Analytical Chemistry of Impurities in Amino Acids Used as Nutrients: Recommendations for Regulatory Risk Management.

Proteinogenic amino acids are natural nutrients ingested daily from standard foods. Commercially manufactured amino acids are added to a wide range of nutritional products, including dietary supplements and regular foods. Currently, the regulatory risk management of amino acids is conducted by means of setting daily maximum limits of intake. However, there have been no reported adverse effects of amino acid overdosing, while impurities in low-quality amino acids have been identified as causative agents in several health hazard events. This paper reviews the analytical chemistry of impurities in amino acids and highlights major variations in the purity of commercial products. Furthermore, it examines the international standards and global regulatory risk assessment of amino acids utilized in dietary supplements and foods, recommending (1) further research on analytical methods that can comprehensively separate impurities in amino acids, and (2) re-focusing on the regulatory risk management of amino acids to the analytical chemistry of impurities.

Detection of impurities in dietary supplements containing L-tryptophan.

Impurities in nine dietary supplements containing L-tryptophan were evaluated using an HPLC methodology. In five tested products, the total impurities were higher than the thresholds described in the Food Chemical Codex or implemented in the EU for pharmaceutical grade L-tryptophan. In addition, liquid chromatography-mass spectrometry was used to specifically test for the presence of 1,1'-ethylidenebis-L-tryptophan (EBT). None of the tested products contained detectable amounts of EBT. High amounts of unidentified impurities in some dietary supplements point to potential health risks.

Subchronic Tolerance Trials of Graded Oral Supplementation with Phenylalanine or Serine in Healthy Adults.

Phenylalanine and serine are amino acids used in dietary supplements and nutritional products consumed by healthy consumers; however, the safe level of phenylalanine or serine supplementation is unknown. The objective of this study was to conduct two 4-week clinical trials to evaluate the safety and tolerability of graded dosages of oral phenylalanine and oral serine. Healthy male adults (n = 60, 38.2 ± 1.8y) completed graded dosages of either phenylalanine or serine supplement (3, 6, 9 and 12 g/d) for 4 weeks with 2-week wash-out periods in between. Primary outcomes included vitals, a broad spectrum of circulating biochemical analytes, body weight, sleep quality and mental self-assessment. At low dosages, minor changes in serum electrolytes and plasma non-essential amino acids glutamine and aspartic acid concentrations were observed. Serine increased its plasma concentrations at high supplemental dosages (9 and 12 g/day), and phenylalanine increased plasma tyrosine concentrations at 12 g/day, but those changes were not considered toxicologically relevant. No other changes in measured parameters were observed, and study subjects tolerated 4-week-long oral supplementation of phenylalanine or serine without treatment-related adverse events. A clinical, no-observed-adverse-effect-level (NOAEL) of phenylalanine and serine supplementation in healthy adult males was determined to be 12 g/day.

International Regulations on Amino Acid Use in Foods and Supplements and Recommendations to Control Their Safety Based on Purity and Quality.

We examined international regulatory developments related to the use of proteinogenic amino acids in human nutrition and concluded that the current risk-assessment practices tend to focus exclusively on setting maximum daily limits. In this brief review we argue that controlling the standards of purity and ingredient quality are the key safety issues that should be considered during risk assessment. Moreover, if maximum intake limits on amino acids are implemented, they should be defined using a well-established rationale for the health risks associated with high intakes. This would avoid setting limits that are so low that they render the dietary supplements ineffective and which, therefore, could mislead the consumer. We further suggest that there should be greater regional concordance in how the use of amino acids as ingredients is regulated and use the capacity of industry to oversee pre-competitive issues, such as standards of purity and scientific research on the safety of generic ingredients. Our arguments are based on clinical safety scientific research and oversights of amino acid purity standards conducted in the last decade by the not-for-profit international association, the International Council on Amino Acid Science.

Proposals for Upper Limits of Safe Intake for Methionine, Histidine, and Lysine in Healthy Humans.

Based on research presented during the 10th Amino Acid Assessment Workshop, no observed adverse effect levels (NOAELs) for supplemental methionine at 46 mg/(kg·d) (∼3.2 g/d), for supplemental histidine at 8.0 g/d, and for supplemental lysine at 6.0 g/d have been proposed. These NOAELs are relevant to healthy adults and are applicable only to high-purity amino acids administered in fortified foods or dietary supplements. Because individuals are exposed to the above supplemental amino acids in the context of complex combinations of essential amino acids or individually in dietary supplements for various physiologic benefits, such as body fat reduction, skin conditioning, mental energy increase, or herpes simplex treatments, the above safety recommendations will make an important contribution to regulatory and nutritional practices.

Tolerance to graded dosages of histidine supplementation in healthy human adults.

BACKGROUND: Histidine is an essential amino acid with health benefits that may warrant histidine supplementation; however, the clinical safety of histidine intake above the average dietary intake (1.52-5.20 g/d) needs to be vetted. OBJECTIVES: We aimed to determine the tolerance to graded dosages of histidine in a healthy adult population. METHODS: Healthy adults aged 21-50 y completed graded dosages of histidine supplement (4, 8, and 12 g/d, Study 1) (n = 20 men and n = 20 women) and/or a 16-g/d dosage of histidine (Study 2, n = 21 men and n = 19 women); 27 participants (n = 12 men and n = 15 women) completed both studies. After study enrollment and baseline measures, participants consumed encapsulated histidine for 4 wk followed by a 3-wk recovery period. Primary outcomes included vitals, select biochemical analytes, anthropometry, serum zinc, and body composition (via DXA). RESULTS: No changes in vitals or body composition occurred with histidine supplementation in either study. Plasma histidine (measured in subjects who completed all dosages for Studies 1 and 2) was elevated at the 12- and 16-g/d dosages (compared with 0-8 g/d, P < 0.05) and blood urea nitrogen increased with dosage (P = 0.013) and time (P < 0.001) in Study 1 and with time in Study 2 (P < 0.001). In Study 1, mean ferritin concentrations were lower in 12 g/d (46.0 ng/mL; 95% CI: 34.8, 60.9 ng/mL) than in 4 g/d (51.6 ng/mL; 95% CI: 39.0, 68.4 ng/mL; P = 0.038). In Study 2, 16 g/d increased mean aspartate aminotransferase from baseline (19 U/L; 95% CI: 17, 22 U/L) to week 4 (24 U/L; 95% CI: 21, 27 U/L; P < 0.001) and mean serum zinc decreased from baseline (0.75 µg/dL; 95% CI: 0.71, 0.80 µg/dL) to week 4 (0.70 µg/dL; 95% CI: 0.66, 0.74 µg/dL; P = 0.011). CONCLUSIONS: Although values remained within the normal reference ranges for all analytes measured, in all dosages tested, the human no-observed adverse effect level was determined to be 8 g/d owing to changes in blood parameters at the 12-g/d dosage.This trial was registered at clinicaltrials.gov as NCT04142294.

Tolerance to increased supplemented dietary intakes of methionine in healthy older adults.

Background: l-Methionine (Met) is an essential amino acid for humans and is important for protein synthesis and the formation of polyamines and is involved in the synthesis of many metabolites, including homocysteine. Free-Met supplements have been claimed to have multiple positive effects; however, it remains unclear what the exact tolerance level is. With aging, Met metabolism changes, and increased plasma homocysteine is more apparent. High plasma concentrations of homocysteine are assumed to be associated with a high risk of developing atherosclerosis.Objective: We estimated the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL) of supplemented, oral, free Met in healthy older adults by examining the increase in plasma homocysteine as the primary determinant.Design: We provided capsules with free Met to 15 healthy older adult subjects for 4 wk at climbing dosages of, on average, 9.2, 22.5, 46.3 and 91 mg · kg body weight-1 · d-1 with washout periods of 2 wk between each intake. Before, at 2 and 4 wk during, and 2 wk after each dosage, we studied a complete panel of biochemical blood variables to detect possible intolerance to increased Met intake. Plasma homocysteine and body composition were measured, and tolerance, quality of life, and cognitive function were assessed via questionnaires.Results: Plasma homocysteine was elevated with the highest dose of supplemented Met. The estimated NOAEL of supplemented Met was set at 46.3 mg · kg body weight-1 · d-1, and the estimated LOAEL of supplemented Met was set at 91 mg · kg body weight-1 · d-1 (on the basis of the actual intakes) in subjects independent of sex. No signs of intolerance were observed via questionnaires or other blood variables at the LOAEL. There were no meaningful changes in body composition.Conclusions: On the basis of plasma homocysteine, the NOAEL of supplemented Met intake is 46.3 and the LOAEL is 91 mg · kg body weight-1 · d-1 in healthy older adults. Both the NOAEL and LOAEL are not associated with meaningful effects on health and wellbeing. This trial was registered at clinicaltrials.gov as NCT02566434.

Proposals for Upper Limits of Safe Intake for Arginine and Tryptophan in Young Adults and an Upper Limit of Safe Intake for Leucine in the Elderly.

On the basis of research presented during the 9th Amino Acid Assessment Workshop, a No Observed Adverse Effect Level (NOAEL) for diet-added arginine (added mostly in the form of dietary supplements) of 30 g/d and an upper limit of safe intake (ULSI) for diet-added tryptophan (added mostly in the form of dietary supplements) of 4.5 g/d have been proposed. Both recommendations apply to healthy young adults. The total dietary leucine ULSI proposed for elderly individuals is 500 mg · kg-1 · d-1 All 3 recommendations are relevant only to high-quality amino acid-containing products with specifications corresponding to those listed in the US Pharmacopeia Because the above amino acids are extensively utilized as dietary supplements for various real or perceived benefits, such as vasodilation, spermatogenesis, sleep, mood regulation, or muscle recovery, the above safety recommendations will have an important impact on regulatory and nutritional practices.

Food-added monosodium glutamate does not alter brain structure or antioxidant status.

The International Glutamate Technical Committee (IGTC) comments on a recent publication in "Pathophysiology" entitled "Evidence of alterations in brain structure and antioxidant status following "low-dose" monosodium glutamate ingestion" (authored by Onaolapo, Onaolapo, Akanmu and Gbola) [1]. In particular, IGTC highlights that, in the view of published scientific literature [2-12], the methods of this study were inappropriate and did not support conclusions drawn by the authors.

A 4-week toxicity study of methionine in male rats.

To examine 4-week toxicity of l-methionine (methionine), 5-week-old Fisher strain male rats were fed on diets containing 0, 0.1, 0.3, 0.9, 2.7 (w/w) of added methionine. Although no deaths were recorded, the highest dose of methionine (2.7% [w/w] of diet) reduced food intake and significantly suppressed growth rate. Growth suppression was characterized by an increase in hemolysis, splenic, and hepatic accumulation of hemosiderin, hemolytic anemia, and promotion of hematopoiesis. Other changes observed in the highest methionine intake group were a decrease in white blood cell count, thymus atrophy, and histological abnormalities in the adrenal gland and testis. Small, but significant, growth suppression, accompanied by some minor changes in plasma biochemical parameters, was also seen in rats fed on a test diet containing 0.9% (w/w) of additional methionine. Thus, no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) of diet-added methionine were determined at 0.3% and 0.9% (w/w), corresponding to 236 and 705 mg/kg/d body weight, respectively. Since the basal diet contained protein-bound methionine at 0.5% (w/w), NOAEL and LOAEL of total dietary methionine were estimated at 0.8% and 1.4% (w/w) of diet.

Long-term ingestion of monosodium L-glutamate did not induce obesity, dyslipidemia or insulin resistance: a two-generation study in mice.

The use of monosodium glutamate (MSG) as a flavor enhancer spans more than 100 y and there are many studies indicating the safety of general use of MSG. Recently, however, Collison et al. (2010) reported a two-generation study with a low dose of MSG that caused abdominal obesity, insulin resistance and dyslipidemia in mice. Due to public health concerns over metabolic syndrome, their report merits careful analysis. The present study attempted to repeat the Collison et al. findings. Groups of male or female C57BL/6J mice were fed a control diet or one supplemented with high-fructose corn syrup (HFCS) at a level of 20%. Drinking water control was provided or treatment groups were given 0.064% MSG solution (w/v). Diets and MSG administration continued throughout mating and during gestation and lactation periods. To further investigate the effects of ingestion of MSG, the offspring were continued on the same dosing conditions until they reached 32 wk of age. MSG administration in mice fed a normal or a HFCS diet throughout gestation and for 32 wk after birth, did not affect growth, girth size, abdominal fat weight or body composition. This study reports that MSG did not trigger insulin resistance, dyslipidemia or hepatic steatosis, regardless of the diet, not reproducing the results of the above-mentioned study (Collison et al., 2010).

Effect of lysine supplementation on health and morbidity in subjects belonging to poor peri-urban households in Accra, Ghana.

BACKGROUND: Lysine affects diarrhea and anxiety via effects on serotonin receptors, enhanced intestinal repair, and sodium chloride-dependent opioid peptide transport. OBJECTIVE: The objective was to investigate the effects of lysine supplementation on morbidity, growth, and anxiety in children and adults of peri-urban areas of Accra, Ghana. DESIGN: In a double-blind randomized trial, the effect of lysine supplementation (1 g lysine/d) compared with that of placebo was examined in 2 groups of men, women, and children (n = 271). Primary outcomes included diarrheal and respiratory morbidity, growth, and anxiety and complement C3, C-reactive protein, serum cortisol, transferrin, and ferritin values. Independent-sample t tests, odds ratios, generalized estimating equations, 4-parameter sinusoid regression, and generalized linear models were used. RESULTS: Thirty percent of men, 50% of women, and 15% of children were at risk of lysine inadequacy. Supplementation in children reduced diarrheal episodes [19 lysine, 35 placebo; odds ratio (OR): 0.52; 95% CI: 0.29, 0.92; P = 0.046] and the total number of days ill (21 lysine, 47 placebo; OR: 0.44; 95% CI: 0.26, 0.74; P = 0.034). Mean days ill per child per week (0.058 ± 0.039 lysine, 0.132 ± 0.063 placebo; P = 0.017) were negatively associated with weight gain with control for baseline weight and study group (P = 0.04). Men had fewer coryza episodes (23 lysine, 39 placebo; OR: 0.60; 95% CI: 0.36, 1.01; P = 0.05), total number of days ill (lysine: 130; placebo: 266; OR: 0.51; 95% CI: 0.28, 0.93; P = 0.03), and mean days ill per person per week (lysine: 0.21 ± 0.23; placebo: 0.41 ± 0.35; P = 0.04). Serum ferritin (P = 0.045) and C-reactive protein (P = 0.018) decreased in lysine-supplemented women but increased in placebo-supplemented women. CONCLUSION: Lysine supplementation reduced diarrheal morbidity in children and respiratory morbidity in men in Ghana.

Impact of lysine-fortified wheat flour on morbidity and immunologic variables among members of rural families in northwest Syria.

BACKGROUND: Previous studies have shown an effect of lysine fortification on nutrition and immunity of poor men, women, and children consuming a predominantly wheat-based diet. OBJECTIVE: To examine the lysine value of diets and the effect of lysine fortification on functional protein status, anthropometry, and morbidity of men, women, and children in rural Syria. METHODS: At baseline of a two-phase study using 7-day household food intake inventories (n = 98), nutrient availabilities per adult male equivalent were estimated. In the intervention phase, a 16-week double-blind trial, households (n = 106) were randomly assigned to control and lysine groups. Hematologic and anthropometric data were collected from men (n = 69; 31 control, 38 lysine), women (n = 99; 51 control, 48 lysine), and children (n = 69; 37 control, 32 lysine) at baseline, 12 weeks, and 16 weeks. Total CD3 T lymphocytes as well as T lymphocytes bearing the receptors CD4, CD8, and CD56, IgM, IgG, IgA, complement C3, C-reactive protein, serum albumin, prealbumin, transferrin, retinol-binding protein, hemoglobin, and hepatitis B surface antigen were determined. Health status and flour usage were monitored. Paired- and independent-sample t-tests and chi-square tests were performed. RESULTS: Mean nutrient availability per adult equivalent was 2,650 +/- 806 kcal, 70.1 +/- 26.4 g protein, 65 +/- 14% cereal protein, and 41.9 +/- 0.8 mg lysine per gram of protein. Complement C3 was significantly higher in men receiving lysine than in controls (p < .05). Among women, there were significant differences between the control and lysine groups in diarrhea period prevalence (total number of diarrheal episodes during the period of intervention divided by the total number of observations), (20 in the control group, 6 in the lysine group; p = .014), the mean number of days ill (0.4 +/- 0.7, control, 0.14 +/- 0.4, lysine, p = 0.03), and the number of diarrheal episodes per person per year (1.39 in the control group, 0.47 in the lysine group). No other significant differences between the lysine and the control groups were observed. CONCLUSION: Lysine fortification of wheat flour demonstrated a positive effect on diarrheal morbidity in women. The effect could be attributed to an improvement in protein utilization but possibly also to a direct effect of lysine in gastrointestinal tract. Studies in populations with higher diarrheal prevalence and significant dietary lysine deficiency are needed to determine whether the reported effects on diarrheal prevalence are replicable and whether they are pharmacological or nutritional. It would be particularly desirable to study the effect of lysine on diarrhea in preschool children, who have much higher morbidity and mortality rates from this disease than school-age children or adults.

Oral treatment with L-lysine and L-arginine reduces anxiety and basal cortisol levels in healthy humans.

Dietary supplementation with an essential amino acid L-lysine has been shown to reduce chronic anxiety in humans with low dietary intake of L-lysine. A combination of L-lysine and L-arginine has been documented to normalize hormonal stress responses in humans with high trait anxiety. The present study was carried out in one hundred eight healthy Japanese adults. The aim of study was to find out whether a week-long oral treatment with L-lysine (2.64 g per day) and L-arginine (2.64 g per day) reduces trait and stress-induced state anxiety and basal levels of stress hormones. We confirmed that, without regard to gender, the amino acid treatment significantly reduced both trait anxiety and state anxiety induced by cognitive stress battery. In addition, we found that the treatment with L-lysine and L-arginine decreased the basal levels of salivary cortisol and chromogranin-A (a salivary marker of the sympatho-adrenal system) in male subjects. These results of this double-blind, placebo controlled and randomized study confirm the previous findings in humans and animals and point to a combination of L-lysine and L-arginine as a potentially useful dietary intervention in otherwise healthy humans with high subjective levels of mental stress and anxiety.

Exercise-dependent preference for a mixture of branched-chain amino acids and homeostatic control of brain serotonin in exercising rats.

In this article we discuss studies showing that rats are able to regulate their intake of BCAA depending on the level of exercise, and that they will choose a solution of BCAA over water during times of intense exercise. We found that the voluntary intake of a solution made of BCAA, L-arginine and L-glutamine positively correlated with the timing and volume of exercise during the dark (active) period of the circadian rhythm. In the second behavioral protocol in which rats were fed BCAA fortified diet (2.0%, wt:wt), we observed voluntarily increased volume of physical activity beginning from d 4 of feeding on. In the second, neuro-behavioral, part of the study we measured the brain content of 5-hydroxytryptamine (5-HT) as well as plasma amino acid profiles in well-trained exercising rats to test a hypothesis that BCAA may alleviate central aspects of fatigue. A solution made of BCAA, L-arginine, and L-glutamine applied before running elevated the BCAA/tryptophan plasma ratio at the end of and after running, and decreased 5-HT release in the lateral hypothalamus and amygdala after running, when compared with the controls. The exercise-related shift in the fluid preference toward a BCAA-based solution suggests an ergogenic benefit. The forced-running study shows the lateral hypothalamus and possibly amygdala might be the critical brain regions implied in the central effects of a BCAA-based solution.

Subchronic treatment with amino acid mixture of L-lysine and L-arginine modifies neuroendocrine activation during psychosocial stress in subjects with high trait anxiety.

The aim of the present study was to evaluate possible modulatory effect of the treatment with L-lysine and L-arginine on neuroendocrine activation during psychosocial stress in healthy subjects with relatively high trait anxiety in a randomized, double blind placebo controlled trial. In 29 healthy subjects at the upper limit of the normal range of a trait anxiety scale, a mixture of L-lysine and L-arginine (3 g each/day) was administered for 10 days followed by exposure to a psychosocial stress procedure based on public speech. Hormone levels, cardiovascular activation and skin conductance were measured. Amino acid treatment resulted in enhanced adrenocorticotropic hormone, cortisol, adrenaline and noradrenaline levels and galvanic skin responses during stress compared to those in placebo-treated group. Increases in the heart rate and blood pressure in response to public speaking task were not influenced by amino acid treatment. Results of the present study support the hypothesis that L-lysine in combination with L-arginine, which may induce anxiolytic effects, modify hormonal responses during psychosocial stress in humans. Such action may represent a normalization of hormone levels to the pattern observed previously in subjects with low trait anxiety.

Prolonged oral treatment with an essential amino acid L-leucine does not affect female reproductive function and embryo-fetal development in rats.

L-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of L-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7-17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0-20). L-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of L-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that L-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.