Safety assessment of L-Arg oral intake in healthy subjects: a systematic review of randomized control trials.

L-Arg is a nonessential amino acid but has many physiological roles. Accordingly, L-Arg has been used in various fields, but there is only limited information available about its safety upon overdose. Generally, the no-observed adverse effect level (NOAEL) is used when setting the upper amount for chemical substances. Recently, systematic reviews have been used to assess the safety as well as the effectiveness and usefulness of them. Therefore, we conducted an assessment of the safety of the oral intake of L-Arg in healthy subjects using gastrointestinal symptoms as an index. We limited the study design to only double-blind randomized controlled trials and searched PubMed, Cochrane Library, EBSCOhost, and Ichushi-Web from inception until May 2021. Assessment of the quality of studies was conducted using the Cochrane Collaboration tool and Jadad score, and the random effects model was used for data analysis. Ultimately, 34 studies were selected for inclusion in this work. The dosage of L-Arg used in the studies ranged from 2000 to 30,000 mg/day (or/one-time dose), and the treatment duration was 1-84 days. The increased risk of gastrointestinal symptoms associated with L-Arg intake from 23 studies (647 participants in total) in which such symptoms were reported was 0.01 (95% confidence interval: - 0.02-0.04), which was not significant difference. NOAEL was estimated as 7531 mg/ one-time dose using a weighted change-point regression model (UMIN000046133).Registration and protocol: Umin.ac.jp as UMIN000046133.

Supplementation of Dihomo-γ-Linolenic Acid for Pollen-Induced Allergic Symptoms in Healthy Subjects: A Randomized, Double-Blinded, Placebo-Controlled Trial.

Dihomo-γ-linolenic acid (DGLA) is an n-6 polyunsaturated fatty acid that has been shown to have anti-inflammatory and anti-allergic effects in mice and cell study. To date, however, no human intervention study has examined the effects of DGLA. Therefore, we investigated the effects of DGLA on pollen-induced allergic symptoms in healthy adults. We performed a randomized, double-blind, placebo-controlled, parallel-group study comprising healthy Japanese men and women. Each subject received four 250 mg capsules providing 314 mg DGLA/day (DGLA group, n = 18) or olive oil (placebo group, n = 15) for 15 weeks. The primary outcomes, classification of the severity of allergic rhinitis symptoms (CSARS), and the Japanese Rhino-conjunctivitis Quality of Life Questionnaire (JRQLQ) served as symptom scores during the pollen season. In the DGLA group, the cedar pollen associated symptoms of sneezing and a blocked nose in the CSARS were significantly lower than those in the placebo group (p < 0.05, p < 0.01, respectively). Significant trends were observed the symptoms of runny nose in the CSARS and total symptom score (TSS) in the JRQLQ for cedar pollen (p < 0.1). To our knowledge, this is the first study to report the effects of DGLA in humans, and the results suggest that DGLA is effective in reducing allergic symptoms caused by pollen.

A change-point regression approach for estimating no observed adverse effect level from systematic review.

Systematic reviews can be used not only to evaluate the efficacy and usefulness of a drug or food ingredient, but also as a safety assessment method. One of the aims of safety assessment is to estimate the no observed adverse effect level and the lowest observed adverse effect level. However, no methodology to statistically estimate the no observed adverse effect level from systematic review results has yet been reported. Estimation of the no observed adverse effect level involves a search for the dose above which adverse events occur is even exploration of the thresholds in dose response. To search for the dose above which adverse events occur, we examined an estimation method using the weighted change-point regression model, which includes the weights of each study used for systematic reviews in the model. This model could be applied to safety data of an omega-3 study in the form of a systematic review. We demonstrated that the dose response to omega-3 intake regarding adverse events had a threshold value and that the no observed adverse effect level could be estimated using the developed model.

Integrative Approaches of Bioassay and Computational Analysis for Discovering Potential Bioactive Compounds and Predictive Toxicity.

Changes in eating habits are brought about by drastic changes in lifestyle and environment, and, it has been pointed out, are strongly involved in the increase in neurological diseases and onset of cancer in younger adult ages. There is a wide variety of chemical substances in food, and there is a need to analyze the effects of complex exposures on complex mechanisms of action and to develop methods for evaluating and predicting them. The power of molecular nutrition needs to create an integrated approach to human nutrition in line with the grand social challenges of diet-related illnesses. The current article aims to explore some of these areas where integration is appropriate. Therefore, in this symposium, we will introduce the contents of four performers who are conducting cutting-edge research. 1) Chemoprevention by vitamin A and its derivatives, 2) Toxicity prediction of natural compounds from a developing database of bioactive gradients from Kampo medicine, 3) Toxicity prediction of chemicals using pluripotent stem cells. 4) Detection of bioactive compounds in "Aji" or "Umami" in food. By detecting and predicting the biological activity and toxicity of chemical substances such as nutrients in foods, it will be possible to provide better molecular information on dietary components. In addition, we will introduce next-generation health and prevention methods.

"Nutrient-Repositioning"-Unexpected Amino Acid Functions.

Repositioning is usually used to indicate drug repositioning, or the finding of new disease applications for existing, approved drugs. Nutrients can be ingested for nutritional as well as therapeutic purposes, acting much the same as drugs. Amino acids are organic compounds that possess both amino and carboxy group functionalities and are best known as building blocks of proteins in living organisms. Recent studies of individual amino acids have revealed them to be functional ingredients of new therapeutics, promoting health in addition to nutrition. Here, we propose "nutrient-repositioning", the discovery of effects different from the existing effects of nutrients. This review summarizes some recent discoveries of unexpected amino acid functions, especially in BCAAs, histidine and serine.

Effect of Docosahexaenoic Acid and Eicosapentaenoic Acid Supplementation on Sleep Quality in Healthy Subjects: A Randomized, Double-Blinded, Placebo-Controlled Trial.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)-omega-3 fatty acids with various functions-influence sleep in children and young adults. However, only limited studies on their effects on sleep in middle- and old-aged adults have been reported. Therefore, we investigated the effects of DHA and EPA on sleep quality in subjects aged ≥ 45 years. We performed a randomized, placebo-controlled, double-blinded, parallel-grouped study, in which we randomly assigned 66 healthy Japanese males and females. Each individual received six 480 mg capsules containing 576 mg DHA and 284 mg EPA per day (DHA/EPA group, n = 33), or corn oil (placebo group, n = 33), for 12 weeks. Before and after the intervention, the Oguri-Shirakawa-Azumi sleep inventory MA version (OSA-MA) and the sleep state test were conducted. In the DHA/EPA group, factor III (frequent dreaming) scores among the OSA-MA scores were significantly improved compared to the placebo group. Additionally, sleep state tests revealed that sleep efficiency improved in the DHA/EPA group. To our knowledge, this study is the first to report that DHA/EPA improves sleep quality in middle- and old-aged individuals, even at doses lower than those administered in previous studies.

Dietary Alaska Pollack Protein Induces Acute and Sustainable Skeletal Muscle Hypertrophy in Rats.

Our previous studies suggested that Alaska pollack protein (APP) intake increases skeletal muscle mass and that it may cause a slow-to-fast shift in muscle fiber type in rats fed a high-fat diet after 56 days of feeding. In this study, we explored whether dietary APP induces acute and sustainable skeletal muscle hypertrophy in rats fed a normal-fat diet. Male 5-week-old Sprague-Dawley rats were divided into four groups and fed a purified ingredient-based high-fat diet or a purified ingredient-based normal-fat diet with casein or APP, containing the same amount of crude protein. Dietary APP significantly increased gastrocnemius muscle mass (105~110%) after 2, 7 days of feeding, regardless of dietary fat content. Rats were separated into two groups and fed a normal-fat diet with casein or APP. Dietary APP significantly increased gastrocnemius muscle mass (110%) after 56 days of feeding. Dietary APP significantly increased the cross-sectional area of the gastrocnemius skeletal muscle and collagen-rich connective tissue after 7 days of feeding. It decreased the gene expression of Mstn /Myostatin, Trim63/MuRF1, and Fbxo32/atrogin-1, but not other gene expression, such as serum IGF-1 after 7 days of feeding. No differences were observed between casein and APP groups with respect to the percentage of Type I, Type IIA, and Type IIX or IIB fibers, as determined by myosin ATPase staining after 7 days of feeding. In the similar experiment, the puromycin-labeled peptides were not different between dietary casein and APP after 2 days of feeding. These results demonstrate that APP induces acute and sustainable skeletal muscle hypertrophy in rats, regardless of dietary fat content. Dietary APP, as a daily protein source, may be an approach for maintaining or increasing muscle mass.

Estimation of inter-individual variability of protein requirement by indicator amino acid oxidation method.

The indicator amino acid oxidation (IAAO) method is a recently developed method to determine the protein requirement and is particularly useful for analyzing human subjects because of its minimal invasiveness. IAAO study is performed using two-phase regression analysis, with the break-point between these phases being the estimated average requirement. However, this method requires that the break-point lie within a certain range in advance, which is in practice difficult. Recently, the change-point regression model (CPRM) has been proposed to be more effective for two-phase regression analysis. There is also a need to re-evaluate the value corresponding to the recommended dietary allowance. Calculation of the recommended dietary allowance requires data on the average requirement and the inter-individual variability of this requirement. However, no inter-individual variability values have been reported in the IAAO method. The aim of this study was thus to estimate the inter-individual variation in protein requirement using CPRM. From seven IAAO studies, the inter-individual variability was estimated as a coefficient of variation of about 20%. The coefficient of variation of the protein requirement determined by IAAO study was wider than the ordinary coefficient of variation obtained from the nitrogen balance test.

Therapeutic effects of sertraline on improvement of Ovariectomy-induced decreased spontaneous activity in mice.

We have already reported that ovariectomized (OVX) rats reduced the spontaneous activity during the dark period due to the decease of serotonin release in the amygdala. In this study, we examined the potential of sertraline, a selective serotonin reuptake inhibitor, on the recovery of less spontaneous activity seen in mice with OVX-induced despair-like behaviors. Female 9-week old ICR mice were underwent either OVX or sham surgery. Sertraline (10 mg/kg/day, s.c.) or saline were started to administer to each group for 8 weeks (6 times/week) from the 8th week after OVX. Each spontaneous activity of mouse was evaluated during the dark period (19:00-07:00) using an infrared sensor. Moreover, mRNA expression levels of tryptophan hydroxylase (TPH) and X-box binding protein 1 (XBP1) were measured in the hippocampus and prefrontal cortex using by a real-time PCR method. We found out that the OVX-induced despair-like behaviors were improved by the continuous administration of sertraline. After treatment of OVX, our real-time PCR data showed that sertraline significantly suppressed the upregulation of XBP1 expression levels in both hippocampus and prefrontal cortex, although this suppression of the downregulation of TPH expression levels was seen in only hippocampus. These results suggest that sertraline improves the decrease in spontaneous activity induced by OVX assessed by the hippocampus suppressing decreased serotonin synthesis in the serotonergic neuron.

Lactoferrin Suppresses Decreased Locomotor Activities by Improving Dopamine and Serotonin Release in the Amygdala of Ovariectomized Rats.

BACKGROUND: Decreases in female hormones not only affect bone metabolism and decrease bone mass, but also affect the central nervous system, causing brain disorders such as depression and dementia. Administration of estradiol by hormone replacement therapy can improve dementia, while reduced estradiol in ovariectomized (OVX) model rats can reduce both bone density and locomotor activity. The antidepressant fluvoxamine, which is widely used in clinical practice, can improve this effect on locomotor reduction. Similarly, lactoferrin (LF) can reportedly improve inhibitory locomotion due to stress. OBJECTIVE: In this study, we examined the effect of LF on neurite outgrowth in vitro and in vivo using PC12 cells and rats, respectively. METHODS: We performed an in vivo study in which 8-week-old female OVX rats were administered LF five days a week for 6 weeks from the day after surgery. After administration was completed, spontaneous locomotor activity in the dark period, immobility time in a forced swim test, and release amount of dopamine and serotonin in the brain were measured. RESULTS: LF was found to have a neurite outgrowth function in PC12 cells. Moreover, LF was found to improve OVX-induced decreases in locomotor activity and increases in immobility time in the forced swim test. Furthermore, the administration of LF elicited significant recovery of decreased dopamine and serotonin release in the brains of OVX group rats. CONCLUSION: These results strongly suggest that LF improved OVX-induced decreases in momentum during the dark period and, moreover, that release of dopamine and serotonin in the brain was involved in this effect.

Comprehensive Safety Assessment of l-Lysine Supplementation from Clinical Studies: A Systematic Review.

BACKGROUND: Despite the widespread use of l-lysine in dietary supplements, the safety information pertinent to excessive l-lysine ingestion is limited and, to the best of our knowledge, there is no published systematic review of safety. OBJECTIVE: The objective of this study was to assess the clinical safety of l-lysine supplementation of a regular diet. METHODS: We searched PubMed, Cochrane Library, Ichushi Web, and EBSCOhost using the relevant keywords, "l-lysine" and "clinical trial." To investigate all adverse events observed during intervention trials, we included all intervention studies with orally ingested l-lysine without restricting background factors, environment, study designs, and sample sizes. RESULTS: We identified 71 articles, which included 3357 study subjects. The l-lysine doses ranged from 16.8 to 17.5 g/d, and the dosing period ranged from 1 to 1095 d. The observed adverse events were mainly subjective gastrointestinal tract symptoms; however, the risk analysis for incidence of gastrointestinal symptoms was not statistically significant (risk ratio of 1.02). CONCLUSION: The provisional no-observed-adverse-effect level in healthy human subjects was based on gastrointestinal symptoms and identified at 6.0 g/d. The review protocol was registered at umin.ac.jp as UMIN000028914 before the beginning of the study.

Dietary Alaska pollack protein improves skeletal muscle weight recovery after immobilization-induced atrophy in rats.

The promotion of muscle recovery after immobilization is important to preserve an optimum health status. Here, we examined the effect of dietary Alaska pollack protein (APP) on skeletal muscle weight after atrophy induced by hind limb immobilization using plaster immobilization technique. Rat left limb was casted with a wetted plaster cast under anesthesia. After 2 weeks of feeding, the cast was removed and the rats were divided into three groups, namely, a baseline group, high-fat casein diet group, and high-fat APP diet group. After 3 weeks of feeding, the skeletal muscles (soleus, extensor digitorum longus [EDL], and gastrocnemius) were sampled. The estimated weight gains of soleus, gastrocnemius, and EDL muscle in the immobilized limbs were significantly larger in the rats fed with APP diet as compared with those fed with casein diet. In soleus muscle, dietary APP increased the expression of Igf1 and Myog genes in the immobilized limbs after the recovery period.

Safety assessment of L-lysine oral intake: a systematic review.

Currently, the use of amino acids in supplements and functional foods is increasing globally. However, there are no guidelines for the upper limit of ingestion for the safe use of these amino acids. Safety evaluation of chemical substances is generally performed through non-clinical and clinical studies. However, amino acids that have these safety data are limited. Therefore, we used a systematic review approach for evaluating the safety of amino acids. In the present study, we evaluated the safety of L-lysine added to an ordinary diet in humans. Using PubMed, Cochrane Library, Ichushi Web, and EBSCOhost as search databases, we comprehensively searched human studies on oral ingestion of L-lysine. Ultimately, 71 studies were selected for evaluation. Of these, 12 studies were of relatively high quality with Jadad scores ≥ 3. The dose range of L-lysine in the selected studies was 16.8-17,500 mg/day, and the range of dosing period was 1-1095 days. The observed adverse events were mainly subjective symptoms related to the gastrointestinal tract such as nausea, stomachache, and diarrhea. The provisional no-observed-adverse-effect level obtained based on these gastrointestinal symptoms was 6000 mg/person/day. Integrated analysis of the risk for developing gastrointestinal symptoms revealed that the risk ratio was 1.02 (95% CI, 0.96-1.07; p = 0.49); thus, no significant increase was observed. (UMIN000028914).

A 100-m Sprint Time Is Associated With Deep Trunk Muscle Thickness in Collegiate Male Sprinters.

Introduction: One reason athletes train their trunk muscles is that the body's trunk stability has been shown to prevent injury. However, the relationship between body trunk muscle thickness, particularly that of deep muscles, and athletic performance remains to be clarified. Purpose: We aimed to explore the relationship between 100-m sprint performance and the sizes of the trunk stabilizing muscles, the psoas major muscle (PM), transversus abdominis (TA), and multifidus muscle (MM), in collegiate sprinters. Methods: Fourteen male sprinters belonging to a university athletics club participated in this study. The thicknesses of the TA and MM were measured using an ultrasonic diagnostic apparatus (ProSound C3; Aloka, Tokyo, Japan). The cross-sectional area of the PM was assessed by a magnetic resonance imaging apparatus (Vantage Elan; Toshiba Medical Systems, Tokyo, Japan). The relationship between these anthropometric parameters and the 100-m sprint time was analyzed by Spearman's correlation coefficient, multi- regression analysis, and the change-point regression model. Results: The sizes (mean ± SD) of the muscles were: PM, 43.074 ± 7.35 cm2; TA, 4.36 ± 0.72 mm; and MM, 3.99 ± 0.48 cm. The mean 100-m sprint time was 11.00 ± 0.48 s. Spearman's correlation analysis revealed that the 100-m sprint time had a significant moderate negative correlation with TA (ρ = -0.691, p < 0.01) and a low negative but not significant correlation with MM (ρ = -0.327, p = 0.28), whereas PM did not show a significant or in-negligible correlation. The change-point regression model found the change-points in the 100-m sprint time and the thickness of the TA and MM at 4.70 mm (95% CI: 4.00-5.43 mm) and 3.84 cm (95% CI: 3.28-4.31 cm), respectively. The sprint time decreased with an increase in the thickness of the muscles up to the change-points, whereas it did not change even if the muscles became thicker than the change-points. The change-points were consistently observed when the thickness of the muscles was normalized by body mass. Conclusion: Sprint performance for 100-m was found to be associated with TA and MM thickness in a biphasic manner. As muscle thickness increased, the sprint time decreased, followed by a plateau phase.

The Effect of a High-Fat Diet on the Development of Abdominal Aortic Aneurysm in a Vascular Hypoperfusion-Induced Animal Model.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Most cases of AAA remain asymptomatic until rupture, and the mortality rate of patients with AAA rupture is very high. Currently, the relation between dietary habits and AAA development remains unknown. In this study, we evaluated the effects of a high-fat diet on the development of AAA in a vascular hypoperfusion-induced animal model. The risk of AAA rupture and AAA diameter in the high-fat group significantly increased compared with those in the control group. The number and size of adipocytes in the vascular wall in the high-fat group significantly increased as compared with those in the control group. Additionally, the collagen-positive sections in the areas with adipocytes significantly decreased as compared with those without adipocytes. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12, and macrophage-positive areas in the parts with adipocytes also significantly increased as compared with those without adipocytes. These data suggested that AAA rupture risk increased through accelerating chronic inflammation due to the accumulation of adipocytes in the vascular wall in the high-fat group.

Fish protein hydrolysate exhibits anti-obesity activity and reduces hypothalamic neuropeptide Y and agouti-related protein mRNA expressions in rats.

Fish protein is a source of animal protein that is consumed worldwide. Although it has been reported that the intake of Alaska pollack protein (APP) reduces body fat accumulation and increases muscle weight in rats, the mechanisms underlying these effects are poorly understood. As a possibility, peptides released from APP in the gastrointestinal tract are important to the functions of APP. In the present study, we examined the effects of APP hydrolysate digested artificially with pepsin and pancreatin on white adipose tissue and skeletal muscle. We found that APP hydrolysate group shows significantly lower weight of white adipose tissue and higher weight of soleus muscle than the control group. We also found that APP hydrolysate group reduces food intake and mRNA expressions of neuropeptide Y and agouti-related protein in the hypothalamus compared with the control group. These results may imply that APP hydrolysate exhibits anti-obesity activity by the reduction of appetite and the enhancement of basal energy expenditure by skeletal muscle hypertrophy in rats. The downregulation of orexigenic gene by APP hydrolysate in the hypothalamus may contribute to the reduction of appetite. These results suggest that the effect of APP on anti-obesity and muscle hypertrophy may be induced by peptides released from APP in the gastrointestinal tract.

Adipocyte in vascular wall can induce the rupture of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a vascular disease involving the gradual dilation of the abdominal aorta. It has been reported that development of AAA is associated with inflammation of the vascular wall; however, the mechanism of AAA rupture is not fully understood. In this study, we investigated the mechanism underlying AAA rupture using a hypoperfusion-induced animal model. We found that the administration of triolein increased the AAA rupture rate in the animal model and that the number of adipocytes was increased in ruptured vascular walls compared to non-ruptured walls. In the ruptured group, macrophage infiltration and the protein levels of matrix metalloproteinases 2 and 9 were increased in the areas around adipocytes, while collagen-positive areas were decreased in the areas with adipocytes compared to those without adipocytes. The administration of fish oil, which suppresses adipocyte hypertrophy, decreased the number and size of adipocytes, as well as decreased the risk of AAA rupture ratio by 0.23 compared to the triolein administered group. In human AAA samples, the amount of triglyceride in the adventitia was correlated with the diameter of the AAA. These results suggest that AAA rupture is related to the abnormal appearance of adipocytes in the vascular wall.

Eicosapentaenoic and docosahexaenoic acids-rich fish oil supplementation attenuates strength loss and limited joint range of motion after eccentric contractions: a randomized, double-blind, placebo-controlled, parallel-group trial.

PURPOSE: This study investigated the effect of eicosapentaenoic and docosahexaenoic acids-rich fish oil (EPA + DHA) supplementation on eccentric contraction-induced muscle damage. METHODS: Twenty-four healthy men were randomly assigned to consume the EPA + DHA supplement (EPA, n = 12) or placebo (PL, n = 12) by the double-blind method. Participants consumed EPA + DHA or placebo supplement for 8 weeks prior to exercise and continued it until 5 days after exercise. The EPA group consumed EPA + DHA-rich fish oil containing 600 mg EPA and 260 mg DHA per day. Subjects performed five sets of six maximal eccentric elbow flexion exercises. Changes in the maximal voluntary contraction (MVC) torque, range of motion (ROM), upper arm circumference, muscle soreness as well as serum creatine kinase, myoglobin, IL-6, and TNF-α levels in blood were assessed before, immediately after, and 1, 2, 3, and 5 days after exercise. RESULTS: MVC was significantly higher in the EPA group than in the PL group at 2-5 days after exercise (p < 0.05). ROM was also significantly greater in the EPA group than in the PL group at 1-5 days after exercise (p < 0.05). At only 3 days after exercise, muscle soreness of the brachialis was significantly greater in the PL group than in the EPA group (p < 0.05), with a concomitant increase in serum IL-6 levels in the PL group. CONCLUSION: Eight-week EPA + DHA supplementation attenuates strength loss and limited ROM after exercise. The supplementation also attenuates muscle soreness and elevates cytokine level, but the effect is limited.

A novel Alaska pollack-derived peptide, which increases glucose uptake in skeletal muscle cells, lowers the blood glucose level in diabetic mice.

We found that the tryptic digest of Alaska pollack protein exhibits a glucose-lowering effect in KK-Ay mice, a type II diabetic model. We then searched for glucose-lowering peptides in the digest. Ala-Asn-Gly-Glu-Val-Ala-Gln-Trp-Arg (ANGEVAQWR) was identified from a peak of the HPLC fraction selected based on the glucose-lowering activity in an insulin resistance test using ddY mice. ANGEVAQWR (3 mg kg(-1)) decreased the blood glucose level after intraperitoneal administration. Among its fragment peptides, the C-terminal tripeptide, Gln-Trp-Arg (QWR, 1 mg kg(-1)), lowered the blood glucose level, suggesting that the C-terminal is critical for glucose-lowering activity. QWR also enhanced glucose uptake into C2C12, a mouse skeletal muscle cell line. QWR did not induce the phosphorylation of serine/threonine protein kinase B (Akt) and adenosine monophosphate-activated protein kinase (AMPK). We also demonstrated that QWR lowered the blood glucose level in NSY and KK-Ay, type II diabetic models.