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.

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.

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.

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.

Thirteen-week oral toxicity study of L-arginine in rats.

The amino acid L-arginine (Arg) has been used extensively in dietary and pharmacological products. This study evaluated toxicological and behavioral effects of Arg produced by Ajinomoto Co. (Tokyo, Japan) during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received only a standard diet. All diets were administered ad libitum for 13 continuous weeks. To examine recoverability of any potential effects, the administration period was followed by a 5-week-long recovery, during which only a standard diet was provided. In male and female rats in each concentration group, treatment-related changes were not observed for clinical signs, body weights, diet consumption, ophthalmology, gross pathology, organ weight, or histopathology. An elevated level of plasma glucose was detected in some male rats (5.0%, w/w) during the analysis conducted in the fifth week of administration; however, the degree of the change was within the physiological range, and no changes were observed at the end of the administration period. In the same group, an increase in hemoglobin, together with a tendency toward an increase in the red blood cell counts, was found, but the change was considered toxicologically insignificant. The no-observed-adverse-effect level (NOAEL) for Arg was estimated at 5.0% (w/w) for both genders (males, 3.3 +/- 0.1 g/kg/day; females, 3.9 +/- 0.2 g/kg/day).

Thirteen-week oral toxicity study of L-glutamine in rats.

L-Glutamine (Gln) is a semiessential amino acid used in enteral feeding in critically ill patients, and is contained in numerous dietary supplements available to the general public. This study evaluated toxicological effects of Gln in male and female Sprague-Dawley rats. Gln produced by Ajinomoto Co. (Tokyo, Japan) was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w), respectively. A control group of rats received only a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine recoverability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. Throughout the administration and recovery periods, no deaths were observed, and no changes in diet consumption, ophthalmologic findings, gross pathology, and histopathology were detected. Several changes in urine parameters (total protein, urine pH, and a positive incidence (+/-) of ketone bodies) were observed in the 2.5% and 5.0% groups at the end of the administration period. Minor increases were found in hematology parameters for the 5.0% group (platelet count, gamma-globulin, lactate dehydrogenase [LDH]), but all changes were within physiological range. No effects of administration were observed in the 1.25% group. The no-observed-adverse-effect level (NOAEL) for Gln was estimated at 1.25% for both genders (males 0.83 +/- 0.01 g/kg/day; females, 0.96 +/- 0.06 g/kg/day).

Thirteen-week oral toxicity study of L-lysine hydrochloride in rats.

L-Lysine hydrochloride (Lys) is an essential amino acid in humans and animals, and it is used in animal feeds, in prevention of herpes simplex recurrence, and cereal fortification in some developing countries. This study evaluated toxicological and behavioral effects of Lys during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. In male and female rats in each concentration group, treatment-related changes were not observed in the clinical signs, body weights, diet consumption, water intake, ophthalmology, gross pathology, organ weights, or histology. A Lys-related drop in serum concentration and an increase in urine excretion of chlorides was a compensatory reaction to the ingested hydrochloride. No functional, biochemical, or histological changes in renal function were found. The no-observed-adverse-effect level (NOAEL) for Lys was estimated at 5.0% for both genders (male, 3.36 +/- 0.12 g/kg/day; female, 3.99 +/- 0.28 g/kg/day).

Thirteen-week oral toxicity study of branched-chain amino acids in rats.

Branched-chain amino acids (L-isoleucine, L-valine, and L-leucine) are being increasingly used in sport supplements. This study evaluated toxicological and behavioral effects of L-isoleucine (Ile), L-valine (Val), and L-leucine (Leu) during a dosing study with male and female Sprague-Dawley rats. The amino acids were incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. No significant, dose-related effects on body weight were found in rats fed a Leu- and Ile-supplemented diet. Val mixed into a diet at 5.0% (w/w) decreased slightly, but significantly body weight gain in females, but not males. Ile (5.0% w/w) affected the urine electrolytes, protein, ketone bodies, urine glucose, and urobilinogen in both genders, yet the observed changes remained mostly within the range observed in controls. The random findings in hepatology and ophthalmology at the 13-week sacrifice were not considered toxicologically relevant to effects of the tested amino acids. No significant changes in organ weights were recorded. We estimate the no-observed-adverse-effect level (NOAEL) for Ile at 2.5% for both genders (male, 1.565 +/- 0.060 g/kg/day; females, 1.646 +/- 0.095 g/kg/day), Val at 5.0% for males (3.225 +/- 0.135 g/kg/day) and 2.5% for females (1.853 +/- 0.060 g/kg/day), and Leu at 5.0% for both genders (males, 3.333 +/- 0.101 g/kg/day: females, 3.835 +/- 0.257 g/kg/day).

Lysine fortification reduces anxiety and lessens stress in family members in economically weak communities in Northwest Syria.

Lysine is a limiting amino acid in diets based on wheat as the staple. In experimental animals, prolonged dietary lysine inadequacy increases stress-induced anxiety. If observed in humans, such a result would have a strong implication for the relationship between nutrition and communal quality of life and mental health. As part of a 3-month randomized double-blind study, we tested whether lysine fortification of wheat reduces anxiety and stress response in family members in poor Syrian communities consuming wheat as a staple food. In the lysine-fortified group, the plasma cortisol response to the blood drawing as a cause of stress was reduced in females, as was sympathetic arousal in males as measured by skin conductance. Lysine fortification also significantly reduced chronic anxiety as measured by the trait anxiety inventory in males. These results suggest that some stress responses in economically weak populations consuming cereal-based diets can be improved with lysine fortification.

A diet fortified with L-lysine and L-arginine reduces plasma cortisol and blocks anxiogenic response to transportation in pigs.

We studied the effects of diet fortified with L-lysine HCl (Lys) and L-arginine (Arg) on stress (transportation) responses in male finishing pigs (Landrace x LargeWhite x Duroc). Pigs (n = 16) were randomly divided into two equally sized groups so that the average starting body weight in the groups was identical. For 1 week immediately preceding the transportation, the first group of pigs received a control diet while the second group received a Lys and Arg fortified diet. Plasma aminogram, cortisol and body weight were evaluated. Behavior of pigs was measured with the help of a video camera, recorded for 2 h at the same time, as on the day, before a day and immediately after transportation. The study revealed main stimulatory effects of transportation and main inhibitory effect of Lys and Arg on plasma cortisol (P < 0.05) without transportation x treatment interactions. Pigs fed with Lys and Arg diet tend to have higher body weight at the end of the experiment, when compared to their normally fed counterparts, but the difference did not reach a significant level (P < 0.21). Lys and Arg diet significantly inhibited stress-induced increase in locomotion (P < 0.05), without affecting feeding pattern. Transportation stress decreased plasma Lys and Arg. This decrease was reversed in the fortified group, and what is more the plasma Lys and Arg levels were significantly higher than in controls (P < 0.05). Lys and Arg enhanced plasma urea production (P < 0.05), without regards to stress. The behavioral results indicate a reduction in stress-induced anxiety in pigs fed with Lys and Arg fortified diet, that parallels similar observations in research with rats and broilers. The mechanism probably involves a decreased plasma cortisol, and/or normalized plasma Lys, Arg levels.

Preference for a solution of branched-chain amino acids plus glutamine and arginine correlates with free running activity in rats: involvement of serotonergic-dependent processes of lateral hypothalamus.

Rats were given free access to a running wheel, food, water, and a solution composed of branched-chain amino acids plus glutamine and arginine (the "BCAA-based" solution). A positive relationship between dark-period running distance and preference for the BCAA-based solution was observed. Serotonin release in the lateral hypothalamus, the central nucleus of amygdala and the medial nucleus raphe in overnight fluid-deprived rats during their first subsequent free drinking was also measured. A lowered serotonin release in the lateral hypothalamus characterized the rats that consumed the BCAA-based solution. No drink-related changes were observed in the amygdala. A separate group of rats was trained on a treadmill. Following the training period, plasma amino acids and brain serotonin release were measured during running. The BCAA-based solution infused before running elevated the branched chain amino acids/tryptophan plasma ratio at the end of, and after, running. Additionally, a decreased lateral hypothalamus serotonin release was seen 80 min after running, when compared with water-infused rats. No fluid-related changes in the amygdala were observed. The exercise-related shift in the fluid preference towards a BCAA-based solution suggests an ergogenic benefit. The forced-running study shows the lateral hypothalamus as a critical region in the effects of a BCAA-based solution.