Download 2nd Amino Acid Workshop

2nd Amino Acid Workshop
Application of the Concepts of Risk Assessment to the Study
of Amino Acid Supplements1
Yuzo Hayashi2
NPO Communication Center for Food and Health Sciences, Tokyo 135-0004, Japan
KEY WORDS: amino acids dietary reference intake (DRI) tolerable upper intake level
(UL) metabolomics
The execution of any given risk assessment may be
hampered by various uncertainties resulting from deficiencies
or critical gaps in the necessary information. When this occurs,
plausible assumptions are made based on prevailing scientific
thought, taking these uncertainties into account, so that the
assessment can be completed (3). Therefore, risk assessment is
regarded as a complex mixture of currently available data and
assumptions based on prevailing scientific thought.
Traditional concepts of risk assessment
Risk assessment is the use of available scientific information
to characterize potentially adverse effects associated with exposure of humans to an agent under known or expected conditions (1). Methodologically, it consists of four steps, namely,
hazard identification, hazard characterization, exposure assessment and risk characterization (2).
Hazard identification is primarily the process of identifying
the effects that are considered to be adverse, irrespective of the
dose needed or the specific mechanisms involved to elicit the
effect. Hazard characterization is the step to provide a quantitative and qualitative estimate of the adverse effects so that the
dose-response relationship and the mode of action for these
effects can be established. Exposure assessment involves the
evaluation of the modes, magnitudes, duration and time of
actual or anticipated exposure, and the number and nature of
persons who are likely to be exposed. Risk characterization, the
final step of risk assessment, is the estimation of the likelihood
of adverse effects in the human population as a consequence of
the exposure by taking into consideration the results of the
hazard identification, hazard characterization and exposure
assessment.
Application of risk assessment for nutrients
Traditional concepts and methodologies for risk assessment
have been developed and long used for food additives, contaminants and pesticide residues. How to apply these concepts and
methodologies for nutrients is the current challenge.
Practically, risk assessment is intended to provide the
scientifically sound basis for regulatory or nonregulatory action
to manage the risks from the agent to humans (risk management) (4). Regulatory management for safe and effective use of
nutrients includes the establishment of standards for intake
levels. The Food and Nutrition Board (FNB)3 of the Institute of
Medicine (IOM) has proposed the Dietary Reference Intakes
(DRIs) which are comprised of the Estimated Average
Requirement (EAR), the Recommended Dietary Allowance
(RDA), the Adequate Intake (AI) and the Tolerable Upper
Intake Level (UL). The UL, a terminology corresponding to the
Acceptable Daily Intake (ADI) for food additives, is defined by
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: esato@icaas-org.
com.
3
Abbreviations used: FASEB, Federation of the American Societies for
Experimental Biology; FNB, The Food and Nutrition Board; IOM, The Institute of
Medicine; NOAEL, no-observed-adverse-effect-level; UF, uncertainty factor; UL,
tolerable upper intake level.
0022-3166/03 $3.00 Ó 2003 American Society for Nutritional Sciences.
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ABSTRACT Risk assessment is defined as the use of available scientific information to characterize potentially
adverse effects associated with exposure of humans to an agent under known or expected conditions. Practically,
risk assessment is intended to provide the scientifically sound basis for regulatory or nonregulatory action to manage
the risks in humans from the agent. Therefore, the final goal of risk assessment for supplementary use of amino acids
is to provide scientific evidence and scientific logic for the establishment of tolerable upper intake levels (UL) for
amino acids. At present, however, execution of risk assessment for amino acids is hampered by deficiencies in
necessary scientific information, particularly, experimental or clinical/epidemiological data related to the estimation of
no-observed-adverse-effect-levels (NOAEL), and scientific principles for the allocation of uncertainty factors (UF) in
extrapolation from experimental/clinical data to the general human population. This paper attempts to identify the
scientific data and scientific thoughts/methodologies required for deriving UL or assessing the margin of safety for the
supplementary use of amino acids. J. Nutr. 133: 2021S–2024S, 2003.
SUPPLEMENT
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the IOM as ‘‘the highest level of a daily nutrient intake that is
likely to pose no risk of adverse effects to almost all individual in
the general population ’’ (5).
The IOM has applied a risk assessment model for establishing UL for micronutrients. Briefly, as in the case of an
ADI for a food additive, a UL for a micronutrient is established
on the basis of the most critical No-Observed-Adverse-EffectLevel (NOAEL) in toxicological studies by applying an uncertainty factor (UF):
NOAEL
:
UF
Theoretically, this kind of traditional model could be applicable to the establishment of ULs for amino acids. At present, however, the establishment of ULs of amino acids has not
occurred, primarily due to insufficient scientific information,
particularly, experimental or clinical/epidemiological data related to the estimation of NOAELs and to insufficient scientific
principles for the allocation of UF for extrapolation from
experimental/clinical data to the general human population.
UL 5
Consumed proteins are incorporated and utilized in the body
as amino acids. Thus, information from investigations of high
protein intakes may be useful for the basic understanding of
hazards or potentially adverse effects in humans due to excess
intakes of amino acids. Historical records of extremely high
protein intakes in humans show that daily intakes of protein
above 45% on an energy basis in adults are associated with
weakness, nausea, diarrhea and ultimately death, whereas daily
intakes of 20–35% protein are without ill effects (6).
Various biochemical and physiological responses can be
associated with high protein intakes. A high intake of protein
provides excess amounts of amino acids in the body and influences metabolic capacities to oxidize amino acids and synthesize urea. Urinary excretion of urea nitrogen is indicative of
total protein intake (7,8). A high protein diet has been shown
in human and animal studies to increase calcium excretion.
Presumably due to the increase in glomerular filtration rate and
the decrease in renal reabsorption of calcium (9).
According to a cross-sectional study in a healthy Japanese
population, the correlation of urinary calcium excretion with
animal protein intake is significantly positive in both sexes
whereas that with plant protein is not. Calcium excretion is also
correlated with daily urinary excretion of urea. A significant
correlation is also observed between daily calcium excretion
and daily urinary excretion of sulfate. The correlation in 50–79
y old subjects remains significant even after adjustment for sex,
age, body weight, sodium excretion and calcium intake. From
these findings the authors suggest that excess protein, especially
that rich in sulfur-containing amino acids, in habitual diets may
augment calcium excretion in the urine, particularly in the
elderly (10). Some data suggest that high protein diets lead to
increased urinary calcium loss (8), which could be hazardous
particularly for females due to its involvement with bone health
(11).
It is known that bone growth is stunted in protein-energy
malnutrition (12), and the outcome of hip fracture is improved
with protein supplements to the typical elderly victim of osteoporotic fracture (12). If an increase in urinary calcium loss is
assumed to be the principal mechanism by which harm might
be produced by a high protein intake, excess protein will not
harm the skeleton if the calcium intake is adequate (13). A
dietary calcium to protein ratio .20 mg (calcium): 1 g (protein)
probably provides adequate protection for the skeleton (13).
Like other natural food components, amino acids are
assumed to be nontoxic at the level occurring in the diet.
The area of concern is the intake of large amounts of amino
acids as supplements. Supplementation of several amino acids
may be beneficial in healthy individuals (11), but as seen in the
outbreak of the L-tryptophane-associated eosinophilia-myalgia
syndrome (EMS), potential risk is also present. Generally intake
of large quantities of individual amino acids is not recommended because much more information is needed (11).
The first step of risk assessment or hazard identification is
usually performed on the basis of animal toxicity tests and/or
clinical/epidemiological studies in humans. In animal toxicity
tests, administration of large amounts of individual amino acids
has caused various kinds of adverse effects such as growth
retardation by glycine (14), hyperkalemia and hypermagnesemia by arginine (15), hypercholesterolemia and hepatomegaly
by histidine (16), pancreatic damage and kidney enlargement
by methionine (17), corneal lesions by tyrosine (18), neurotoxicity by cysteine (19) or glutamic acid (20). In human clinical
studies, except for cases of hypersensitivity or intolerance,
administration of amino acids has been associated with no
adverse effect or mild changes of the values of laboratory tests.
The differences in responses to amino acids between animals
and humans as described in the literature are assumed to be
attributable mostly to the difference in dosage levels used and
eventually to differences in blood concentrations or target
tissue concentrations between animals and humans.
Hazard characterization
Hazard characterization is the step of defining the doseresponse relationship and the mechanism/site of action on the
basis of data from toxicity tests, toxicodynamic studies and
toxicokinetic studies. NOAELs of food additives or contaminants are established based on dose-response data in longterm toxicity tests. In the case of amino acids, however, such
data are extremely limited.
Potential adverse effects associated with excessive intakes
of amino acids are mechanistically classified into three
categories (21):
1) Specific/inherent amino acid toxicity: Exertion of toxicity
specific to individual amino acids such as corneal lesions
in young growing rats ingesting large amounts of tyrosine.
2) Amino acid antagonism: Exertion of adverse effects due
to competition among a structurally related group of
amino acids such as the branched chain amino acid
antagonism or the lysine-arginine antagonism.
3) Amino acid imbalance: Various definitions have been
proposed; (1) growth retardation of animals consuming
diets containing an excessive amount of one amino acid
(22); (2) adverse effects caused by any modification of
the dietary amino acid pattern (23); and (3) effects of
surpluses of indispensable amino acids other than one
that is limiting for growth or maintenance (21).
Specific amino acid toxicities are the critical factor for the
establishment of NOAELs and UFs. As shown in Table 1,
the correlative analysis of intake levels, blood concentrations and the incidence or severity of an adverse effect is useful
for extrapolation of animal data to the situation in humans.
In contrast, the establishment of ways to maintain an appropriate amino acid composition of total dietary and supplementary amino acid intake is a practical protection against
adverse effects due to amino acid antagonism and amino acid
imbalance.
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Lessons from high protein intakes
Hazard identification
RISK ASSESSMENT OF AMINO ACIDS
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TABLE 1
Relationship between dose of monosodium glutamate (MSG) for inducing hypothalamic lesions and plasma glutamate levels
in infant, weanling and adult mice (from reference 24)
Treatment of MSG
Age
Route
Dose (g/leg)
No. of mice affected
Peak level of plasma
glutamate (mmol/100ml)
Infant (10 d)
Nontreatment
p o2
po
po
—
0.5
0.73
0.8
0/8
0/8
2/8
4/8
17 6 11
62 6 6
104 6 183
—
Weanling (23 d)
Nontreatment
s c4
sc
sc
—
0.5
0.73
1.0
0/8
0/8
2/8
6/8
10
278
385
760
6
6
6
6
1
15
323
52
Adult (3–4 mo)
Nontreatment
s c4
sc
sc
—
1.0
1.23
1.5
0/8
0/8
4/8
4/8
10
539
631
841
6
6
6
6
1
25
303
105
Mean 6 SE.
Oral administration by a 10% aqueous solution.
Lowest effect dose (LED) of MSG for hypothalamic lesions and peak value of plasma glutamate at LED.
Subcutaneous injection in a 4% aqueous solution.
Exposure assessment
Exposure data are generated from monitoring, modeling results or reasoned estimates (25). At present, however, available
exposure data for amino acids are limited. Based on 3 reference
documents, namely, Standard Tables of Food Composition in
Japan (Resources Council, Science and Technology Agency in
Japan, 1986), The Amino Acid Composition of Foods in Japan
(Resources Council, Science and Technology Agency in Japan,
1986), and Food Group–Based Nutrient Intakes, National
Nutrition Survey (Ministry of Health and Welfare, Japan,
1995), daily intakes of individual amino acids in Japanese have
been calculated as follows (in mg/d); Ile 3338, Leu 6452, Val
4415, Met 1919, Cys 1328, Lys 5273, Phe 3770, Tyr 2794, Trp
965, Thr 3187, His 2479, Arg 5037, Ala 4277, Asx 7490, Glx
14716, Gly 3799, Pro 4845, Ser 3706 (T. Kimura, Personal
Communication, October, 2002).
A number of studies have estimated the requirements of
individual amino acids based on metabolic demands in various
age groups (26). Correlative analyses of exposure data from
food intakes monitoring with the requirement estimates based
on metabolic demands will provide a scientific basis for a foodbased guideline for adequate intakes of amino acids.
Risk characterization and future research
Practically, the goal of risk characterization for amino acids is
to establish ULs or NOAELs and UFs by taking into consideration the results of the hazard identification, hazard characterization and exposure assessment.
In a FASEB report published in 1992 (27), the expert panel
concluded that the available data provided only a limited basis
for safety evaluation of supplementary use amino acids, and
that a systemic approach to safety testing is needed. The FNB
IOM document on dietary reference intakes (28) published in
2001, also concludes that the available data are insufficient for
deriving ULs of amino acids for healthy humans. Therefore it
may be useful to summarize scientific data, scientific thought
and future research projects required for deriving NOAELs,
UFs and ULs for amino acids.
Animal studies. Idealistically, properly-designed long-term
animal studies are necessary for determination of NOAELs for
individual amino acids. Also scientific strategies or scientific
thoughts need to be established for the evaluation of long-term
effects based on data from short-term studies and toxicodynamic/toxicokinetic studies.
Adverse effects on neurological processes represent a primary
concern for supplementary use of amino acids. Thus, neurotoxicological testing programs such as a Functional Observational
Battery (FOB), a collection of noninvasive nervous system tests
to evaluate manifestations of neurologic dysfunction (29),
should be incorporated into animal studies of amino acids.
Metabolic and physiological effects. High concentrations
of certain amino acids such as glutamate, phenylalanine,
cysteine or arginine are known to cause various, usually mild,
effects on metabolic functions, hormonal secretion, immune
function, or electrolyte balance (27). It is important to study
possible adverse outcomes associated with the long term continuation of these effects.
Subgroups potentially at higher risk for adverse effects from
use of amino acid supplements. In the FASEB report, the
expert panel members identified several subgroups potentially
at higher risk for possible adverse health effects resulting from
supplementary use of amino acids, these include (1) infants,
children and adolescents; (2) pregnant and lactating women;
(3) elderly individuals; (4) persons homozygous or heterozygous
for inborn errors of amino acid metabolism; and (5) individuals
with low intakes of protein.
More data are needed on indispensable amino acid requirements for infants, children, adolescents, the elderly and
pregnant women (28).
Application of new technologies for metabolomics. The
occurrence of adverse effects resulting from intakes of large
amounts of an amino acid is attributable to high concentrations
of the amino acid in the target tissues or generation of toxic
metabolites/abnormal patterns of metabolites due to overload
of the principal metabolic pathway. Thus, application of newly
developed technologies for metabolomics will provide useful
information related to mechanisms of adverse effects in association with large intakes of an amino acid.
Approach for safety studies in humans. The available data
are obviously deficient for deriving ULs for amino acids.
However it should be considered that most of these data are
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1
2
3
4
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obtained from toxicity testing of amino acids performed in
accordance with the guidelines for food additives. Therefore,
as an alternative approach, human studies carefully designed
in accordance with safety standards inherent in the Phase I
Clinical Trial for pharmaceuticals may be useful to produce
critical data to assess the margin of safety for amino acids in
supplementary use.
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