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Supplement: 2nd Amino Acid Workshop

The Safety Testing of Amino Acids¹

Clinical Pharmacology Group, University of Southampton, Biomedical Sciences Building, Southampton SO16 7PX, UK

² To whom correspondence should be addressed. E-mail: agr{at}soton.ac.uk.

	ABSTRACT

TOP ABSTRACT CONCLUSIONS LITERATURE CITED

 
The risk assessment of compounds added to foods or taken as supplements is usually based on hazard characterization studies performed in animal test species. A large default uncertainty factor, or margin or exposure, is usually required to allow for possible species differences and human variability in the toxicokinetics and toxicodynamics of the compound. The development of biomarkers offers the potential to rationalize the risk assessment of amino acids, and to refine the extrapolation of data from animals to humans. The use of high resolution nuclear magnetic resonance spectroscopy applied to readily accessible biological fluids, such as urine and plasma, offers great potential for the identification of toxicologically relevant biomarkers in animal studies that can then be applied to studies in humans.

KEY WORDS: • amino acids • risk assessment • biomarkers • metabonomics • toxicokinetics

There is a well-established paradigm for the characterization of risks associated with the ingestion of chemicals in food or in the diet (1). Although risk assessment is an iterative process, it is usually considered to consist of four principal components:

i) hazard identification, in which the adverse effects (hazards) that can be produced by the chemical are investigated; hazard identification usually involves in vitro investigations and high dose in vivo studies in experimental animals,

ii) hazard characterization, in which the relevance of the detected adverse effects to humans is considered, and the dose-response relationship is assessed,

iii) exposure assessment, in which the predicted human intake is determined, and

iv) risk characterization, which integrates hazard characterization and exposure assessment to determine a level of exposure that would be without a significant risk, or the risk associated with a specific level of exposure.

In contrast to food additives and pesticides, there is an extensive database on the biodisposition and effects of amino acids in humans, although the majority of the data relate to the nutritional consequences of deficiency and not the potential for adverse effects. When data from studies in humans are available on the safety of doses of single amino acids, then these would be used for hazard characterization, and the safe dose in humans would be based on these human data. There have been numerous studies in experimental animals in which diets containing excess or inadequate levels of specific amino acids have been investigated; however these have been designed to investigate nutritional and/or metabolic consequences and not the possible adverse effects at high doses. There are well-defined procedures necessary for hazard identification and characterization using in vitro studies and studies in experimental animals with defined methods for the numbers of animals, tissue sampling and histopathological techniques that are necessary to ensure that the results are suitable for hazard characterization (4–6).

Despite the enormous database available related to the biological effects of amino acids in humans and animals, hazard identification and characterization related to the intake of specific amino acids are likely to have to rely on animal studies undertaken specifically for risk assessment purposes. A critical consideration for amino acids will be an assessment of the adequacy of the uncertainty factor that is applied to establish a "safe intake", or the adequacy of the "margin of safety" between the NOAEL and the anticipated human exposure. The application of the usual default uncertainty factor of 100 to the NOAEL for an adverse effect is likely to give an "approved" intake of the amino acid that would be devoid of any biological activities, and if applied to the total intake could result in deficiency of an essential amino acid.

A problem in the risk assessment of single amino acids, or mixtures of amino acids, added to foods or used as food supplements, is likely to be the determination of an adequate margin of safety, and its scientific justification. Data to assist in defining species differences and/or human variability in toxicokinetics and/or toxicodynamics will be important in the justification of lower than normal margins of safety. The development of toxicokinetic or toxicodynamic information relevant to human risk assessment requires the generation of data from studies in humans, either in vitro or in vivo, to reduce the uncertainties associated with either interspecies extrapolation or human variability. Obviously any studies in humans will be restricted to nontoxic doses, and must conform to the safety standards inherent in the Phase I studies on pharmaceutical compounds.

One possible approach that could provide data that would clarify the extrapolation processes would be the development of suitable biomarker data.

Risk assessment using the amino acid or its metabolite as biomarkers that correlate with the dose-response for the adverse effect detected in animal studies

Relating any adverse effects detected, for example in 90-d animal studies, to changes in biomarkers would provide a scientific basis for the replacement of the default uncertainty factor by a more logical and compound-specific assessment factor.

The biomarker would have to be studied at a range of doses in animals, including intakes producing the adverse effect, so that the dose-response for changes for the biomarker could be correlated with the dose-response for the adverse effect. To show that changes in the biomarker represented a surrogate for the adverse effect, the biomarker would need to be studied under conditions that affected the toxicity of the amino acid, for example different species, routes etc. Understanding the mode of action of the amino acid in producing the adverse effect could be used to support the validity of the biomarker.

An essential part of the use of biomarker data would be the need for data from closely related studies in humans, but at the anticipated intake/dose levels. This would necessitate the development of biomarkers in readily accessible body fluids such as urine or plasma (7).

Any changes in the metabolism and plasma kinetics of the amino acid with increase in dose in animals, from nontoxic to toxic doses, should be studied. Changes from linear to nonlinear kinetics may reflect saturation of metabolic pathways (8). An abnormal pattern of metabolites at high doses could be responsible for the adverse effect; alternatively, saturation of the metabolic process per se may be the cause of the adverse effect via changes in other substrates, in which case the abnormal pattern of metabolites would represent a biomarker of saturation. When the adverse effect is associated with nonlinear kinetics, then the plasma or urinary concentrations of the amino acid itself and/or its metabolites may be sufficient to act as a biomarker of the adverse effect. If nonlinearity is established in animals, and it is related to the adverse effect, then definition of the linear kinetic range in humans would provide considerable understanding about the adequacy of any available margin of safety.

However, if the metabolism and kinetics are linear, then plasma or urinary concentrations of the amino acid itself and/or its metabolites will simply be biomarkers of exposure and/or internal dose, and cannot be related to the adverse effect. Under such circumstances, a more comprehensive analysis of metabolic status may help to identify a toxicologically relevant biomarker. Metabonomic studies provide the prospect of developing biomarkers that can be used to define species differences and also to investigate human variability. Because amino acids are normal body constituents, there will be normal variability between healthy adults, and this variability can provide a context for the interpretation of any altered profiles detected after dosage with a single amino acid.

The use of metabonomics to define biomarkers in animal and human studies, for use in risk assessment

The great advantage of metabonomics over genomics, transcriptomics and proteomics is that it could be applied to in vivo studies in humans and the monitoring of changes in animals over relevant dose ranges. There is a developing literature on metabonomics which indicates that it might be of potential value in the risk assessment of amino acids.

Application of high resolution proton nuclear magnetic resonance spectroscopy (1H-NMR) at 600 MHz or 750 MHz has been used to define the changes in urinary parameters in experimental animals under a variety of experimental conditions. A major advantage of the use of such technologies is that a special formulation of the amino acid would not be needed and the signals would represent the total amino acid, both endogenous plus exogenous.

Application of 1H-NMR has been used to define the changes in urinary parameters after toxic doses of chemicals in experimental animals. Published examples include changes in plasma amino acids and urinary excretion products after uranyl nitrate in rats (9), hydrazine in rats (10,11), paracetamol in rats (12) and ifosfamide in humans (13,14), and well as studies on known hepato- and nephro-toxins (15–17).

The technique can be adapted to study changes in tissue samples such as liver and kidney (18,19), testes (20) and adrenal (21) in addition to urine and plasma. Such data from in vitro mechanistic studies and in vivo studies in animals would be of value in defining the underlying metabolic basis for the use of the biomarker. 1H-NMR has been applied to study subtle differences such as strain differences in metabolism (22), the influence of exercise and hydration in humans (23) and the influence of the oestrous cycle (24). However, the technique requires sophisticated instrumentation, and the 1H-NMR spectra are very complex; each contains many thousands of resonances with a high dynamic range. Consistent methods of pattern recognition (25,26) and of reducing this wealth of data to manageable proportions are essential (23).

The technique holds great potential in relation to amino acids, and it has been used to study inborn errors amino acid of metabolism (27–29), the effects of Alzheimer's disease on the amino acids in the cerebrospinal fluid (30), and dystrophic tissue in Duchenne muscular dystrophy (31).

Overall, the application of 1H-NMR techniques to urine and plasma offers a rapid method of developing biomarkers of adverse effects and of changes in amino acid homeostasis.

	CONCLUSIONS

TOP ABSTRACT CONCLUSIONS LITERATURE CITED

 
The risk characterization procedures that are used to assess the safety of compounds such as food additives may not be readily applicable to high intakes of individual amino acids. Adequate hazard identification requires the use of established toxicological testing protocols, such as 90-d feeding studies performed to good laboratory practice, to ensure that all potentially relevant adverse effects are recognized. Dosage selection will need to take into account possible nutritional imbalances that would not occur at lower intakes. The use of large uncertainty factors, which are a normal part of hazard characterization for threshold effects, might lead to a conclusion that greater than normal intakes of an amino acid may not be appropriate. Greater understanding of the dose-response relationships for any high dose metabolic changes could provide a rationale for the use of lower than normal uncertainty factors. High resolution nuclear magnetic resonance spectroscopy offers a valuable method for identifying and defining metabolic abnormalities produced by high doses of individual amino acids in animal feeding studies. Related studies at lower doses in humans would refine hazard characterization, and could justify the use of low uncertainty factors.

	FOOTNOTES

 
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.

³ Abbreviations used: ADI, acceptable daily intake; 1-NMR, proton nuclear magnetic resonance spectroscopy; NOAEL, no observed adverse effect level; RfD, reference dose; TDI, tolerable daily intake.

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TOP ABSTRACT CONCLUSIONS LITERATURE CITED

 

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