Tolerable amounts of amino acids for human supplementation: summary and lessons from published peer-reviewed studies.

Amino acid supplementation may be indicated to correct for insufficient amino acid intake in healthy individuals, and in specific physiological or pathophysiological situations. However, there is a concern to not supplement beyond the tolerable upper intake level (UL) by determining parameters of no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) for each amino acid. Since the NOAEL and LOAEL values are at least one order of magnitude different when comparing the values obtained in rats and humans, the aim of this review is to evaluate to what extent the amino acid UL measured in the rat model, when referenced to the dietary usual consumption (UC) and dietary requirement (RQ) for indispensable amino acids, may be used as an approximation of the UL in humans. This review then compares the ratios of the NOAEL or LOAEL over UC and RQ in the rat model with the same ratios calculated in humans for the nine amino acids (arginine, serine, glycine, histidine, leucine, lysine, methionine, phenylalanine, and tryptophan) for which this comparison can be done. From the calculations made, it appears that for these 9 amino acids, the calculated ratios for rats and humans, although rather different for several amino acids, remains for all of them in the same order of magnitude. For tryptophan, tyrosine, and valine, the ratios calculated in rats are markedly different according to the sex of animals, raising the view that it may be also the case in humans.

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.

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.

Rice endosperm protein slows progression of fatty liver and diabetic nephropathy in Zucker diabetic fatty rats.

We previously reported that rice endosperm protein (REP) has renoprotective effects in Goto-Kakizaki rats, a non-obese diabetic model. However, whether these effects occur in obese diabetes remains unclear. This study aimed to clarify the effects of REP on obese diabetes, especially on fatty liver and diabetic nephropathy, using the obese diabetic model Zucker diabetic fatty (ZDF) rats. In total, 7-week-old male ZDF rats were fed diets containing 20 % REP or casein (C) for 8 weeks. Changes in fasting blood glucose levels and urinary markers were monitored during the experimental period. Hepatic lipids and metabolites were measured and renal glomeruli were observed morphologically. HbA1c levels were significantly lower in rats fed REP, compared with C (P<0·05). Compared with C in the liver, REP prevented lipid accumulation (total lipid, TAG and total cholesterol, P<0·01). Liver metabolome analysis indicated that levels of metabolites associated with glycolysis, the pentose phosphate pathway and carnitine metabolism were significantly greater in the REP group than in the C group (P<0·05), suggesting activation of both glucose catabolism and fatty acid oxidation. The metabolite increases promoted by REP may contribute to suppression of liver lipid accumulation. Urinary excretion of albumin and N-acetyl-ß-d-glucosaminidase was significantly reduced in rats fed REP for 8 weeks (P<0·01). In addition, there was a distinct suppression of mesangial matrix expansion and glomerular hypertrophy in response to REP (P<0·01). Thus, REP had preventive effects on obese diabetes, fatty liver and diabetic nephropathy.

Protective effect of composite earthworm powder against diabetic complications via increased fibrinolytic function and improvement of lipid metabolism in ZDF rats.

Thrombosis is the leading cause of mortality globally. It is not only a complication but also a risk factor for progression of diabetes. However, alternative oral therapies and prophylaxis with less adverse effect for thrombosis have not been well studied. In this study, composite powder containing earthworm (CEP) was used and its fibrinolytic activity was measured. CEP was found to have a high urokinase-type plasminogen activator like activity in an in vitro assay. It also had significantly shortened euglobulin clot lysis time (ECLT) at 4 and 24 h after ingestion in Sprague Dawley rats. Zucker Diabetic Fatty rats were used to assess the effect of CEP on diabetes and diabetic nephropathy. After 10 weeks of feeding, CEP significantly shortened ECLT and attenuated HbA1c, hepatic lipid accumulation, and urinary albumin excretion and improved glomerular mesangial matrix score. Therefore, CEP may have beneficial effects on diabetes and diabetic nephropathy.

Diversity of amino acid signaling pathways on autophagy regulation: a novel pathway for arginine.

Autophagy is the intracellular bulk degradation process to eliminate damaged cellular machinery and to recycle building blocks, and is crucial for cell survival and cell death. Amino acids modulate autophagy in response to nutrient starvation and oxidative stress. We investigated the relevance of reactive oxygen species (ROS) production on the regulation of autophagy using amino acids, both as a mixture and individually, in rat hepatoma H4-II-E cells. Nutrient starvation elevated ROS production and stimulated autophagy. Treatment with complete (CAA), regulatory (RegAA) and non-regulatory (NonRegAA) amino acid mixtures showed significant suppression of ROS production, whereas only CAA and RegAA exhibited significant suppression of autophagy, suggesting a dissociation of the two responses. The effects of individual amino acids were examined. Leucine from RegAA decreased ROS production and suppressed autophagy. However, methionine and proline from RegAA and arginine, cystine and glutamic acid from NonRegAA suppressed autophagy with an opposite increase in ROS production. Other amino acids from the NonRegAA group showed stimulating effects on ROS production without an autophagic response. Arginine's effect on autophagy suppression was not blocked by rapamycin, indicating an mTOR-independent pathway. Inhibitor studies on arginine-regulated autophagy may indicate the involvement of NO pathway, which is independent from ROS and mTOR pathways.

A quick signal of starvation induced autophagy: transcription versus post-translational modification of LC3.

Autophagy is the major intracellular lysosomal bulk degradation pathway induced by nutrient starvation and contributes to the elimination of damaged organelles and protein aggregates to recycle building block and is essential for cell survival. Microtubule-associated protein 1 light chain 3 (LC3) plays an indispensable role in macroautophagy formation and is a molecular marker for the process. Here, we show that autophagy increased through quick robust signaling from starvation by enhanced levels of LC3, LC3-EGFP (enhanced green fluorescent protein) punctate, and bulk proteolysis in rat hepatoma H4-II-E cells and fresh rat hepatocytes. After the addition of amino acids to the starvation condition, a similar quick signal appeared by significant reduction of the LC3 ratio and bulk proteolysis. Interestingly, we observed that post-translational modification of LC3 conversion occurred even long before the changes happened in the level of LC3-mRNA (messenger RNA) expression. A similar coordinated but diverse effect on LC3 was confirmed by using autophagy and lysosomal inhibitors. These results indicated that during starvation the initial robust signal to the cytoplasm can induce autophagy activity through modification at the protein level, whereas after depleting readily available autophagy proteins the signal goes to the DNA transcription level to maintain the autophagy capacity of cells.

Rice protein ameliorates the progression of diabetic nephropathy in Goto-Kakizaki rats with high-sucrose feeding.

The effect of rice protein (RP) on diabetic nephropathy in non-obese, spontaneous type 2 diabetic Goto-Kakizaki (GK) rats was investigated.GK rats at 7 weeks of age were fed 20% RP or casein (C) in standard or high-sucrose diets for 10 weeks. Plasma total cholesterol,TAG, alkaline phosphatase (ALP), adiponectin, creatinine and urinary albumin excretion (UAE) were measured and renal histology was evaluated. Compared with C, RP lowered plasma TAG and improved plasma adiponectin levels in GK rats fed the standard diet (P<0·05), and also lowered total cholesterol and ALP in high-sucrose-fed GK rats (P<0·05). RP markedly suppressed the sharp increase in UAE when GK rats were fed high-sucrose diets (P<0·05), and prevented glomerular mesangial matrix expansion in the deep renal cortex near the corticomedullary junction (P<0·05). These results strongly indicate that dietary RP can ameliorate the progression of diabetic nephropathy at an early stage compared with C.

A proposal for an upper limit of leucine safe intake in healthy adults.

Based on recent research, an upper limit of safe intake (ULSI) for leucine is proposed for healthy adults: 0.53 g/(kg·d). Because leucine has been used as a dietary supplement for many years in people practicing exercise and sport, further study with long-term exposure to leucine in this specific subpopulation should be performed to eventually adjust the ULSI.

Sake lees fermented with lactic acid bacteria prevents allergic rhinitis-like symptoms and IgE-mediated basophil degranulation.

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

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.

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells.

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 microM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, alpha-lipoic acid, exerted autophagy suppression, while H(2)O(2) stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H(2)O(2), rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.

Improvement in the in vivo digestibility of rice protein by alkali extraction is due to structural changes in prolamin/protein body-I particle.

Rice prolamin, constituting type-I protein body (PB-I), is indigestible and causes deterioration of rice protein nutritional quality. In this study, the in vivo digestibility of rice protein isolates was investigated by tracing their intraluminal transit in the gastrointestinal (GI) tract of rats by western blotting and by observing the structures excreted in the feces by electron microscopy. Two types of rice protein isolates, produced by alkali extraction (AE-RP) and by starch degradation (SD-RP), were compared. The protein patterns in the isolates were similar, but their digestion in the GI-tract showed striking differences. In the AE-RP group, 13-kDa prolamin (13P) quickly disappeared in the lower GI tract and was not excreted in the feces. By contrast, in the SD-RP group, 13P accumulated massively and nearly intact PB-Is were excreted. These results indicate that the in vivo digestibility of prolamin can be improved by alkali extraction through structural changes to it.

Reduction in dietary lysine increases muscle free amino acids through changes in protein metabolism in chickens.

Taste is crucial to meat quality, and free Glu is an important taste-active component in meat. Our recent study showed that the short-term feeding of a low-Lys diet increases the concentration of free Glu and other free amino acids in chicken muscle and improves its taste. Here, we investigated the mechanisms by which the feeding of a low-Lys diet increases free Glu in chicken muscle. Two groups (n = 10 per group) of 28-day-old female Ross strain broiler chickens were fed diets with a graded Lys content of 90% or 100% of the recommended Lys requirement (according to National Research Council [1994] guidelines) for 10 D. Free amino acid concentrations and the mRNA abundance of protein metabolism-related genes were measured in breast muscle, and breast muscle metabolome analysis was conducted. Free Glu in muscle was increased by 51.8% in the Lys 90% group compared with the Lys 100% group (P < 0.01). Free threonine, glutamine, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, and 3-methyl-histidine concentrations in breast muscle were also increased in the Lys 90% group (P < 0.05). Metabolome analysis also showed that free amino acids were increased in the Lys 90% group. The mRNA abundance of µ-calpain, caspase-3, and 20S proteasome C2 subunit were increased in the Lys 90% group (P < 0.05). Moreover, the free Glu concentration in muscle was correlated with mRNA abundance of µ-calpain (r = 0.74, P < 0.01), caspase 3 (r = 0.69, P < 0.01), 20S proteasome C2 subunit (r = 0.65, P < 0.01), and cathepsin B (r = 0.52, P < 0.05). Our study suggests that the feeding of a low-Lys diet to chickens increased the free Glu content of breast muscle by promoting protein degradation.

Effects of rice proteins from two cultivars, Koshihikari and Shunyo, on hepatic cholesterol secretion by isolated perfused livers of rats fed cholesterol-enriched diets.

BACKGROUND/AIMS: The aim of the present study was to clarify the effect of rice proteins, with different contents of glutelin and prolamin, on the regulation of hepatic cholesterol output pathways and the development of hypocholesterolemia in rats. METHODS: Seven-week-old male Wistar rats were fed 2 types of rice protein from either the cultivar Koshihikari (RRP) or the cultivar Shunyo (SRP), or casein as a control, for 2 weeks (n = 6 for each group). Each diet was supplemented with 1% cholesterol and 0.25% sodium cholate. Using an isolated perfused liver, hepatic secretion of cholesterol into bile and the circulation was measured during a 4-hour perfusion. RESULTS: Total hepatic cholesterol secretions into the circulation were significantly reduced by both rice proteins (p < 0.05), and hepatic cholesterol secretions into very-low-density lipoproteins were also effectively decreased by RRP and SRP. In contrast, bile flow and biliary output of bile acids were significantly stimulated by RRP and SRP (p < 0.05). CONCLUSIONS: These results demonstrate that the key metabolic pathways of hepatic cholesterol are modified by both rice proteins leading to similar hypocholesterolemic effects. The increased excretion of biliary bile acids associated with a decreased output of hepatic cholesterol into the circulation suggests a functional reciprocal interrelationship between both of the hepatic cholesterol secretory pathways in the rice-protein-fed rats, regardless of rice protein type.

Physiological Multifunctions of Rice Proteins of Endosperm and Bran.

Although it is considered a staple food, rice intake is under serious debate for its physiological usefulness, especially for diabetic patients, because of starch content. However, rice protein, the second major component of rice, has gained attention recently for its newly-discovered functions, which were previously unknown. Rice protein, a plant protein, shows multiple beneficial functions on lipid metabolism and diabetes and its complications, nephropathy, fatty liver and osteoporosis. Rice proteins of endosperm and bran, an ingredient of white rice and an unused product of brown rice, respectively, are valuable components for human health.

The 7th workshop on the assessment of adequate intake of dietary amino acids: summary of general discussion.

Extensive discussion sessions were held at the end of each of the 2 d of the workshop. Through the course of the workshop, it became clear that there were different opinions on how to use uncertainty factors to obtain upper levels of intake from no observed adverse effect levels of a particular nutrient and that the selection of an appropriate uncertainty factor would be rather arbitrary. Much of the discussion centered around the potential for using metabolic limits, expressed as the level of intake at which the major pathway of metabolism may approach saturation and at which the amino acid is metabolized by alternative pathways, as a measurable early or surrogate marker for amino acid excess and possible toxicity. After extensive discussion on various conditions that would need to be satisfied for metabolic limits to be used as markers of excessive intake of amino acids, there was a general consensus that methods such as measuring oxidation limits are an attractive approach that merit future investigation. It was noted that there are many data on the clinical use of glutamine, whereas data for proline are very scarce. There was recognition that regardless of the available data, there is regulatory pressure for setting upper levels of intake for amino acids and that much more data are required.

Effect and proposed mechanism of vitamin C modulating amino acid regulation of autophagic proteolysis.

Autophagy is an intracellular bulk degradation process, induced under nutrient starvation. Failure of autophagy has been recognized as a contributor to aging and multiple age related neurodegenerative diseases. Improving autophagy is a beneficial anti-aging strategy, however very few physiological regulators have been identified. Here, we demonstrate that vitamin C is a nutritional stimulator of autophagy. Supplementation of fresh hepatocytes with vitamin C increased autophagic proteolysis significantly in the presence of amino acids in a dose- and time-dependent manner, although no effect was observed in the absence of amino acids. In addition, inhibitor studies with 3-methyladenine, chloroquine, leupeptin and ß-lactone confirmed that vitamin C is active through the lysosomal autophagy and not the proteasome pathway. Furthermore, the autophagy marker LC3 protein was significantly increased by vitamin C, suggesting its possible site of action is at the formation step. Both the reduced (ascorbic acid, AsA) and oxidized form (dehydroascorbic acid, DHA) of vitamin C exhibited equal enhancing effect, indicating that the effect does not depend on the anti-oxidation functionality of vitamin C. To understand the mechanism, we established that the effective dose (50 µM) was 15× lower than the intracellular content suggesting these would be only a minor influx from the extracellular pool. Moreover, transporter inhibitor studies in an AsA deficient ODS model rat revealed more accurately that the enhancing effect on autophagic proteolysis still existed, even though the intracellular influx of AsA was blocked. Taken together, these results provide evidence that vitamin C can potentially act through extracellular signaling.