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The Journal of Nutrition

7th Amino Acid Assessment Workshop

The 7th Workshop on the Assessment of

Adequate Intake of Dietary Amino Acids:

Summary of General Discussion

1,2

Takeshi Kimura,3* Andrew G. Renwick,4 Motoni Kadowaki,5 and Luc A. Cynober6

3Ajinomoto Co., Inc., Quality Assurance and External Scientific Affairs Department, 104-8315 Tokyo, Japan; 4School of Medicine,

University of Southampton, SO16 7PX Southampton, UK; 5Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata

University, 950-2181 Niigata, Japan; and 6Clinical Chemistry Laboratory, Hotel-Dieu Hospital/Laboratory of Biological Nutrition,

Paris Descartes University, 75270 Paris, France

Abstract

Extensive discussion sessions were held at the end of each of the 2 d of the workshop. Through the course of the

workshop, it became clear that there were different opinions on how to use uncertainty factors to obtain upper levels of

intake from no observed adverse effect levels of a particular nutrient and that the selection of an appropriate uncertaintyfactor would be rather arbitrary. Much of the discussion centered around the potential for using metabolic limits,

expressed as the level of intake at which the major pathway of metabolism may approach saturation and at which the

amino acid is metabolized by alternative pathways, as a measurable early or surrogate marker for amino acid excess and

possible toxicity. After extensivediscussion on various conditions that would need to be satisfied for metabolic limits to be

used as markers of excessive intake of amino acids, there was a general consensus that methods such as measuring

oxidation limits are an attractive approach that merit future investigation. It was noted that there are many data on the

clinical use of glutamine, whereas data for proline are very scarce. There was recognition that regardless of the available

data, there is regulatory pressure for setting upper levels of intake for amino acids and that much more data are

required. J. Nutr. 138: 2050S2052S, 2008.

Introduction

The first day took the form of an open symposium on upperlevels of intake for nutrients organized jointly by the Interna-tional Council on Amino Acid Science and the International LifeSciences Institute Japan, and the second day was a closedconference centered on the available data for glutamine andproline. The discussions at the end of each day focused mainlyon the possibilities and difficulties in setting upper levels ofintake for individual amino acids, because many do not havewell-established toxicity and fewer clinical data are availablecompared with vitamins or minerals. Furthermore, it wasrecognized that even for vitamins and minerals for which there

are enough clinical data to obtain no observed adverse effectlevels (NOAEL) in humans, there is no international consensuson what uncertainty factors to apply in setting upper levels ofintake. Consequently, there was extensive discussion of thepotential for using measurable parameters as a way to obtainupper levels of intake. The discussions from both days aresummarized in this report.

Issues in determining upper levels of nutrient intake

Information was presented on the movement in various coun-tries and international bodies toward requiring the identificationof upper levels of intake for most nutrients, generally coupled

with the setting of dietary reference intakes. The currentmethodology for setting upper levels of intake for nutrients isbased on a risk assessment model with hazard identification,hazard characterization, dietary intake assessment, and riskcharacterization as components (1). Examples of setting upperlevels of intake for vitamins and minerals were presentedthrough the course of the workshop (25) and it became clearthat although the same data had been used for risk assessment byvarious groups, there were differences in approaches to selectinguncertainty factors to convert the NOAEL into upper levels ofintake. With respect to amino acids, previous workshops haveshown that an extensive database was needed to undertake riskassessments for higher intakes and, consequently, there was

interest in whether there are ways in which data could be

1 Published in a supplement to The Journal of Nutrition. Presented at the

conference The Seventh Workshop on the Assessment of Adequate and Safe

Intake of Dietary Amino Acids held November 23, 2007 in Tokyo. The

conference was sponsored by the International Council on Amino Acid Science

(ICAAS). The organizing committee for the workshop was David H. Baker,

Dennis M. Bier, Luc A. Cynober, Yuzo Hayashi, Motoni Kadowaki, Sidney M.

Morris Jr, and Andrew G. Renwick. The supplement coordinators were David H.

Baker, Dennis M. Bier, Luc A. Cynober, Motoni Kadowaki, Sidney M. Morris Jr,

and Andrew G. Renwick. Supplement coordinator disclosures: all of the

coordinators received travel support from ICAAS to attend the workshop.2 Authordisclosures: T. Kimura, is ICAAS CEO; A. G. Renwick,M. Kadowaki,and

L. A. Cynober, received travel support from ICAAS to attend the workshop.

* To whom correspondence should be addressed. E-mail: takeshi_kimura@

ajinomoto.com.

2050S 0022-3166/08 $8.00 2008 American Society for Nutrition.

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obtained that have direct relevance to setting upper levels ofintake.

Metabolic levels of intake and measurable markers

The issue of whether metabolic limits, such as a dose-dependentinflection point in the generation of CO2 from a stable-isotope-labeled amino acid (2,3) or in the urinary excretion of vitamins(4), could serve as biological markers for upper levels of intakefor these nutrients was discussed at length. It was proposed thata metabolic limit could be expressed as the level of intake at

which the major pathway(s) of metabolism approach saturationand at which the amino acid is metabolized by alternativepathways. The basic concept is that in general, the nutrientsthemselves are not toxic per se, as observed in a number of casesof inborn errors of amino acid metabolism where extremely highamino acid plasma concentrations were not associated withadverse effects (6), but that certain metabolites in the mainpathways or metabolites originating from alternate pathwaysmay have had higher toxicity. There was agreement that for eachamino acid, its major routes of metabolism and also anymetabolic pathways thought to be linked to adverse effects needto be considered in defining a metabolic upper level of intake. Itwas pointed out that it would not be possible to develop a

common metabolic limit for all amino acids, because theircatabolic pathways are very different. For example, the toxicitiesseen for methionine excess stem from the specific pathwaysassociated with methionine catabolism and therefore would notapply to other amino acids. It was noted that although toxicitiescan be shown at very high doses in animals, it is unethical toperform experiments in humans that result in adverse effects;consequently, many of the data regarding amino acid toxicity inhumans have come from inadvertent dosing. It was noted that ametabolic limit would be a surrogate or early marker forpossible toxicity at higher intakes and could be investigatedethically in humans. It was suggested that precautionary upperlevels of intake could be defined if a metabolic limit were viewed

as a predictor of adverse metabolic effects and treated as if itwere the lowest observed adverse effect level. To use metaboliclimits as surrogates for upper levels of intake, it was noted that itwill be necessary to show that the metabolic limits are reachedbefore any toxicological signs are manifested; it was pointed outthat such examples exist in the pharmaceutical area (7).

There was extensive discussion on how metabolic limits couldbe measured in humans. It was pointed out that it would bedifficult to undertake experiments with methionine in humansbecause of the likelihood of well-known toxicity being expressedat high doses. It was suggested that oxidation may notbe theonlymarker for metabolic limits and that in the case of methionine,metabolites such as S-adenosyl methionine could be a marker. Itwas noted that animal data on the correlations between toxicity

and any postulated metabolic limits are necessary before pro-ceeding to human studies. Questions were raised regarding thesimilarities of amino acid metabolism between species and it wasnoted that, apart from the metabolism of certain amino acidisomers, metabolism across species is generally similar, althoughonly primates and humans use glutamine for the conjugation oforganic acids for detoxification and excretion (8). However, animportant difference between humans and other animals inamino acid utilization is the fact that humans grow at muchslower rates than most other animals.

Upper levels of intake for health and disease

The issue of the differences in metabolism between health and

disease and the impact on upper levels of intake was discussed. It

was noted that there are examples of disease states where thereare clear differences in the metabolism of amino acids; forexample, it was reported that the requirement for branched-chain amino acids increases by 80% in children with liverdisease. Based on the influence of glutamine deficiency onstimulation of AMP-activated protein kinase activity (9) heat-shock proteins (10), and glutathione metabolism (11), it hasbeen hypothesized that only patients having lower plasmaglutamine levels may benefit from glutamine supplementation(12,13). This is in accordance with a study showing that

glutamine depletion is an independent factor in addition toacute physiology and chronic health evaluation II score forthe prediction of mortality in intensive care unit patients atadmission (14). There are also differences in the effects of short-term and long-term supplementation. Although there werequestions on the relevance of the application of upper levels ofintake for healthy individuals to diseased patients, there wasmuch support for the view that differences in susceptibilityshould be treated as a continuum with values for the healthyindividual serving as reference points. It was also noted that theconcept of measuring metabolic limits could be applied todiseased patients as well. On the other hand, upper levels ofintake are usually set for the healthy general population and the

inclusion of subjects with disease in the same data set as healthysubjects would increase the variability too much, so that anyuncertainty factor used to set the upper level of intake would beunnecessarily excessive for the general population. The need toconsider whether the amino acid was taken with food or withoutfood was also pointed out. People taking tryptophan supple-ments because they think it helps them sleep through serotoninsynthesis and people taking lysine for herpes-induced cold soreswere given as examples of when amino acid supplements weretaken on an empty stomach. The issue of whether metaboliclimits may need to be exceeded for information on certainclinical effects to be gained, such as the evaluation of pituitaryfunction by acute infusion of a high dose of arginine, was also

discussed. It was noted that it would be possible to measure thevarious metabolic pathways for arginine to see if any proposedmetabolic limit would need to be exceeded to gain beneficialeffects; for example, there was work on the balance betweennitric oxide synthesis and arginase in lung tissues of mice withcystic fibrosis or other lung diseases that could provide usefulinsights (15,16).

Relevance of animal data

The issue of extrapolation of animal data to humans was dis-cussed. It was noted that in the current process of upper level ofintake evaluations, animal data are considered only when there isdifficulty in hazard identification or dose-response assessmentusing the available human data (17). There were differences of

opinion on the relevance of animal data to human upper level ofintake determination. Whereas some had the opinion that animalexperiments do not predict the efficacy of amino acids and thatthe extrapolation from animals to humans may be limited toadverse effects, others expressed that amino acid requirements forhuman neonates could be estimated from amino acid require-ments in pigs by using the total daily protein intake data as areference. It was reported that this has been successfully applied tomethionine and threonine requirements and that the requirementsforthese amino acids areproportional to the protein intake.It wassuggested that the same principle might also apply to setting uppermetabolic limits. However, it was pointed out that for glutamine,there are vast species differences in muscle glutamine concentra-

tions (18) and doubts were raised as to the validity of a simple

Adequate intake of dietary amino acids 2051S

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model based on percentages of protein intake being applicable toall amino acids. On the other hand, others pointed out thatalthough the basal levels may be different, various species showsimilar percentage reductions in glutamine levels after burninjuries, so that differences in basal levels do not necessarily reflectdifferent metabolic requirements (19).

Glutamine and proline

There was much discussion regarding glutamine and it wasnoted that in the clinical setting, at least 40 g/d has been given to

patients whose glutamine clearances are slower than those ofhealthy individuals (20,21). These patients showed no adverseeffects after glutamine dosing and so it was suggested that thesedata could be used for setting upper levels of intake for healthyindividuals. Others noted that it was difficult to see any specifictoxicities associated with excess glutamine in animal experi-ments. This made it difficult to undertake hazard identification,a problem for risk analysis, and it was suggested that wheretoxicity could not be observed at any dose, the highest observedintake with appropriate data could be set as the upper level onintake. There was some discussion on the differences betweenglutamine and glutamate. It was noted that there are reports thatfeeding 147 g/d monosodium glutamate was without any

adverse effects (22). It was noted that data on proline are sparsecompared with most amino acids. There was a discussion on thedifference in metabolism between neonates and adults withregard to arginine synthesis, with proline being the only precur-sor for arginine in the neonate, but glutamate and glutaminebeing used as substrates in adults (23). It was noted that inneonates, only one-half the required arginine could be producedfrom proline (2426).

The discussion ended with the general consensus that regard-less of the available data, there is regulatory pressure for settingupper levels of intake for amino acids and that much more dataare required. The following points were generally recognized: 1)there are differences in opinions on what uncertainty factors toapply to NOAEL to obtain upper levels of intake; 2) the use ofmetaboliclimits as an early marker or surrogatemarker for upperlevels of intake merits investigation; 3) measuring oxidationlimits is an attractive approach for obtaining metabolic limits foramino acids; and 4) there is a considerable deficiency of data forcertain amino acids, such as proline, and much more data areneeded in general for considering upper levels of intake.

Other articles in this supplement include references (2732).

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13. Berg A, Rooyackers O, Norberg A, Wernerman J. Elimination kineticsof L-alanyl-L-glutamine in ICU patients. Amino Acids. 2005;29:2218.

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19. Stinnett JD, Alexander JW, Watanabe C, MacMillan BG, Fischer JE,Morris MJ, Trocki O, Miskell P, Edwards L, et al. Plasma and skeletalmuscle amino acids following severe burn injury in patients andexperimental animals. Ann Surg. 1982;195:7589.

20. Tjader I, Rooyackers O, Forsberg AM, Vesali RF, Garlick PJ, WernermanJ. Effects on skeletal muscle of intravenous glutamine supplementationin ICU patients. Intensive Care Med. 2004;30:26675.

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22. Bazzano G, DElia JA, Olson E. Monosodium glutamate: feeding oflarge amounts in man and gerbil. Science. 1970;169:12089.

23. Bertolo RF, Burrin DG. Comparative aspects of tissue glutamine andproline metabolism. J Nutr. 2008;138:20329.

24. Brunton JA, Bertolo RF, Pencharz PB, Ball RO. Proline amelioratesarginine deficiency during enteral but not parenteral feeding in neonatalpiglets. Am J Physiol. 1999;277:E22333.

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30. Barbul A. Proline precursors to sustain mammalian collagen synthesis.J Nutr. 2008;138:20214.

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