasma concentration is high enough to result in saturation of the transporter. Frequently nonlinear (zero-order) due to the roles of specific transporters designed to maintain a constant body load.

Kindly prepared by A. Renwick for 1st AAAW, Tokyo, June 2001.

measurement of apoptosis, signaling pathways and membrane activities, for example) in real time with quantitative metabolic kinetics during different states (fasting, prandial, postprandial). As this effort is undertaken the crucial influence of heterogeneity of response should also be recognized. In addition, it can be anticipated that there will be an emergence of new signaling pathways, transporters/receptors, molecular targets, mRNA and protein expression patterns, all of which have ramifications for subpopulation analysis and the application of pharmacogenomics. Basically, there is need to develop reliable functional markers of amino acid adequacy and excess in human subjects. Such markers might include such characteristics as those suggested, for example, by Benzie (22). They should: i) be accessible for measurement; the marker must be present in body fluids or cells that can be sampled or imaged in some way, ii) be in a form and quantity that can be measured objectively and reproducibly; suitable analytical tools and methods must be available, and iii) reflect a change in the target tissue or fluid that has a direct impact on health, i.e., they must relate to a physiological or pathological endpoint for that amino acid. It seems likely that combinations of markers (23), including molecular, biochemical and physiological (22) and those that measure a) exposure, b) response or toxic effect and c) susceptibility (24) will be needed to better and more completely characterize the spectrum of response to intakes of amino acids.

Coda There is much to be learned about the metabolic and functional consequences of specific and especially relatively high intakes of amino acids in humans subjects. Hence, this workshop will build on the ideas and issues identified at the 1st AAAW, as noted above. The aim is to eventually maximize the contribution that exogenous amino acid intake(s) can make to promoting health and attenuating disease processes.

LITERATURE CITED 1. Harper, A. E. (1974) Amino acid excess. In: Nutrients in Processed Foods: Proteins (White, P. L. & Fletcher, D. C., eds.). 49–59. Publishing Sciences Group, Acton, MA. 2. Harper, A. E., Benevenga, N. J. & Wohlhueter, R. M. (1970) Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50: 428–558. 3. Young, V. R. & Fukagawa, N. K. (1988) Amino acid interactions: a selective review. In: Nutrient Interactions (Bodwell, C. E. & Erdman, J. W., Jr., eds.). 27–71. Marcel Dekker, New York. 4. FAO/WHO. (Food and Agriculture Organization of the United Nations/ World Health Organization) (1982) Evaluation of Certain Food Additives and Contaminants. Twenty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no. 683, World Health Organization, Geneva, Switzerland. 5. FAO/WHO. (Food and Agriculture Organization of the United Nations/ World Health Organization) (1995) The Application of Risk Analysis to Food Standard Issues. Recommendations to the Codex Alimentarius Commission (ALINORM 95/9, Appendix 5), World Health Organization, Geneva, Switzerland. 6. NRC. (National Research Council) (1983) Risk Assessment in the Federal Government: Managing the Process. National Academy Press, Washington, DC. 7. NRC. (National Research Council) (1994) Science and Judgment in Risk Assessment. Committee on Risk Assessment of Hazardous Air Pollutants. Board on Environmental Studies and Toxicology. National Academy Press, Washington, DC. 8. WHO. (World Health Organization) (1987) Principles for the Safety Assessment of Food Additives and Contaminants in Food. Environmental Health Criteria 70. World Health Organization, Geneva, Switzerland. 9. FNB/IOM. (Food and Nutrition Board/Institute of Medicine) (1998) Dietary Reference Intakes. A Risk Assessment Model for Establishing Upper Intake Levels for Nutrients. National Academy Press, Washington, DC. 10. Young, V. R., Yu, Y. M. & Borgonha, S. (2000) Proteins, peptides and amino acids in enteral nutrition: overview and some research challenges. In: Proteins, Peptides and Amino Acids in Enteral Nutrition (Furst, «« P. & Young, V. R., eds.). Nestle´ Nutrition Workshop Series Clinical & Performance Program. Vol 3, pp. 1–23, Nestec, Vevey, Switzerland. 11. Chen, J., Kuhlencordt, P., Astern, J. & Huang, P. L. (2003) Effects of chronic treatment with L-arginine on atherosclerosis in apoE knockout and apoE/ iNOS double knockout mice. Arterioscler. Thromb. Vasc. Biol. 23: 97–103. 12. Loscalzo, J. (2003) Adverse effects of supplemental L-arginine in atherosclerosis: consequences of methylation stress in a complex catabolism? Arterioscler. Thromb. Vasc. Biol. 23: 3–5. 13. Stead, L. M., Au, K. P., Jacobs, R. L., Brosnan, M. E. & Brosnan, J. T. (2001) Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate. Am. J. Physiol. Endocrinol. Metab. 281: E1095–E1100. 14. Reeds, P. J. (2000) Dispensable and indispensable amino acids for humans. J. Nutr. 130: 1835S–1840S.

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15. Cynober, L. A. (2002) Plasma amino acid levels with a note on membrane transport: characteristics, regulation and metabolic significance. Nutrition 18: 761–766. 16. Benevenga, N. J. & Harper, A. E. (1967) Alleviation of methionine and homocysteine toxicity in the rat. J. Nutr. 93: 44–52. 17. Ip, C. & Harper, A. E. (1973) Effects of dietary protein content and glucagon administration on tyrosine metabolism and tyrosine toxicity in the rat. J. Nutr. 103: 1594–1607. 18. Cottington, E. M., LaMantia, C., Stabler, S. P., Allen, R. H., Tangerman, A., Wagner, C., Zeisel, S. H. & Mudd, S. H. (2002) Adverse event associated with methionine loading test. A case report. Arterioscler. Thromb. Vasc. Biol. 22: 1046–1050. 19. de-Jonge, W. J., Kwikkers, K. L., te Velde, A. A., Van Deveiter, S. J. H., Nolte, M. A., Mebius, R. E., Ruijter, J. M. & Lamers, M. C. (2002) Arginine deficiency affects early b cell maturation and lymphoid organ development in transgenic mice. J. Clin. Invest. 110: 1539–1548.

20. de Jonge, W. J., Hallemeesch, M. H., Kwikkers, K. L., Ruijter, J. M., Gierde Vries, C., van Roon, M. A., Meijer, A. J., Marescau, B., De Deyn, P. P., Deutz, N. E. P. & Lamers, W. H. (2002) Over-expression of arginase I in enteroctyes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle and lymphoid development. Am. J. Clin. Nutr. 76: 128–140. 21. Rowling, M. J., McMullen, M. H., Chipman, D. C. & Schalinske, K. L. (2002) Hepatic glycine N-methyltransferase is up-regulated by excess dietary methionine in rats. J. Nutr. 132: 2545–2550. 22. Benzie, I. F. F. (1999) Vitamin C: prospective functional markers for defining optimal nutritional status. Proc. Nutr. Soc. 58: 469–476. 23. Lesko, L. J. & Atkinson, A. J., Jr. (2001) Use of biomarkers and surrogate endpoints in drug development and regulatory decision-making: criteria, validation and strategies. Annu. Rev. Pharmacol. Toxicol. 41: 347– 366. 24. Timbrell, J. A. (1988) Biomarkers in toxicology. Toxicology. 129: 1–12.