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Supplement: 3rd Amino Acid Workshop

Potential Approaches to the Assessment of Amino Acid Adequacy in Rats: A Progress Report¹

^* Institute of Life Sciences and Research Institute for Health Fundamentals, Ajinomoto Co., Inc., Kawasaki, Japan

² To whom correspondence should be addressed. E-mail: takeshi_kimura{at}ajinomoto.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
We report on research progress on two approaches that may be useful in determining the upper adequacy range for macronutrients such as amino acids. One approach was to attempt to identify "toxic metabolites" that were responsible for toxicity or biomarkers for the toxicity of excessive intake of an amino acid in rats. We found that there was hepatic toxicity that was specifically associated with L-cystine excess, but not with L-cysteine excess. We analyzed urine samples from rats fed basal diets or L-cystine or L-cysteine excess diets and identified 25 peaks from gas chromatography mass spectrometry analysis that were specific for L-cystine excess and also correlated with toxicity markers. Another approach was to try to identify "metabolic limits" by measuring CO₂ arising from amino acid excess. Uniformly ¹³C labeled L-leucine was used as tracer, in diets with added L-leucine fed to rats, and ¹³CO₂ arising from its metabolism was collected over 24 h and the fraction of the ingested L-leucine that was exhaled as CO₂ was calculated. The fractional exhalation of ¹³CO₂ increased with increasing L-leucine dose, but showed an inflexion point at 8.9 g/kg body weight, after which it reached a plateau. This suggested that >8.9 g/kg BW, the catabolism of L-leucine changed and this approximately coincided with the dose above which a statistically significant decrease in body weight was seen.

KEY WORDS: • dietary reference intakes • tolerable upper intake level • ADI • GC/MS • cluster analysis

Introduction

In animal studies, the effects of ingesting excess amino acids have been shown to vary with the type of amino acids as well as with other factors such as the protein level of the diet, the coingestion of other amino acids, and the route of administration (1–4). Among the amino acids comprising protein, sulfur amino acids are known to adversely affect growth at the lowest dose, whereas branched-chain amino acids are not associated with significant adverse effects on growth at the same dose. We are in the process of studying two different types of approaches for these two types of amino acids, to understand better how to assess the range of adequate intake for metabolites such as amino acids, and a progress report for each approach is presented here.

It is clear that the use of a safety factor of 1/100, commonly used in the safety assessment of food additives, is not suitable for extrapolating the results of animal data to humans in the case of many nutrients. For example, the no adverse effect level (NOAEL)³ for rats determined from chronic toxicity studies for glycine was <1800 mg/kg body weight (5). Multiplication of this value with a safety factor of 1/100 gives <18 mg/kg body weight. If this value is applied to a human weighing 60 kg, the acceptable daily intake (ADI) would be <1.08 g/d. However, the average glycine intake from ordinary food can be calculated as 3.6 g/d in Japan from official statistics (6,7). Consequently, the intake of glycine through our daily diet is already more than three times the calculated ADI. Even if the 1.08 g/d is taken to mean the amount that can be ingested in addition to the average intake, this is <30% above the average intake of glycine from protein; individuals ingesting 30% more protein than the average, which would represent a substantial part of the population, would be above the calculated ADI. It could be said that this contradiction is inherent in the application of a safety evaluation system developed for synthetic and xenobiotic substances to the evaluation of nutrients (8). The 100-fold safety factor represents the product of a 10-fold safety factor to allow for the difference in animal species times another 10-fold for individual differences. It would seem unreasonable to apply this idea developed for the safety evaluation of xenobiotics to nutrients that are utilized by all animals. Against this backdrop, the current safety evaluation of nutritional food supplements places great emphasis on clinical data for humans (9). The metabolism of amino acids and their effects are influenced by genetic factors, age, nutritional status, simultaneous intake of other amino acids and nutrients, and the mode of intake among other factors. It would be extremely difficult to take all of the abovementioned safety-related factors and conditions fully into account in human clinical trials due to ethical as well as economical reasons. Thus, there should be a place for animal studies in the assessment of adequate intakes of macronutrients such as amino acids, especially in identifying under various conditions, toxicological endpoints and early biomarkers of toxicity that may be measurable in humans. We have been examining the possibility of identifying markers of toxicity in blood, urine, and breath and describe below attempts to identify such markers in urine and breath.

To use a pharmacokinetic approach to the risk assessment of amino acid excess, the identification of metabolites responsible for toxicity, or biomarkers that correlate well with toxicity are necessary (10–12). Data from inborn errors of metabolism would indicate that, in a number of cases such as hypermethionninemia (13), histidinemia (14), and hyperlysinemia (15), the amino acid itself is not directly responsible for toxicity, because no adverse symptoms were observed in patients with extremely high plasma levels of these amino acids. In contrast, there are reports of adverse effects due to the overloading of these amino acids in animals and in humans (16,17). It would thus suggest that metabolites in the normal catabolic pathways, usually found at low concentrations, or abnormal metabolites arising from the overloading of certain metabolic pathways may be responsible for toxicity, at least of a number of amino acids. If such metabolites could be identified, then it may be possible to use a pharmacokinetic approach to the risk assessment of amino acid excess. However, the identification of such metabolites may pose a problem, as there are many metabolites resulting from the catabolism of amino acids, and especially because there is incomplete knowledge of the minor pathways of catabolism resulting from the range of specificities of the various enzymes involved. We have developed analytical methods based on cluster analysis of multivariate correlations (CAMC) that could be used to identify metabolites whose behavior correlates to toxicity endpoints (18). We report here on the use of CAMC on gas chromatography mass spectrometry (GC/MS) metabolic profiling data to identify potential candidate metabolites for the adverse effects of excess feeding of an amino acid to rats.

On the other hand, it is questionable that a "toxic metabolite" is responsible for the adverse effects caused by excessive ingestion for all amino acids, as the toxicology of only a few amino acids seem to exhibit specific organ toxicity (4). There may be mechanisms of toxicity not involving "toxic metabolites", such as the depletion of certain other key essential nutrients resulting from the overloading of a single metabolite. If the adverse effects of amino acid excess can be attributed to the depletion of a particular metabolite, then it may also be possible to apply a pharmacokinetic approach to the risk assessment of amino acid excess, or it may be possible to add the depleted metabolite in question to alleviate the adverse effects. However, it may not be possible to identify "toxic metabolites" or "depleted metabolites" if these occur only within specific organs or tissues. It could be argued that as the dose of a particular nutrient increases, the rate of production of abnormal metabolites would increase when certain rate-limiting steps in catabolism are saturated. The increase in concentration of particular saturating metabolites may lead them to be processed in an abnormal manner by enzyme systems whose main activity is for a different metabolite. Most enzymes are not completely specific and have a range of specificities for related compounds, the activities for the side reactions depending on substrate concentration. The accumulation of a saturating metabolite should be coupled with the decrease in the rate of catabolism represented by the generation of CO₂ derived from the saturating metabolite. We therefore focused on the possibility of using the CO₂ generated from the catabolism of excessive amino acids as a marker for a "metabolic limit." CO₂ generated from labeled amino acids has been used successfully for determining the lower range of adequacy (nutritional requirements) of essential amino acids. These are based on the lower inflection point in the dose response curve for the CO₂ generated from varying doses of the amino acid in question or from the catabolism of another amino acid (19,20). The observation of an upper inflection point in the dose response curve for CO₂ generated from phenylalanine has been reported (21). We have examined the upper inflection point for ¹³CO₂ derived from [U-¹³C]-L-leucine in rats fed excessive amounts of L-leucine and compared it to the toxicology data to see if this measure could be used as a biomarker.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Animals

The study received prior approval from Ajinomoto's Institutional Animal Care and Use Committee. Male Fischer rats (F-344, Charles River Japan, Atsugi, Japan) were maintained in a controlled condition of 12-h light, 12-h dark, at 23 ± 1 °C, and at 55 ± 10% humidity. The animals were housed in stainless steel cages and had free access to commercially produced nonpurified diet (Oriental Yeast Co., Ltd., Tokyo, Japan) or a purified diet and tap water. Purified diets were prepared based on the AIN-93G composition (22) with some modification, in which both dextrinized cornstarch and sucrose were substituted by cornstarch (modified AIN-93G). For the studies with sulfur amino acids, when the animals reached 6–7 wk of age, the diet was changed to the experimental ones, the modified AIN-93G basal control diets or diets in which a part of the cornstarch was replaced with L-cysteine or L-cystine [4.8 or 7.2% (w/w = 15% or 21% of total N intake, respectively)]. Inferior venous blood samples were collected on day 5 from the rats fed basal or the experimental diets containing 4.8% (w/w) of additional L-cysteine (2.9 ± 0.2 g/kg BW/d, N = 6) or L-cystine (2.4 ± 0.2 g/kg BW/d, N = 6) for serum biochemical analysis. Portal blood samples during ingestion were collected on day 3 of the experiment from the rat fed with basal or the experimental diet containing 4.8% (w/w) of additional L-cysteine (2.3 ± 0.4 g/kg BW/d, N = 6) or L-cystine (2.0 ± 0.2 g/kg BW/d, N = 6) for plasma cystine measurement. The rats given basal or the experimental diets containing 7.2% (w/w) of additional L-cysteine (3.2 ± 0.4 g/kg BW/d, N = 6) or L-cystine (2.6 ± 0.3 g/kg BW/d, N = 9) for 5 d were used for cystine measurement of postprandial portal plasma and for the analysis of urinary metabolites. For the L-leucine studies, rats were fed commercially produced nonpurified diet for 1 wk and then the modified AIN-93G diet for 1 wk, then switched to experimental diets in which L-leucine was added in graded levels of 0, 2.5, 5, 7.5, 10, 15, 20, 30% (w/w = 0, 8, 15, 21, 26, 34, 41, 51% of total N intake) in the modified AIN-93G diet in place of cornstarch and trained to complete each meal within 9 h (0900–1800 h) for 10–14 d. For the labeled diets [U-¹³C]-L-leucine (>98%; Cambridge Isotope Laboratories, Andover, MA) was mixed into the additional L-leucine at the constant ratio of 5% of total L-leucine. The labeled diets (equal amounts of food intakes of the day before) were given to the rats (n = 2–4 per diet) independently placed in tight-sealed boxes at 0900 h to collect the expired CO₂ for the subsequent 24 h. The mean values of the additional L-leucine ingestion were 2.2, 3.4, 4.5, 7.6, 13.1, 15.7, and 20.0 g/kg BW/d in the individual diet groups, respectively.

Carbon dioxide collection and analysis

Breaths of rats in the sealed boxes were aspirated by vacuum pumps independently at the flow rate of 1 L/min, and the expired breath CO₂ was collected for 24 h by passing the aspirated air through NaOH solution. CO₂ in the air was removed from the air supplied to the sealed boxes by passing through both soda lime columns and 2N NaOH solution columns. The absorbed CO₂ was precipitated as BaCO₃ by the method of Benevenga et al. (23) with total CO₂ production (mol/d) calculated from the recovery of BaCO₃. To obtain the isotope ratio (¹³CO₂/¹²CO₂) of each breath sample, the CO₂ regenerated by the addition of trichloroacetic acid to BaCO₃ was measured by an isotope selected nondispersive infrared spectrometer (UBiT-IR300, Otsuka Electronics Co., Ltd., Osaka, Japan). For each CO₂ sample placed in the sample side of the inlet, ¹³CO₂ was measured against a standard gas sample of known ¹³CO₂ content in the reference side of the inlet. The ¹³CO₂ production originating from dietary [U-¹³C]-L-leucine was calculated by subtracting the ¹³CO₂ production from the rats fed basal diets without isotope after standardization of the values by the three-fourth power of the body weight (W^0.75). Under the assumption that the oxidation of [U-¹³C]-L-leucine was equal to that of natural L-leucine, the proportion of oxidized carbon of additional dietary L-leucine [Ox (%)] was calculated as follows:

where P (¹³CO₂) is the ¹³CO₂ production originating from tracer, and I ([U-¹³C]-L-leucine) is the amount of dietary intake of [U-¹³C]-L-leucine (mmol). The constant 6 accounts for the number of ¹³C in a single molecule of [U-¹³C]-L-leucine.

Serum and plasma analysis

Serum samples were obtained after placing the blood at rest for 1–2 h. Aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activities in the sera were measured using a biochemistry automatic analyzer TBA-120FR (Toshiba Medical Systems, Tochigi, Japan). Plasma samples were obtained using ethylenediaminetetraacetatic acid (EDTA; NONCLOT-D, Daiichi Pure Chemicals, Tokyo, Japan) as anticoagulant, mixed with two volumes of 5% (w/w) trichloroacetic acid and centrifuged to remove protein as precipitate immediately after plasma collection. The samples were kept at 4°C during all steps to minimize chemical reactions of thiol metabolites. The amino acid concentrations were measured by an automatic amino acid analyzer (L-8800, Hitachi, Tokyo, Japan). Briefly, amino acids separated by cation-exchange chromatography were detected spectrophotometrically after post column reaction with nihydrin reagent. Although use of EDTA is not recommended for amino acid analysis because of the contaminants reacting with ninhydrin (24), we have confirmed that EDTA usage does not give any additional peaks in our amino acid analysis.

Preparation of urine for GC/MS

Urine samples were processed for GC/MS analysis based on the method as reported previously (25). Metabolic cages were used to collect 24-h urine samples from the rats fed with the basal or experimental diet containing 7.2% of additional L-cysteine or L-cystine for 5 d as mentioned above. The samples were filled to 12 mL, centrifuged to remove debris, and deproteinized with 10-kDa cutoff filters. The filtrates were brought to pH 2–3 with HCl and incubated for 4 h in the presence of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride to derivatize ketoacids to generate O-(pentafluorobenzyl)oximes. Acid insoluble metabolites were obtained after the centrifugation of the specimens as precipitate (fraction A). The supernatants were applied to 1-mL bed volume of cation-exchange column (Dowex 50Wx8, H+ form; Bio-Rad, Hercules, CA). Cation-binding metabolites were eluted with 3 M ammonia solution (fraction B). Organic acids including ketoacid derivatives were extracted from the pass-through fraction of the cation-exchange chromatography by ethylacetate (fraction C). All three fractions (A, B, and C) were dried under vacuum. The dried residues were dissolved in 50 µL of trimethylsilylation reagent mixture [N-methyl-N-trimethylsilyltrifluoroacetamide and trimethylchlorosilane, 100:1 (v/v)] and incubated overnight at room temperature to form trimethylsilyl derivatives.

GC/MS analysis

A Hewlett-Packard GC/MS system (Palo Alto, CA) consisting of a Model 6890N gas chromatography equipped with HP-5MS column (30 m x 0.25 mm i.d., Hewlett Packard) and a Model 5973N mass spectrometer was used for the analysis. The GC temperature was increased from 60°C to 280°C at a rate of 5°C/min. Positive ions generated by electron impact ionization were monitored by scan mode (m/z: 50–700).

Statistical analysis

All data are expressed as means ± SD. Statistical significance among the groups was determined either by Tukey's honestly significant difference test after an ANOVA for multiple comparisons. Regression analyses of the relations between dietary intake of additional L-leucine and proportion of its carbon oxidation were performed on MS Excel 2000 (Microsoft Corporation, Redmond, WA), and a change in the relation was evaluated by the broken-line analysis of Anderson and Nelson (26).

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Urine analysis

The ingestion by F344 rats of diets containing 4.8% L-cystine caused significantly higher serum ASAT and ALAT levels compared to animals fed basal diets, whereas rats ingesting 4.8% L-cysteine did not show these differences (Fig. 1), indicating that the observed toxicity was specific to the ingestion of L-cystine. Histopathological examination indicated perilobular necrosis of the liver, only in animals receiving excess L-cystine (not shown). It has been reported that L-cystine is converted to L-cysteine by the intestine (27) and increases in plasma L-cysteine concentrations were observed after dosing with either L-cystine or L-cysteine (not shown). However, we found that portal levels of L-cystine were not significantly different between rats fed L-cysteine or L-cystine (Tukey's test; Fig. 2). This was the case whether the blood samples were collected during meal ingestion or postprandially. It had been reported that plasma heavy metals and proteins could promote autooxidation of plasma thiols (28). We, however, found that L-cysteine and L-cystine were both stable in the acidified deproteinized supernatant for a week at 4°C, and the effects of autooxidation of plasma cysteine were minimized by placing blood samples immediately at 4°C and deproteinizing immediately after plasma collection. Recently, we have developed a GC/MS based method to quantify plasma cysteine, cystine, and protein-bound cysteine using ¹⁵N-cysteine as an internal standard after protection of free thiol moieties by iodoacetic acid (29). Plasma cystine concentrations measured by this new method showed quite high correlation with the values by amino acid analyzer in this study (r = 0.98) and results that are in agreement with this study were obtained. The fact that portal levels of L-cystine were not significantly different between the L-cystine–fed animals and L-cysteine–fed animals suggested that the hepatic toxicity seen was not due to the L-cystine molecule itself but possibly due to a metabolite of L-cystine produced by the intestine. To try to identify metabolites that might be responsible for the toxicity, the urine of rats ingesting excess L-cystine, L-cysteine, and basal diets were analyzed by GC-MS after derivatization. Data from individual animals were analyzed with proprietary software that identified 1466 peaks. The peaks for the L-cystine group were assigned an index of 1 and the rest were assigned an index of 0. Correlation coefficients between peak heights and their respective indices were calculated and 253 peaks showing statistically significant correlations with the indices were identified. The arbitrary heights of these peaks from each rat were analyzed together with the corresponding value for serum ASAT and ALAT for each animal, using the previously reported cluster analysis of multivariate correlations method (18) to obtain a dendrogram of the correlations between the behavior of each of the peaks across individual animals of the three dosing groups. The analysis indicated that 25 peaks clustered together with ASAT, ALAT (Fig. 3). Preliminary analysis of the identities of the 25 peaks indicate such substances as ethanolamine, oxo-proline, 2,4-hydroxypyrimidine, ß-aminoisobutyric acid, and - aminobutyric acid. There is a possibility that many of the peaks are generated as the result of the toxicity rather than being the cause, as the causal relationship between the pathological state and resultant abnormal metabolites are probably stronger than the causal relationship between the "toxic metabolite" and the pathological state. To deal with this problem, we intend to study the urinary metabolite profiles during the early stages of dosing, before a significant rise in ASAT and ALAT are observed, and to analyze the early metabolic profile data against the ASAT and ALAT values seen at a later stage for the same animals, thus eliminating the contributions from the metabolite changes resulting from the hepatic toxicity.

View larger version (13K): [in this window] [in a new window]   FIGURE 1  L-cystine–specific increase in serum ASAT and ALAT. Dietary intake of excessive L-cystine (4.8% (w/w), 2.9 ± 0.2 g/kg BW/day, N = 6) for 5 d led to the threefold elevation of serum ASAT and ALAT compared to basal control (N = 6), whereas excessive intake of L-cystine (4.8% (w/w), 2.4 ± 0.2 g/kg BW/d, N = 6) did not change these levels significantly. Data are expressed as mean ± SD. (a, b) p < 0.05 between a and b, the same characters represent lack of statistical significance (Tukey's test).

View larger version (21K): [in this window] [in a new window]   FIGURE 2  L-cystine concentrations in portal plasma. Portal plasma during ingestion was collected on day 3 of the experiment from rats fed with basal or experimental diet containing 4.8% (w/w) of additional L-cystine or L-cysteine (left graph). Postprandial portal plasma (at least 4 h after the last meal) was collected from the rat fed with basal or the experimental diet containing 7.2% (w/w) of additional L-cystine or L-cysteine for 5 d (left graph). Data are expressed as mean ± SD. (a, b) p < 0.05 between a and b, the same characters represent lack of statistical significance (Tukey's test).

View larger version (16K): [in this window] [in a new window]   FIGURE 3  Cluster analysis of multivariate correlations. The peak heights of urinary metabolites were analyzed together with the values of serum ASAT and ALAT for the respective animals. Multivariate analysis was performed on the data to give the first correlation coefficient matrix. Multivariate analysis was performed on the first matrix to give a matrix of correlation coefficients representing the similarity of the behavior of a particular metabolite (peak) to other metabolites within the total data set. The correlation coefficients from the second matrix were subtracted from 1, so that better correlations were represented by smaller numbers. Cluster analysis was performed on the final data matrix to give the dendrogram. Twenty-five peaks were identified in the same cluster containing ASAT and ALAT.

In the range of L-leucine additions from 0 to 30% in diets, the ratio of ¹³CO₂/¹²CO₂ in the breath increased linearly (r² = 0.99), reflecting the increase in ingested labeled L-leucine (not shown). However, when the percent oxidation of the L-leucine ingested was calculated, it was found that oxidation reached a plateau phase at >10% of L-leucine in diets (Fig. 4). We evaluated the biphasic changes by applying broken-line analysis to the plot of L-leucine oxidation (%) against the actual intakes of additional L-leucine (g L-leucine/kg body weight). As a result, a phase of linear increase, followed by a plateau phase, was shown and the inflection point between the two phases was found to be at the dose of 8.9 g L-leucine/kg BW (Fig. 4). It might be thought that the capability for oxidation of excessive L-leucine (or -ketoisocaproate, -KIC) was altered around the intersection in its biphasic changes. Concentrations of L-leucine and the other amino acids in blood plasma, taken at the termination of the 24-h breath test, were not significantly different (P > 0.05) among the L-leucine diet groups. These results showed that there was no accumulation of L-leucine in the blood stream. Therefore, additional and excessive L-leucine was thought to be deaminated and decarboxylated to isovaleryl-CoA via -KIC, or directly incorporated into proteins. In a different study with the same strain of rats and composition of basal diets, rats fed diets containing 10% L-leucine, equivalent to an actual intake of 8.2 g L-leucine/kg BW, did not show any effects on body weight gain but rats fed diets containing 15% L-leucine, equivalent to an intake of 12.4 g L-leucine/kg, did show statistically significant growth inhibition compared to animals fed basal diet (not shown). The fact that the dose at the CO₂ inflection point obtained by breath analysis was between the two doses suggests that the upper inflection point may serve as an early marker of reduction in body weight gain due to excess L-leucine. It is not possible to deduce from this experiment alone the direct mechanism for the reduction in body weight gain. The fact that a plateau was reached for the percent oxidation of added L-leucine implies that incompletely oxidized metabolites of L-leucine might be increased with increasing doses of L-leucine, and especially above the inflection point dose of 8.9 g L-leucine/kg BW. To address the mechanism, further investigations regarding excretion of ¹³C, from the metabolism of [U-¹³C]-L-leucine, into urine and feces, and the contribution of each position in the carbon skeleton of L-leucine to oxidation or metabolism, are in progress.

View larger version (17K): [in this window] [in a new window]   FIGURE 4  Broken-line analysis for oxidation of additional L-leucine fed to rats. Experimental diets containing various dosages of additional L-leucine were fed to the rats individually. Intake of additional L-leucine (g/kg BW) in each rat and the corresponding proportion of L-leucine oxidation (% of additional L-leucine carbon in the diet) were plotted (N = 18). Fitted line for linear phase: Y = 2.44X + 35.7; for plateau phase: Y = –0.01X + 57.5. Arrow indicates the amount of L-leucine intake (8.9 g/kg BW) at the intersection between linear and plateau phases.

We believe the two approaches presented here represent possible ways to address the various issues associated with analyzing excess amino acid or macronutrient intake. It is hoped that further research into these approaches will allow us to understand the mechanisms of the effects of excess amino acid and macronutrient intake, and aid the process of risk assessment for determining the adequate range of intakes for amino acids and other macronutrients.

	FOOTNOTES

 
¹ Presented at the conference "The Third Workshop on the Assessment of Adequate Intake of Dietary Amino Acids" held October 23–24, 2003 in Nice, France. 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 as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Vernon R. Young, Dennis M. Bier, Luc Cynober, Yuzo Hayashi, and Motoni Kadowaki.

³ Abbreviations used: ADI, acceptable daily intake; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; BW, body weight; CAMC, cluster analysis of multivariate correlations; GC/MS, gas chromatography mass spectrometry; NOAEL, no adverse effect level; -KIC, -ketoisocaproate.

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