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Determination of the tolerable upper intake level of leucine in acute dietary studies in young men
Abstract
Leucine has been suggested to improve athletic performance. Therefore, the branched-chain amino acids (BCAAs), especially leucine, are popular as dietary supplements in strength-training athletes; however, the intake of leucine in excess of requirements raises concerns regarding adverse effects. Currently, the tolerable upper intake level (UL) for leucine is unknown.
The objective of the current study was to determine the UL for leucine in adult men under acute dietary conditions.
Five healthy adults (20-35 y) each received graded stepwise increases in leucine intakes of 50, 150, 250, 500, 750, 1000, and 1250 mg · kg(-1) · d(-1), which corresponded to the Estimated Average Requirement (EAR) and the EAR ×3, ×5, ×10, ×15, ×20, and ×25 in a total of 29 studies. The UL of leucine was identified by the measurement of plasma and urinary biochemical variables and changes in leucine oxidation by using l-[1-(13)C]-leucine.
A significant increase in blood ammonia concentrations above normal values, plasma leucine concentrations, and urinary leucine excretion were observed with leucine intakes >500 mg · kg(-1) · d(-1). The oxidation of l-[1-(13)C]-leucine expressed as label tracer oxidation in breath (F(13)CO(2)), leucine oxidation, and α-ketoisocaproic acid (KIC) oxidation led to different results: a plateau in F(13)CO(2) observed after 500 mg · kg(-1) · d(-1), no clear plateau observed in leucine oxidation, and KIC oxidation appearing to plateau after 750 mg · kg(-1) · d(-1).
On the basis of plasma and urinary variables, the UL for leucine in healthy adult men can be suggested at 500 mg · kg(-1) · d(-1) or ∼35 g/d as a cautious estimate under acute dietary conditions. This trial was registered at clinicaltrials.gov as NCT00972582.
... Study in young men to determine the UL for leucine. We tested the same model of determining the UL, as proposed above, initially in young men (24). Young men (aged ;27 y) participated in a dose-escalated response study, in which each subject was initially studied at a leucine intake of 50 mg Á kg 21 Á d 21 [because the current leucine Estimated Average Requirement (EAR) is set at 50 mg Á kg 21 Á d 21 ]. ...
... No significant changes were observed for blood and urine biochemical measures. Although leucine has been suggested to act as an insulin secretagogue, we did not observe significant changes in plasma insulin and plasma glucose due to increasing leucine intakes (24). Taking all of the data together, 500 mg Á kg 21 Á d -1 was suggested as the UL for leucine intake under acute dietary conditions (24,25). ...
... Although leucine has been suggested to act as an insulin secretagogue, we did not observe significant changes in plasma insulin and plasma glucose due to increasing leucine intakes (24). Taking all of the data together, 500 mg Á kg 21 Á d -1 was suggested as the UL for leucine intake under acute dietary conditions (24,25). ...
Leucine, a branched-chain amino acid, has been shown to stimulate muscle protein synthesis and has been suggested to play a role in the prevention of age-related muscle atrophy (sarcopenia). Although leucine supplementation may be beneficial, the efficacious dose of leucine is unknown. Before conducting studies with increased doses of leucine, the Tolerable Upper Intake Level (UL) for leucine needs to be determined. The objective of this review is to describe 2 current studies to determine the UL for leucine in young and elderly men. Initially, in young men we tested the conceptual model of determining the maximum oxidative capacity of an amino acid to be an ideal marker for identifying the UL. Leucine oxidation, measured with the use of L-[1-13C]leucine, increased with increasing leucine intakes and reached a plateau at higher intakes. Two-phase linear regression analysis identified a breakpoint of 550 mg · kg⁻¹ · d⁻¹ (95% CI: 454, 646 mg · kg⁻¹ · d⁻¹), with a simultaneous increase in blood ammonia concentrations above normal values (35 mmol/L). Recently, a similar study was conducted in elderly men (~72 y old). A breakpoint in leucine oxidationwas observed at 431 mg · kg⁻¹ · d⁻¹ (95% CI: 351, 511 mg · kg⁻¹ · d⁻¹), with blood ammonia concentrations above normal (35 mmol/L) at leucine intakes >550 mg · kg⁻¹ · d⁻¹. Taking the data together, the UL for leucine intake in healthy elderly men could be set at a value similar to young men, at 500 mg · kg⁻¹ · d⁻¹, or ~35 g/d for an individual weighing 70 kg; or, as a cautious estimate, the leucine UL could also be considered as 351 mg · kg⁻¹ · d⁻¹ (the lower 95% CI), which would be ~24.5 g/d for an elderly individual weighing 70 kg. These studies to determine the UL for leucine in humans are acute diet studies, and future studies with additional biomarkers and long-term supplementation of leucine will be necessary.
... Once the oxidative capacity has been reached, it was proposed that plasma amino acid concentrations will rise, including related metabolites. Recently we tested this hypothesis in young adult men (Elango et al. 2012). The objective of the previous study was to identify the UL for leucine intake in young men under acute dietary conditions. ...
... Twophase linear regression analysis identified a breakpoint in F 13 CO 2 at a leucine intake of 550 mg kg −1 day −1 . Thus, taking all the data together, as a cautious estimate, 500 mg kg −1 day −1 was suggested as the UL for leucine intake under acute dietary conditions (Elango et al. 2012;Cynober et al. 2012); whether aging affects the leucine UL is unknown. ...
... Following this baseline study subjects received increased dietary leucine in a graded stepwise intake of 150, 250, 350, 450, 550, 650, and 750 mg kg −1 day −1 corresponding to the EAR, EAR ×3,×7, ×9, ×11, ×13, and ×15 on separate study days. In addition, the leucine test intakes were also chosen based on the previous Leucine UL study in young men (Elango et al. 2012), where the upper limit to oxidize leucine was determined to be 550 mg kg −1 day −1 . In the current study we predicted that there would be an age-related decline in the ability to dispose of leucine and therefore decided to increase leucine intake only up to 750 mg kg −1 day −1 . ...
Leucine, a branched-chain amino acid (BCAA), has been shown to stimulate muscle protein synthesis, and thus has been proposed to prevent age-related muscle atrophy (sarcopenia). Therefore, leucine supplementation may have potential benefits in elderly populations to preserve muscle mass. The tolerable upper intake level (UL) for leucine intake in young men has recently been determined to be 500 mg kg−1 day−1, and increases in blood ammonia concentrations were seen at intake levels above 500 mg kg−1 day−1; the UL for leucine in elderly is unknown. The objective of the current study was to determine the safety of leucine supplementation in healthy elderly men. Six healthy elderly men (72.2 ± 3.5 years) received graded stepwise increases in leucine intakes ranging from 50 to 750 mg kg−1 day−1, on eight separate study days. Plasma and urinary biochemical variables, including blood ammonia, and an oral primed-continuous protocol of L-1-13C-Leucine was performed. Blood ammonia concentrations above normal values (35 µmol/L) were observed at leucine intakes >550 mg kg−1 day−1. Leucine oxidation measured as a F13CO2 (rate of label tracer oxidation) increased with increasing leucine intakes and started to plateau after 450 mg kg−1 day−1. Two-phased linear regression analysis of the F13CO2 data revealed a breakpoint of 431 mg kg−1 day−1 (R
2 = 0.73), suggesting that the upper limit to oxidize leucine was reached at that point. Taking the data together the upper limit for leucine intake in healthy elderly could be set similar to young men at 500 mg kg−1 day−1 or ~35 g/day for an individual weighing 70 kg.
... Thus, there is a need to identify direct approaches to determine a safe upper limit of leucine intake. Recently, we proposed one such approach (18) and the following article describes our approach with the experimental evidence (19). ...
... We recently reported a study of the effects of graded doses of leucine in healthy young men aimed at defining the UL of leucine in the acute phase (19). Participants received increased dietary leucine in a graded stepwise intake of 50, 150, 250, 500, 750, 1000, and 1250 mg Á kg 21 Á d 21 corresponding to the Estimated Average Requirement and Estimated Average Requirement 33, 35, 310, 315, 320, and 325 on separate study days (26). ...
... F 13 CO 2 increased with increasing leucine intakes up to 500 mg Á kg 21 Á d 21 , after which oxidation remained at a plateau up to intakes of 1250 mg Á kg 21 Á d 21 . Using 2-phase linear regression analysis (34,35), a breakpoint in F 13 CO 2 was identified at 550 mg Á kg 21 Á d 21 (r 2 = 0.85) (19). This breakpoint identifies the maximum oxidative potential in adult humans to dispose of excess dietary leucine intake. ...
Leucine is purported to improve athletic performance. Therefore, the BCAA, especially leucine, are popular as dietary supplements among strength-training athletes. There are, however, concerns regarding possible adverse effects of excessive leucine intake. The objective of the current study was to determine the metabolic and adverse effects of the acute ingestion of very high intakes of leucine supplements. Five healthy men (20-35 y) each received graded stepwise increases in leucine intakes of 50, 150, 250, 500, 750, 1000, and 1250 mg ⋅ kg(-1) ⋅ d(-1) corresponding to the Estimated Average Requirement, and Estimated Average Requirement ×3, ×5, ×10, ×15, ×20, and ×25 to a total of 29 studies. The graded stepwise approach was used rather than a randomization of leucine intake to minimize the possibility of severe adverse effects. Participants were given a maintenance diet for 2 d prior to each leucine level containing 1 g ⋅ kg(-1) ⋅ d(-1) of protein and 1.7× measured the resting metabolic rate. Leucine oxidation was determined using l-[1-13C]-leucine and the appearance of (13)CO(2) (calculated as F(13)CO(2)) in breath. A range of markers was used to monitor for adverse effects, including glucose, insulin, alanine aminotransferase, and ammonia. Plasma leucine concentrations significantly increased beyond an intake of 500 mg ⋅ kg(-1) ⋅ d(-1). The metabolic limit to oxidize leucine was between 550 and 700 mg ⋅ kg(-1) ⋅ d(-1). An increase in blood ammonia concentrations was observed at leucine intakes >500 mg ⋅ kg(-1) ⋅ d(-1). There were no changes in liver alanine aminotransferase. Glucose concentrations fell (P < 0.004) but remained within the normal range and without any change in insulin. This study is the first to our knowledge to directly estimate the safe upper limit of leucine intake in humans and raises concerns that intakes >550 mg ⋅ kg(-1) ⋅ d(-1) or ∼39 g/d may be a risk to health. It is important to note that these are acute studies, where each participant was exposed to graded increases in leucine intake. Longer term adaptation was not studied.
... Safe limits of total BCAA daily intake for healthy adults were estimated in a range from 144 to 210 mg/kg/day based on amino acid oxidation (Riazi et al. 2003). A leucine dose above 150 mg/ kg/day was enough to significantly decrease circulating levels of valine and isoleucine, whereas leucine increased (Elango et al. 2012). The mean BCAA dose used in the included studies was 218.5 ± 160.5 mg/kg/day, including some studies that potentially reached daily leucine intake that may have had effects on decreasing valine and isoleucine circulation or increasing blood ammonia (Foure et al. 2016;Howatson et al. 2012;Kirby et al. 2012;Ra et al. 2013Ra et al. , 2018. ...
... Experimental studies showed that high doses of BCAA have harmfull effects due to oxidative stress (Piscopo et al. 2011;Zhenyukh et al. 2017;Zhenyukh et al. 2018), and expression of inflammatory mediators (Wessler et al. 2019;Zhenyukh et al. 2017;Zhenyukh et al. 2018), and endothelial dysfunction (Wessler et al. 2019;Zhenyukh et al. 2018). A high intake of leucine in BCAA formulations may have decreased valine and isoleucine bioavailability, and other essential amino acids, through a phenomenon called BCAA antagonism (Block and Harper 1984;Elango et al. 2012;Wolfe 2017). Importantly, isoleucine is necessary to maintain anti-inflammatory Treg cells (Ikeda et al. 2017). ...
Branched-chain amino acids (BCAA) are used as a recovery method after exercise-induced muscle damage (EIMD). Although data suggest that BCAA may alleviate the delayed-onset muscle soreness (DOMS) evoked by EIMD, there is no consensus about the most effective supplementation protocol. To investigate the effects of BCAA on DOMS after a single exercise session that caused EIMD, a systematic review and meta-analysis were conducted on the effectiveness of BCAA supplementation to reduce DOMS symptoms in healthy subjects after a single session of EIMD. Randomized clinical trials (RCT) were searched in Medline, Cochrane Library, Science Direct, SciELO, LILACS, SciVerse Scopus, Springer Link journals, Wiley Online Library, and Scholar Google, until May 2021. Ten RCTs were included in the systematic review and nine in the meta-analysis. Seven studies demonstrated that BCAA reduced DOMS after 24 to 72 h. BCAA doses of up to 255 mg/kg/day, or in trained subjects, for mild to moderate EIMD, could blunt DOMS symptoms. However, high variability between studies due to training status, different doses, time of treatment, and severity of EIMD do not allow us to conclude whether BCAA supplementation is efficient in untrained subjects, applied acutely or during a period of pre to post days of EIMD, and at higher doses (> 255 mg/kg/day). The overall effects of BCAA on DOMS after a single session of exercise were considered useful for improving muscle recovery by reducing DOMS in trained subjects, at low doses, in mild to moderate EIMD, and should not be administered only after the EIMD protocol.
... 3) SRs are usually performed to evaluate the safety of novel medicines. Because l-lysine is an amino acid and a nutrient, supplementation may have affected the balance of other amino acids in the body (e.g., 85). Although amino acid imbalance was not reported in the evaluated studies, one cannot preclude that such an amino acid "imbalance" may have happened in some subgroups in certain conditions. ...
... Although amino acid imbalance was not reported in the evaluated studies, one cannot preclude that such an amino acid "imbalance" may have happened in some subgroups in certain conditions. 4) No metabolic studies with l-lysine have been conducted with the direct amino acid oxidation method, such as those previously performed by Elango et al. (85,86) with l-leucine. Direct estimation of metabolic tolerance to supplemental l-lysine is thus lacking. ...
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.
... Given leucine concentrations have been correlated with severity of insulin resistance, our primary goal was to discern if excess leucine could negatively impact insulin signaling. Prior experiments that provided healthy subjects with up to 1250 mg/kg/day of leucine documented plasma leucine concentrations can reach up to 2 mM [42]. Thus, 2 mM was used for the majority of our experiments as we assumed if leucine were to negatively impact insulin signaling, it would be observed at the highest achievable concentrations. ...
... To this point, we had only investigated the effects of an exceedingly high level of leucine on insulin signaling, which while attainable [42], is likely uncommon in the general population. Therefore, we next investigated the effect of more physiologicallyattainable levels of leucine (0.25 mM, 0.5 mM, and 1 mM) on insulin signaling to determine if a concentration-dependent effect occurred. ...
Elevated circulating branched-chain amino acids (BCAA) such as leucine have been consistently correlated with increasing severity of insulin resistance across numerous populations. BCAA may promote insulin resistance through either mTOR-mediated suppression of insulin receptor substrate-1 or through the accumulation of toxic BCAA catabolites. Although the link between circulating BCAA and insulin resistance has been consistent, it has yet to be concluded if BCAA causally contribute to the development or worsening of insulin resistance. This work investigated the effect of leucine both with and without varying levels of insulin resistance on metabolism, metabolic gene expression, and insulin signaling. C2C12 myotubes were treated with and without varied concentrations of leucine up to 2mM for 24 hours both with and without varied levels of insulin resistance. Gene and protein expression were measured via qRT-PCR and western blot, respectively. Mitochondrial metabolism was measured via O2 consumption. Leucine at 2mM increased oxidative metabolism as well as gene expression of mitochondrial biogenesis, which was associated with increased cellular lipid content. Despite increased lipid content of leucine-treated cells, neither acute nor chronic leucine treatment at 2mM affected insulin signaling in insulin sensitive, mildly insulin resistant, or severely insulin resistant cells. Similarly, leucine at lower concentrations (0.25mM, 0.5mM, and 1mM) did not alter insulin signaling either, regardless of insulin resistance. Leucine appears to improve myotube oxidative metabolism and related metabolic gene expression. And despite increased lipid content of leucine-treated cells, leucine does not appear to alter insulin sensitivity either acutely or chronically, regardless of level of insulin resistance.
... 11,25,26 Based on the available evidence, a single bolus consisting of approximately 3 g of leucine and other essential amino acids is sufficient to meet this threshold and stimulate MPS, with an upper intake level of approximately 35 g/d. 22,27,28 The dietary supplementation protocol in this study was divided into 3 phases, all in which a high dose of leucine was consumed by the LBCAA and LEU groups in a single serving. Overall, LBCAA and LEU were unable to elicit an ergogenic response. ...
... 11,22,25,28 Moreover, higher doses of leucine have shown to decrease plasma concentrations of other amino acids. 22,27 Perhaps a disproportionate amount of amino acids as imposed by LBCAA and LEU may have led to an insufficient increase in MPS, thereby preventing any effect from manifesting. 9 Interestingly, a recent study by Teixeira et al 29 suggests that even metabolites of leucine, that is, αhydroxyisocaproic acid and β-hydroxy-β-methylbutyrate, when supplemented fail to mitigate markers of muscle damage or facilitate training-induced performance adaptations. ...
Purpose:
This investigation examined whether enrichment of a standard BCAA supplement with additional leucine or a standalone leucine supplement differentially affects exercise-induced muscle damage and performance recovery compared to a standard BCAA supplement.
Methods:
Twenty-two recreationally-active male and female subjects were recruited and assigned to consume either a BCAA, leucine-enriched BCAA (LBCAA), or standalone leucine (LEU) supplement for 11 days. On the 8th day, subjects performed eccentric-based resistance exercise (ECRE). Lower body mean average power (MAP) and peak power (MPP), plasma creatine kinase (CK), soreness, and pain threshold were measured before and 24h, 48h, and 72 hrs. after ECRE.
Results:
LEU showed decreased MAP (p=0.02) and MPP (p=0.01) from baseline to 48 hrs. post-ECRE while LBCAA and BCAA only trended towards a reduction at 24 hrs. post-ECRE. At 48 hrs. post-ECRE, BCAA showed greater recovery of MPP than LEU (p=0.04). At 24 hrs. post-ECRE, LEU demonstrated a greater increase in plasma CK from baseline than BCAA (p=0.04). Area under the curve for CK was greater in LEU than BCAA (p=0.02) while BCAA and LBCAA did not differ. Only LEU demonstrated increased soreness during rest and under muscular tension at 24 hrs. and 48 hrs. post-ECRE (p<0.05).
Conclusions:
Leucine-enriched BCAA failed to afford any advantages over a standard BCAA supplement for post-exercise muscle recovery while a standalone leucine supplement was comparatively ineffective.
... In our early study the experimental GMP medical food provided 130% of the Leu requirement which was not sufficient relative to the high concentration of Ile within the GMP peptide resulting in a ratio of Leu:Ile of 0.70 and an abnormal postprandial profile of BCAA [7], Figure 1. The BCAA share a common catabolic pathway with branched-chain ketodehydrogenase controlling the irreversible catabolic step that commits the carbon skeletal of BCAA to the TCA cycle [9]. Thus, an imbalance in BCAA intake can stimulate catabolism of BCAA and limit their availability for protein synthesis. ...
... The amount of Leu in currently available Glytactin™ containing GMP medical foods (150 -200 mg Leu/g protein equivalent; PE) was used in our recent outpatient clinical trial. It provided 150 mg Leu/kg/day [2], a level well below the tolerable upper limit for Leu of 500 mg Leu/kg/day [9]. Importantly, a normal plasma AA profile was observed when 30 subjects consumed the GMP medical foods for 3 weeks. ...
... Leu intake in our MMA patient cohort was indeed four to five times higher than the daily recommended intake based on the 2007 Food and Agriculture Organization/World Health Organization guidelines (mean: 195.1 mg/kg/day, range: 30-510 mg/kg/day, compared with the daily recommended intake of 44-50 mg/kg/day; Figure 2d). In some of the patients with MMA, Leu intake reached levels shown to cause hyperammonemia and other side effects when administered to healthy volunteers 26 (Table 2). Of note, target amounts for Leu consumption are not considered in the proposed guidelines for the dietary management of MMA. ...
... Furthermore, in numerous studies performed with human subjects, administration of a Leu load (but not of Val or Ile) by mouth or intravenously results in dramatic decreases in plasma concentrations of Val, Ile, phenylalanine, tyrosine, threonine, and methionine within 1-3 h of administration. 19,26,[32][33][34] While the mechanism(s) behind the lowering effect of Leu on plasma amino acid concentrations are not fully understood, it has been proposed that the inhibitory role of Leu-derived α-ketoisocaproate on the branched-chain ketoacid dehydrogenase-kinase results in activation of branched-chain ketoacid dehydrogenase and increased BCAA oxidation. 19 Original research article In addition, Leu displays a multitude of effects that have not been carefully considered in the management of patients with MMA. ...
PURPOSE:
Medical foods for methylmalonic acidemias (MMAs) and propionic acidemias contain minimal valine, isoleucine, methionine, and threonine but have been formulated with increased leucine. We aimed to assess the effects of imbalanced branched-chain amino acid intake on metabolic and growth parameters in a cohort of patients with MMA ascertained via a natural history study.
METHODS:
Cross-sectional anthropometric and body-composition measurements were correlated with diet content and disease-related biomarkers in 61 patients with isolated MMA (46 mut, 9 cblA, and 6 cblB).
RESULTS:
Patients with MMA tolerated close to the recommended daily allowance (RDA) of complete protein (mut0: 99.45 ± 32.05% RDA). However, 85% received medical foods, in which the protein equivalent often exceeded complete protein intake (35%). Medical food consumption resulted in low plasma valine and isoleucine concentrations, prompting paradoxical supplementation with these propiogenic amino acids. Weight- and height-for-age z-scores correlated negatively with the leucine-to-valine intake ratio (r = -0.453; P = 0.014; R2 = 0.209 and r = -0.341; P = 0.05; R2 = 0.123, respectively).
CONCLUSION:
Increased leucine intake in patients with MMA resulted in iatrogenic amino acid deficiencies and was associated with adverse growth outcomes. Medical foods for propionate oxidation disorders need to be redesigned and studied prospectively to ensure efficacy and safety.
... valine levels after ingestion of each respective amino acid dose-dependently increased up to 90 mg/kg BW. These results suggest that the absorption rate of leucine from the gut into the circulation reached a plateau at a dose of 53 mg/kg BW, although the capacity for leucine in human plasma appears to be much higher (about 2000 μM), as determined by a study that identified the tolerable upper intake level of leucine in young men (Elango et al. 2012). This phenomenon might be associated with the solubility of leucine, which did not completely dissolve at doses of >60 mg/kg BW in test drinks. ...
... Although the elevation of plasma leucine concentrations induced considerably lower concentrations of plasma isoleucine and valine, the mood states of the study participants determined by POMS were not affected by oral leucine at any dose, thus suggesting that the temporal profiles of plasma amino acids induced by leucine ingestion do not affect amino acid status in the brains of healthy men. This is consistent with the findings of a study of up to 1250 mg/kg BW/day of oral leucine to determine tolerable maximal intake (Elango et al. 2012). Mixtures of BCAAs are popular dietary supplements among athletes and are also used as medicines for patients with liver cirrhosis; many of the physiological effects of BCAAs can be attributed to the action of leucine. ...
Physiological conditions in humans affect plasma amino acid profiles that might have potential for medical use. Because the branched-chain amino acids (BCAAs) leucine, isoleucine and valine are used as medicines and supplements, we investigated the acute effects of individual BCAAs (10–90 mg/kg body weight) or mixed BCAAs ingested as a bolus on plasma amino acid profiles in young healthy men. Plasma leucine levels rapidly increased and peaked around 30 min after leucine ingestion. Concentrations of plasma isoleucine, valine and phenylalanine subsequently decreased after ingestion, and those of methionine and tyrosine tended to decrease. The effects of ingested leucine on other plasma amino acids were biphasic, being higher at lower doses (10–20 mg/kg body weight). Isoleucine or valine intake also caused corresponding plasma amino acid concentrations to rapidly elevate, and peaks at 30–40 min after ingestion were much higher than that of plasma leucine after leucine ingestion. However, the increase in plasma isoleucine and valine concentrations essentially did not affect those of other plasma amino acids. The rate of decline among peak plasma BCAA concentrations was the highest for leucine, followed by isoleucine and valine. Oral mixed BCAAs promoted the decline in plasma isoleucine and valine concentrations. These results suggest that plasma leucine is a regulator of the plasma concentrations of BCAAs, methionine and aromatic amino acids.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-3-35) contains supplementary material, which is available to authorized users.
... The choice of indicator amino acid and the essential and recommended characteristics of an indicator amino acid have been discussed frequently in publications (e.g. Elango et al., 2012b;Levesque et al., 2010;Zello et al., 1993) and are critical to sensitively partition the response to graded test intakes between protein synthesis and oxidation. The criteria for selecting the indicator amino acid are: a) it must be an indispensable amino acid; b) it must have a carboxyllabeled carbon that is irreversibly oxidized upon catabolism and is released to 13 CO 2 , which can be quantitatively measured in breath; c) it must have a small, well regulated pool within the body and d) it is not involved in quantitatively signifi cant pathways other than incorporation into protein or oxidation to CO 2 . ...
... <12%) or its expected amino acid bioavailability is less than 60%, then two approaches are possible; either the intake from free amino acids must be more than 120%, or the inclusion of the test protein must provide less than 40% of the total; otherwise the subjects will have a defi cient intake of the test amino acid. Humans have the ability to oxidize large quantities of excess amino acids (Elango et al., 2012b) therefore intake of any amino acids in excess of 120% is not considered to be an issue and therefore this approach is acceptable. Alternatively, inclusion of the test protein can be reduced to less than 40%, and the free amino acids supplemented to 120% or greater to ensure that the intake is adequate for even the least bioavailable amino acid. ...
... The analysis of data from the International Council on Amino Acid Science has resulted in the determination of a tolerable upper intake level for leucine. The recommended limit for leucine supplementation is set at 0.5 g/ kg/day for adults and 0.43 g/kg/day for elderly men over 70 years of age, corresponding to daily intakes of 35 g and 30 g, respectively, for a 70 kg individual [2,57,58]. Exceeding these established values can lead to elevated blood ammonia levels primarily as a result of the rapid transamination rates [2]. This heightened transamination activity is attributed to the higherKm values (Michaelis-Menten constant) of BCAA aminotransferases compared to tissue BCAA, being two to four times greater [19]. ...
Maintaining skeletal muscle mass is important for improving muscle strength and function. Hence, maximizing lean body mass (LBM) is the primary goal for both elite athletes and fitness enthusiasts. The use of amino acids as dietary supplements is widespread among athletes and physically active individuals. Extensive literature analysis reveals that branched-chain amino acids (BCAA), creatine, glutamine and β-alanine may be beneficial in regulating skeletal muscle metabolism, enhancing LBM and mitigating exercise-induced muscle damage. This review details the mechanisms of these amino acids, offering insights into their efficacy as supplements. Recommended dosage and potential side effects are then outlined to aid athletes in making informed choices and safeguard their health. Lastly, limitations within the current literature are addressed, highlighting opportunities for future research.
... One study observed an elevation in leucine excretion and plasma leucine concentration when BCAA was consumed above 0.5g/kg/day. This might have an adverse health effect [14]. From Table 2 it can be said that BCAA consumers in this study are all on the safe side. ...
Nutrition programs, proper guidance, and supplements (hormonal and non-hormonal supplements) that enhance muscle mass could be key factors for bodybuilders to reach their goals with a correct strategy. However, they could involve them in possible adverse health risks. The suit questionnaire form was designed to collect information about bodybuilders' total daily energy intake and supplements to strengthen and boost their muscle mass. It was used to discover whether they were on the right track regarding required energy intake. Moreover, the impact of hormonal and non-hormonal substances on their health has also been investigated. For that, thirty-one advanced bodybuilders as participants were taken (as volunteers); most of them had a long period of training experience and had a muscular body shape. The rest have at least more than four years of bodybuilding experience. Results show that the majority of them didn’t reach the required total daily calorie intake or exceeded by a great margin (3800 kcal). Surprisingly, they also had an unacceptable amount of administrated anabolic androgenic steroids (AAS), some of which are on the banned list by authorized food organizations. According to sports supplements' dose-related effects and health risks, nutrition program strategy and practicing that kind of supplement use may help them build muscle mass but in a very unhealthy way. Bodybuilders seem to be at the possible risk of practicing incorrect paths, because they may be misguided in terms of proper nutritional programs and using muscle enhancers.
... One study observed an elevation in leucine excretion and plasma leucine concentration when BCAA was consumed above 0.5g/kg/day. This might have an adverse health effect [14]. From Table 2 it can be said that BCAA consumers in this study are all on the safe side. ...
Nutrition programs, proper guidance, and supplements (hormonal and non-hormonal supplements) that enhance muscle mass could be key factors for bodybuilders to reach their goals with a correct strategy. However, they could involve them in possible adverse health risks. The suit questionnaire form was designed to collect information about bodybuilders' total daily energy intake and supplements to strengthen and boost their muscle mass. It was used to discover whether they were on the right track regarding required energy intake. Moreover, the impact of hormonal and non-hormonal substances on their health has also been investigated. For that, thirty-one advanced bodybuilders as participants were taken (as volunteers); most of them had a long period of training experience and had a muscular body shape. The rest have at least more than four years of bodybuilding experience. Results show that the majority of them didn’t reach the required total daily calorie intake or exceeded by a great margin (3800 kcal). Surprisingly, they also had an unacceptable amount of administrated anabolic androgenic steroids (AAS), some of which are on the banned list by authorized food organizations. According to sports supplements' dose-related effects and health risks, nutrition program strategy and practicing that kind of supplement use may help them build muscle mass but in a very unhealthy way. Bodybuilders seem to be at the possible risk of practicing incorrect paths, because they may be misguided in terms of proper nutritional programs and using muscle enhancers.
... aquatica) [28]. Protein is a vital nutrient that plays numerous essential roles in the human body, including tissue growth and repair, enzyme and hormone production, immune function, and transport and storage in the animal body [29][30][31]. ...
The rapid and unprecedented expansion of the global population highlights concerns about the sufficiency of food resources to sustain this growth. This study investigates and substantiates the feasibility of renewable food resources in order to meet the nutritional requirements of consumers. Three edible aquatic weeds, helencha (Enhydra fluctuans), malancha (Alternanthera philoxeroides), and kalmi (Ipomoea aquatica), were used to produce edible paper sheets. The composition of the raw aquatic weeds and paper sheet samples was analyzed, including the proximate composition, amino acid content, minerals and heavy metal contents, and bioactive compounds. The dried raw aquatic weeds and paper sheets showed similar proximate compositions, with carbohydrates being the highest component (50.38–64.63%), followed by crude protein (15.25–19.13%), ash (9.30–15.88%), and lipid (1.55–3.43%). The raw weeds and paper sheets were rich in essential minerals like Na, Ca, and Zn with contents ranging from 27.7 mg/100 g to 30.4 mg/100 g, 126.8 mg/100 g to 489.65 mg/100 g, and 4.5 mg/100 g to 16.3 mg/100 g, respectively. Acceptable levels of heavy metals, including Ni, Pb, and Cu, were found. The paper sheets contained seven essential and eight non-essential amino acids. Among the essential amino acids, the phenylalanine content was the highest at 2735.9 mg/100 g in E. fluctuans paper sheets, followed by methionine at 2377.29 mg/100 g in the raw E. fluctuans and histidine at 1972.6 mg/100 g in E. fluctuans paper sheets. A. philoxeroides sheets showed the highest total amino acid content (16,146.81 mg/100 g), while I. aquatica showed the lowest (13,118.67 mg/100 g). The aquatic weed paper sheets were rich in bioactive compounds, and the numbers in E. fluctuans, A. philoxeroides, and I. aquatica paper sheets were 31, 33, and 40, respectively. There were no significant changes in the nutritional content of the aquatic weeds in paper sheet form compared with the raw weeds, which suggests promising prospects for their production and consumption as a source of nutrition and bioactive compounds.
... Hasan [27]. Protein is a vital nutrient that plays numerous essential roles in the human body including tissue growth and repair, enzyme and hormone production, immune function, transport and storage in animal body [28][29][30]. ...
Three edible aquatic weeds, helencha (Enhydra fluctuans), malancha (Alternanthera philoxeroides), and kalmi (Ipomoea aquatica), were used to produce edible paper sheets. The composition of the raw and paper sheet samples was analyzed, including proximate composition, amino acid content, minerals and heavy metal contents, and bioactive compounds. The dried raw and paper sheets showed similar proximate composition, with carbohydrates being the highest (50.38-64.63%), followed by crude protein (15.25-19.13%), ash (9.30-15.88%), and lipid (1.549-3.43%). The weeds and paper sheets were rich in essential minerals like Na, Ca, Zn, and Fe. Acceptable levels of heavy metals, including Ni, Pb, and Cu, were found. The paper sheets contained seven essential and eight non-essential amino acids. A. philoxeroides sheets showed the highest amino acid content (16146.81mg/100g), while I. aquatica showed the lowest (13118.67 mg/100g). The aquatic weeds paper sheets were rich in containing bioactive compounds and the number in E. fluctuans, A. philoxeroides, and I. aquatica paper sheets were 31, 33, and 40, respectively. There were no significant changes in the nutritional content of aquatic weeds in paper sheet form compared to the raw weeds which suggests promising prospects for the production and consumption as a source of nutrition and bioactive compounds.
... 18,20 The dose required to provide neuroprotective effects in the current study was a human equivalent dose of 21 g/d of BCAAs with the 2:1:1: ratio and well below any of the tolerance studies. 30,31 The 21-g dose was used to best reproduce the Aquilani and colleagues studies performed on humans at 19 g/d. ...
Branched-chain amino acids (BCAAs) are known to be neurorestorative after traumatic brain injury (TBI). Despite clinically significant improvements in severe TBI patients given BCAAs after TBI, the approach is largely an unrecognized option. Further, TBI continues to be the most common cause of morbidity and mortality in adolescents and adults. To date, no study has evaluated whether BCAAs can be preventive or neuroprotective if taken before a TBI. We hypothesized that if BCAAs were elevated in the circulation before TBI, the brain would readily access the BCAAs and the severity of injury would be reduced. Before TBI induction with a standard weight-drop method, 50 adult mice were randomized into groups that were shams, untreated, and pre-treated, post-treated, or pre- + post-treated with BCAAs. Pre-treated mice received BCAAs through supplemented water and were dosed by oral gavage 45 min before TBI induction. All mice underwent beam walking to assess motor recovery, and the Morris water maze assessed cognitive function post-injury. On post-injury day 14, brains were harvested to assess levels of astrocytes and microglia with glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA-1) immunohistochemistry, respectively. Pre-treated and pre- +post-treated mice exhibited significantly better motor recovery and cognitive function than the other groups. The pre- + post-treated group had the best overall memory performance, whereas the pre-treated and post-treated groups only had limited improvements in memory compared to untreated animals. Pre- + post-treated brains had levels of GFAP that were similar to the sham group, whereas the pre-only and post-only groups showed increases. Although trends existed, no meaningful changes in IBA-1 were detected. This is the first study, animal or human, to demonstrate that BCAA are neuroprotective and substantiates their neurorestorative benefits after TBI, most likely through the important roles of BCAAs to glutamate homeostasis.
... Ancak, tek başına lösin kullanımı, plazma valin ve izolösinin tükenmesine yol açabilir; bu nedenle, DZAA'lardan herhangi birinin tükenmesini önlemek için üç amino asidin tümünün tüketilmesi gerekir (47). Son zamanlarda, yetişkin erkeklerde lösinin güvenli üst sınırı 550 mg/kg vücut ağırlığı/gün olarak belirlenmiştir; ancak hem diğer popülasyonlar hem de 3 DZAA'nın tümünün bir karışımı için güvenli üst sınırı belirlemek için daha fazla çalışmaya ihtiyaç vardır (48). ...
Vücut geliştirme sporunun popülaritesi gün geçtikçe artmaktadır fakat buna karşılık literatüre bakıldığında kanıta dayalı beslenme önerilerinin sayısının oldukça az olduğu görülmektedir. Bu çalışmada literatürde geçen, vücut ge- liştirme sporcuları için verilen kanıta dayalı güncel beslenme önerileri derlenmiştir. Vücut geliştirme sporuyla ilgilenen bireylerde enerji ihtiyacının doğru hesaplanması, vücut ağırlığında azalma sağlamak için temel hedef olmalıdır. Vücut geliştirme sporcularında kas kaybını önlemek için haftalık en fazla %1’lik vücut ağırlığında azalma önerilmektedir. Vücut geliştirme sporcusu için gerekli enerji hesaplaması yapıldıktan sonra, vücut ağırlığında kilogram başına en az 2.3 g/kg/gün protein alımı önerilmektedir. Aynı zamanda her öğünün 0.4-0.5 g/kg protein içermesi önerilmektedir. Günlük enerji gereksiniminin %15-30’unun yağlardan, %20’sinin proteinlerden ve geri kalanın ise karbonhidratlardan gelecek şekilde beslenme programı düzenlenmelidir. Vücut geliştirme sporcuları için ideal öğün sayısında ise özellikle dayanıklılık egzersizleri süresince günlük 3 ile 6 öğün tercih edilmelidir. Yapılan araştırma sonuçlarına göre öğün saatleri ve sıklığının vücut ağırlığında azalma veya yağsız kütleyi korumak için belirgin bir pozitif etkisinin olmadığı görülmüştür. Müsabakalardan birkaç gün önce uygulanan dehidrasyon programı, elektrolit imbalansına sebep olabileceğinden dikkatli olunması gerekmektedir. Müsabakalara hazırlanılan son dönemlerde karbonhidrat alımındaki artışın olumlu etkileri olabilir, bu sebeple karbonhidrat yüklemesi sporcuya uygun olarak yapılmalıdır. Vücut geliştirme sporcularında supleman kullanımıyla ilgili de farklı tartışmalar bulunmaktadır. Kreatin monohidrat, kafein ve beta alanin alımının müsabakalara hazırlanma süresince olumlu etkileri olduğu görülmüştür. Diğer suplemanlarla ilgili daha fazla çalışmaya ihtiyaç olduğu, güncel literatür verileri dahilinde düşünülmektedir. Son olarak, estetik bir spor çeşidi olan vücut geliştirme sporcularında yeme bozuklukları ve vücut algısı bozukluklarının sık görüldüğü unutulmamalıdır.
... Given BCAA concentrations have been correlated with severity of insulin resistance, our primary goal was to discern if excess BCAA could independently have a negative impact on mitochondrial metabolism. Prior experiments with subjects given valine up to 90 mg/kg [42] or leucine up to 1250 mg/kg/day [43] showed acute individual plasma BCAA concentrations can reach 1-2 mM. With that in mind, we treated cells with a commercially available BCAA mixture (Optimum Nutrition, Downers Grove, IL) which was prepared by dissolving the mixture in prewarmed differentiation media at 20 mM (leucine content) followed by filter sterilization. ...
PurposeBranched-chain amino acids (BCAA) have been shown to enhance several cellular signaling pathways including protein synthesis and mitochondrial biogenesis, yet population data demonstrate a correlation between circulating BCAA and severity of insulin resistance which has been hypothesized to be, in part, a byproduct of BCAA inhibition of mitochondrial function. The purpose of this study is to examine the effect of a BCAA mixture on muscle metabolism and related gene expression in vitro.MethodsC2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2, 2, or 20 mM (based on leucine content) for 6 days. qRT-PCR was used to measure metabolic gene expression. Oxygen consumption and extracellular acidification were used to assess mitochondrial and glycolytic metabolism, respectively. Mitochondrial content was determined via mitochondrial-specific staining.ResultsDespite significantly elevated mitochondrial staining, 6-day BCAA treatment reduced basal mitochondrial metabolism at a supraphysiological concentration (20 mM) in both insulin sensitive and resistant cells. Peak mitochondrial capacity was also reduced in insulin-resistant (but not insulin sensitive) cells. Conversely, basal glycolytic metabolism was elevated following 20 mM BCAA treatment, regardless of insulin resistance. In addition, insulin-resistant cells treated with 20 mM BCAA exhibited reduced gene expression of Ppargc1a, Cytc, Atp5b, Glut4, and several glycolytic enzymes versus insulin sensitive cells treated with 20 mM BCAA.Conclusions
Collectively, these findings suggest BCAA at supraphysiologically high levels may negatively alter mitochondrial metabolism, and concurrent insulin resistance may also diminish peak mitochondrial capacity, as well as impede molecular adaptations that support a transition to a glycolytic preference/compensation.
... 15 In the current study, we did not report plasma amino acid concentrations, but the fact that subjects needed to be supplemented with single amino acids (VAL and ILE) indicates that their plasma values were deficient. High LEU intakes can negatively impact the other two BCAAs (ILEU and VAL), by suppressing their plasma concentrations below normal ranges, 23,24 limiting total protein synthesis, and restricting growth. 21 We are currently performing in vivo stable isotope based experiments in PROP patients and healthy controls to measure protein synthesis to better understand the nutritional effects of an imbalanced BCAA ratio with LEU-enriched medical formula, similar to our recent proof-of concept stable isotope based nutritional studies in pyridoxine-dependent epilepsy. ...
Background
Propionic acidemia (PROP) is an autosomal recessive inherited deficiency of propionyl-CoA carboxylase (PCC) which is involved in the catalytic breakdown of the amino acids valine, isoleucine, methionine, and threonine. PROP nutritional management is based on dietary protein restriction and use of special medical formulas which are free of the offending amino acids, but are enriched in leucine. The resulting imbalance among branched-chain amino acids negatively impacts plasma concentrations of valine and isoleucine, which might impact growth in children with PROP.
Objective and Methods
Our primary objective was to describe dietary protein and calorie intake and their impact on long-term growth outcomes of four PROP patients. This was accomplished through a longitudinal retrospective chart review following the cohort from birth to 18 years.
Results
All children (n = 4) had poor growth outcomes with persistently reduced height-for-age Z scores, and elevated weight and body mass index (BMI) Z scores. Energy intakes for all subjects were within 80% to 120% of the dietary reference intakes for age. All children had low intakes of intact protein compared with current guidelines and were supplemented with medical formula and single l-amino acids (valine and/or isoleucine), which led to the excess consumption of total protein.
Conclusion
Despite adequate total protein and energy intakes, all children had persistently low height Z scores. Restricted intact protein consumption together with the abundant use of medical formula could have affected overall growth. To optimize dietary management in patients with PROP, further research is needed to determine the optimal intake of medical formula relative to intact protein.
... Other authors have reported the importance of BCAA equilibrium in body growth and cellular immunity in animal models [20,21]. In normal adults, high Leu intake was found to increase ammonia levels and to significantly decrease plasma concentration of Val and Ile [22]. ...
Background
Propionic acidemia (PA), an inborn error of metabolism, is caused by a deficiency in propionyl-CoA carboxylase. Patients have to follow a diet restricted in the propiogenic amino acids isoleucine (Ile), valine (Val), methionine (Met) and threonine (Thr); proper adherence can prevent and treat acute decompensation and increase life expectancy. However, chronic complications occur in several organs even though metabolic control may be largely maintained. Bone marrow aplasia and anemia are among the more common.
Materials and methods
In this retrospective study, data for patients with PA being monitored at the Hospital Ramón y Cajal (Madrid, Spain) (n = 10) in the past 10 years were examined to statistically detect relationships between persistent severe anemia outside of metabolic decompensation episodes and dietary practices such as natural protein intake and medical food consumption (special mixture of precursor-free amino acids) along with plasma levels of branched-chain amino acids (BCAA). High ferritin levels were deemed to indicate that a patient had received repeated transfusions for persistent anemia since data on hemoglobin levels at the moment of transfusion were not always passed on by the attending centers.
Results
Three patients had severe, persistent anemia that required repeated blood transfusions. Higher medical food consumption and plasma Leu levels were associated with iron overload. Notably, natural protein intake and plasma Val were negatively correlated with ferritin levels. We also observed an inverse relationship between plasma Val/Leu and Ile/Leu ratios and ferritin.
Conclusion
The present results suggest that severe anemia in patients with PA might be associated with low natural protein intake and BCAA imbalance.
... [22,23,25] and 6-20 g of EAA [24,26]. Leucine intake of CML was approximately 70% below the tolerable upper limit [27,28]. ...
Background
Chocolate milk has gained recent scientific support as a recovery drink. However, it is known that high exercise-demand triggers gastrointestinal discomfort which continues post-exercise, thereby hindering this nutritional strategy. In addition, those who are lactose intolerant cannot benefit from a milk-based beverage. Thus, the aim of this preliminary study was to develop a low-fat, lactose-free, and leucine-enriched chocolate cow milk prototype (CML) representing nutrition-related recommendations for football players, as well as assess athletes’ individual subjective outcomes for gastrointestinal complaints and sensorial acceptability in a field-based setting following strenuous team-sport physical demands.
Methods
This study followed a single group and repeated-measured design with 10 football players (23 ± 2 yrs., 74 ± 14 kg, 174 ± 5 cm) who consumed CML following a 90-min football match simulation protocol (FMP). The total CML intake to achieve 0.150 g leucine·kg [BW]·h − 1 occurred in aliquots of 50, 30 and 20% at 0-, 45- and 75-min post-FMP, respectively. Athletes were evaluated by the prevalence, the type and severity (bloating, nausea, flatulence, and gastric reflux) of gastrointestinal complaints and sensorial acceptability (overall perception, appearance, consistency, and flavour) after drinking each aliquot in a 4-h recovery period.
Results
The CML showed higher scores for “Product Acceptability Index” (88%) and sensorial acceptability (~ 8 in 9-point hedonic scale). Kendall’s W with bootstrapped resample (95%CI) revealed agreement among respondents as “moderate” (overall perception, flavour) to “strong” (appearance, consistency) and with no significant agreement differences between rater response in the timeline analysis (0.57 up to 0.87; p > 0.05). Agresti-Caffo add-4 analysis (95% confidence interval, [95%CI]) revealed no differences in each time-point analysis versus baseline for athletes classified as having severe gastrointestinal symptoms, but confirmed concern with bloating (three athletes showed a transient response at 2-h and only one continued until 3-h; p = 0.051).
Conclusions
These preliminary findings suggest that CML presents good taste and high acceptability by the sampled athletes. Thus, CML may be an alternative sport drink for immediate post-workout supplementation to overcome the energy deficit, offer co-ingested leucine, maintain palatability and adherence including lactose intolerance following a team sport-specific fatigue.
Trial registration
RBR-2vmpz9 , 10/12/2019, retrospectively registered.
... Because l-lysine is an essential amino acid, it is important to determine the upper limit for safe use considering the maximum metabolic limit as an index. Elango et al., estimated the tolerability of single-dose l-leucine to be 500 mg/ kg/day by the direct amino acid oxidation (DAAO) method, blood ammonia levels, and urine l-leucine levels (Elango et al. 2012b;Rasmussen et al. 2016). However, similar studies regarding l-lysine could not be found in the present study. ...
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)
... Prevention and treatment of LBM loss could benefit from direct measurement and monitoring of disturbed protein metabolism. Current methods to measure protein metabolism focus on protein synthesis, which requires blood sampling, muscle biopsies, and/or the use of expensive synthetic labelled amino acids [6][7][8]. Therefore, these methods are not suitable for the clinical setting. A non-invasive bedside method to measure protein metabolism would be more suitable, as direct measurements of the metabolic state could lead to more insight in optimal protein intake and optimal physical activity, which then enables tailored improved treatment for each patient, resulting in improved outcomes of disease. ...
Protein oxidation may play a role in the balance between anabolism and catabolism. We assessed the effect of a protein restricted diet on protein oxidation as a possible reflection of whole body protein metabolism. Sixteen healthy males (23 ± 3 years) were instructed to use a 4-day isocaloric protein restricted diet (0.25 g protein/kg body weight/day). Their habitual dietary intake was assessed by a 4-day food diary. After an overnight fast, a 30 g 13C-milk protein test drink was administered, followed by 330 min breath sample collection. Protein oxidation was measured by Isotope Ratio Mass Spectrometry. To assess actual change in protein intake from 24-h urea excretion, 24-h urine was collected. During the 4-day protein restricted diet, the urinary urea:creatinine ratio decreased by 56 ± 9%, which is comparable to a protein intake of ~0.65 g protein/kg body weight/day. After the protein restricted diet, 30.5 ± 7.3% of the 30 g 13C-milk protein was oxidized over 330 min, compared to 31.5 ± 6.4% (NS) after the subject’s habitual diet (1.3 ± 0.3 g protein/kg body weight/day). A large range in the effect of the diet on protein oxidation (−43.2% vs. +44.0%) was observed. The residual standard deviation of the measurements was very small (0.601 ± 0.167). This suggests that in healthy males, protein oxidation is unaffected after a protein restricted diet. It is uncertain how important the role of fluctuations in short-term protein oxidation is within whole body protein metabolism.
... As an optimal anabolic stimulus [87], 3-4 g/day of leucine (equivalent to 25-33 g/day of WP) seems appropriate whereas intakes of ∼ 35 g/day could be the tolerable safe upper intake level [139]. Supplementation with BCAA is quite safe when the three BCAA leucine, isoleucine and valine are provided in a ratio of ∼ 2:1:1, as supplementation of leucine alone could trigger BCAA imbalance [121]. ...
Purpose
Several supplements are purported to promote muscle hypertrophy and strength gains in healthy subjects, or to prevent muscle wasting in atrophying situations (e.g., ageing or disuse periods). However, their effectiveness remains unclear.
Methods
This review summarizes the available evidence on the beneficial impacts of several popular supplements on muscle mass or strength.
Results
Among the supplements tested, nitrate and caffeine returned sufficient evidence supporting their acute beneficial effects on muscle strength, whereas the long-term consumption of creatine, protein and polyunsaturated fatty acids seems to consistently increase or preserve muscle mass and strength (evidence level A). On the other hand, mixed or unclear evidence was found for several popular supplements including branched-chain amino acids, adenosine triphosphate, citrulline, β-Hydroxy-β-methylbutyrate, minerals, most vitamins, phosphatidic acid or arginine (evidence level B), weak or scarce evidence was found for conjugated linoleic acid, glutamine, resveratrol, tribulus terrestris or ursolic acid (evidence level C), and no evidence was found for other supplements such as ornithine or α-ketoglutarate (evidence D). Of note, although most supplements appear to be safe when consumed at typical doses, some adverse events have been reported for some of them (e.g., caffeine, vitamins, α-ketoglutarate, tribulus terrestris, arginine) after large intakes, and there is insufficient evidence to determine the safety of many frequently used supplements (e.g., ornithine, conjugated linoleic acid, ursolic acid).
Conclusion
In summary, despite their popularity, there is little evidence supporting the use of most supplements, and some of them have been even proven ineffective or potentially associated with adverse effects.
... It has been questioned whether excessive leucine intake may have adverse effects [9]. In an effort to determine the tolerable upper intake level of leucine, Elango et al. [10] provided adult men with graded doses of L-[1-13 C]-leucine ranging from 50 to 1250 mg/kg/day. With increasing leucine intakes, the oxidation of leucine (determined by measuring 13 CO 2 in exhaled breath) increased until a plateau was reached. ...
... The seven tests were -no-treatment Control (breakfast without added test substance), 2 or 4 g of butyrate (sodium salt; Bodybio Inc., Milville, NJ, USA), 2 g of L-carnitine (XPN, QC, Canada), 5 g of L-leucine (XPN, QC, Canada) and 5 or 10 g of O-MAG (SCF Pharma, Ste-Luce, QC, Canada). The doses were chosen according values reported in the literature (Elango, Chapman, Rafii, Ball, & Pencharz, 2012;Kraemer et al., 2008;Rubin et al., 2001) and/or the recommended daily dose on the bottle. For O-MAG, 5 and 10 g doses was chosen because they were comparable to the dose range of MCT commonly used. ...
Butyrate, carnitine and leucine are dietary ingredients that may be ketogenic but whether they are equally or more ketogenic in humans than medium-chain triacylglycerols is unclear. Our objective was to compare the ketogenic effect in healthy adult humans of L-leucine, butyrate, octanoyl-monoacylglycerol (O-MAG) and L-carnitine during a 4 h metabolic study period. Each test substance was taken with a standard breakfast: butyrate (2 or 4 g), O-MAG (5 or 10 g), L-leucine (5 g), or L-carnitine (2 g), and compared to a no treatment Control (n = 13). At the doses provided, butyrate (2 g; 4 g), O-MAG (5 g; 10 g) increased total plasma ketones AUC of 3.5, 5.0, 1.5, and 2.7-fold (P ≤ 0.05), respectively, compared to baseline. L-carnitine did not significantly stimulate ketogenesis relative to Control. Per gram tested, butyrate was the most ketogenic, and has the potential to be an ingredient in a ketogenic functional food or beverage.
... The relative plasma concentrations of the branched-chain AAs with GMP-MFs were Val:Leu:Ile, w3:2:1. This is consistent with the normal reference range and indicates that there was no evidence of an imbalance in the plasma concentrations of branched-chain AAs as a result of the Leu supplementation in GMP-MFs (34,35). ...
Background:
To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe.
Objective:
We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria.
Design:
This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15-49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained.
Results:
The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 μmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (-85 ± 40 μmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 μmol/L, GMP-MFs = 497 ± 34 μmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different.
Conclusions:
GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258.
... Estimated contents of L-valine, L-leucine, and L-isoleucine (g/kg diet) are shown in Table 1. These contents resemble a high-dose supplementation in which adverse outcomes for the monitored variables were not reported [14][15][16]. ...
Background:
Many people believe in favourable effects of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), especially leucine, on muscle protein balance and consume BCAAs for many years. We determined the effects of the chronic intake of a BCAA- or leucine-enriched diet on protein and amino acid metabolism in fed and postabsorptive states.
Methods:
Rats were fed a standard diet, a diet with a high content of valine, leucine, and isoleucine (HVLID), or a high content of leucine (HLD) for 2 months. Half of the animals in each group were sacrificed in the fed state on the last day, and the other half were sacrificed after overnight fast. Protein synthesis was assessed using the flooding dose method (L-[3,4,5-(3)H]phenylalanine), proteolysis on the basis of chymotrypsin-like activity (CHTLA) of proteasome and cathepsin B and L activities.
Results:
Chronic intake of HVLID or HLD enhanced plasma levels of urea, alanine and glutamine. HVLID also increased levels of all three BCAA and branched-chain keto acids (BCKA), HLD increased leucine, ketoisocaproate and alanine aminotransferase and decreased valine, ketovaline, isoleucine, ketoisoleucine, and LDL cholesterol. Tissue weight and protein content were lower in extensor digitorum longus muscles in the HLD group and higher in kidneys in the HVLID and HLD groups. Muscle protein synthesis in postprandial state was higher in the HVLID group, and CHTLA was lower in muscles of the HVLID and HLD groups compared to controls. Overnight starvation enhanced alanine aminotransferase activity in muscles, and decreased protein synthesis in gastrocnemius (in HVLID group) and extensor digitorum longus (in HLD group) muscles more than in controls. Effect of HVLID and HLD on CHTLA in muscles in postabsorptive state was insignificant.
Conclusions:
The results failed to demonstrate positive effects of the chronic consumption of a BCAA-enriched diet on protein balance in skeletal muscle and indicate rather negative effects from a leucine-enriched diet. The primary effects of both diets are an activated catabolism of BCAAs, which leads to an enhanced production of BCKA, alanine and glutamine and their utilization in visceral tissues and an impaired protein synthesis in postabsorptive state, particularly in fast-twitch (white) muscles.
... This amino acid can be ingested in supplement form; however, when determining the safe tolerable upper intake level for leucine intake, trials in youth are limited. One study suggests the upper level for young males aged 20-35 years is 500 mg/kg/day or 35 g/day based on plasma and urinary ammonia and leucine concentrations [32]. This recommendation has not been examined in youth and caution of leucine at these high levels is warranted. ...
Nutrition is an integral component to any athletes training and performance program. In adults the balance between energy intake and energy demands is crucial in training, recovery, and performance. In young athletes the demands for training and performance remain but should be a secondary focus behind the demands associated with maintaining the proper growth and maturation. Research interventions imposing significant physiological loads and diet manipulation are limited in youth due to the ethical considerations related to potential negative impacts on the growth and maturation processes associated with younger individuals. This necessary limitation results in practitioners providing nutritional guidance to young athletes to rely on exercise nutrition recommendations intended for adults. While many of the recommendations can appropriately be repurposed for the younger athlete attention needs to be taken towards the differences in metabolic needs and physiological differences.
Individual amino acids are widely popular as supplements due to various perceived and real health benefits. Currently there are no recommendations set by national health agencies for tolerable upper intake levels (UL) for amino acids due to a lack of well conducted human dose-response trials. In the past decade under the initiative of the International Council on Amino Acid Science (ICAAS), a non-profit organization, a series of UL human clinical studies were conducted. The goal of this narrative review is to summarize the studies on 6 essential amino acids (leucine, tryptophan, methionine, lysine, histidine, phenylalanine), two non-essential amino acids (arginine, serine) and two non-proteinogenic amino acids (ornithine, citrulline) and provide the first set of ULs. A brief background of the concept of the Dietary Reference Intakes (DRI) framework of UL, concept of UL for amino acids, and a perspective of the results are also provided. The data suggests that in relatively healthy adult individuals the tested amino acids are well tolerated, and ULs, or the no-observed-adverse-effect-level (NOAEL), lowest-observed-adverse-effect-level (LOAEL) can be determined. The ULs were for leucine-young (35 g/d), tryptophan (4.5g/d) and leucine-elderly (30g/d); NOAEL and LOAEL for methionine at 3.2 and 6.4g/d, respectively; NOAEL for arginine (30g/d); NOAEL and LOAEL for lysine at 6 and 7.5g/d, respectively; NOAEL and LOAEL for histidine at 8 and 12g/d, respectively; and NOAEL for phenylalanine (12g/d), serine (12g/d), ornithine (12g/d) and citrulline (24g/d). These first set of human UL data is hoped to help national and international agencies set safety standards for supplemental amino acids.
Purpose:
Type 2 diabetes is characterized by reduced insulin sensitivity which correlates with increased circulating BCAA. These experiments investigated the effects of insulin resistance with and without excess BCAA on myotube insulin sensitivity and L-type amino acid transporter-1 (LAT1).
Methods:
C2C12 myotubes were treated with or without excess BCAA for 1 or 6 days, both with and without insulin resistance. Western blot was used to assess insulin sensitivity and LAT1 content. Liquid chromatography-mass spectrometry was used to evaluate BCAA media content.
Results:
Insulin resistance was associated with significantly increased extracellular BCAA accumulation independent of LAT1 content. Conversely, prior BCAA treatment was not associated with extracellular BCAA accumulation regardless of level of insulin sensitivity.
Conclusion:
These data suggest insulin resistance, but not BCAA treatment, promotes extracellular BCAA accumulation independent of changes in LAT1 content, implicating insulin resistance as a causal agent of extracellular BCAA accumulation.
Graphene known as the wonder material and finds its utilization in various applications including sensors.This work reports the solvothermal route and the chemical reflux method synthesis of graphene oxide copper oxide nanocomposite material (GO-CuO). The hitherto prepared GO-CuO nanocomposite was well characterized using the usual spectroscopic and microscopic technique such as Fourier transform infrared (FTIR) spectroscopy, Scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-Vis Spectroscopy (UV), Photoluminescence Spectroscopy(PL) etc. The prepared GO-CuO nanocomposite was found to selectively bind to L-Leucine without any interference from the other naturally occurring L-amino acids as a potential fluorescent chemosensor. The interference studies, effect of time, effect of pH and various equivalence studies were also performed for the GO-CuO nanocomposite with L-Leucine. The antibacterial activity of GO-CuO nanocomposite revealed a minimum inhibitory zone concentration for Methicillin Resistant Staphylococcus aureus to be 200 μg/ml, whereas for A. baumannii it was found to be 100 μg/ml.
AIMs
Branched-chain amino acids (BCAA) are often emphasized in the diets of avid exercisers, yet population data demonstrates a correlation between circulating BCAA and insulin resistance. However, it is unclear if BCAA independently promote insulin resistance in otherwise healthy cells. The purpose of this study is to examine the effect of a BCAA mixture on muscle insulin signaling in vitro in both insulin resistant and sensitive cells. MATERIALS and METHODS: C2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2mM, 2mM, or 20mM (based on leucine content) for either 30 min, 1 day, or 6 days. Western blot was used to assess insulin sensitivity of cells treated with BCAA both with and without concurrent insulin resistance, and, with and without insulin stimulation. RESULTS: BCAA treatment for 1 day significantly reduced basal, but not insulin-stimulated pAkt expression. BCAA treatment for 6 days resulted in significantly reduced basal insulin signaling in health cells and insulin-stimulated insulin signaling in insulin resistant (but not insulin sensitive) cells. CONCLUSION: Similar to previous observations demonstrating BCAA may correlate with insulin resistance during metabolically stressed conditions, we demonstrate excessively high BCAA exposure can negatively influence basal insulin signaling, as well as insulin sensitivity in insulin resistant myotubes. However, given the intentionally high concentrations of BCAA used in this study, the extent to which these observations translate to in vivo models is unclear and warrants further investigation.
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The role of protein oxidation in the regulation of whole body protein metabolism is unknown. Previously, it was observed that vigorous exercise led to increased protein oxidation. To further characterise ¹³C-milk protein oxidation in healthy subjects, the oxidation of ingested ¹³C-protein after an overnight fast was measured using a non-invasive ¹³C-protein breath test. This approach enables the analysis of ¹³C-protein oxidation kinetics and the effect of interfering factors. It was found that the estimated maximal ¹³C-milk protein oxidation was 0.07 g min⁻¹, corresponding to a theoretical maximal oxidation capacity of ≈1.4 g kg body weight⁻¹ d⁻¹. No indications were found for preferential oxidation of non-essential amino acids. Combined ingestion of 30 g ¹³C-whey protein with 30 g glucose resulted in a 19% decrease of ¹³C-whey protein oxidation. It was concluded that exogenous ¹³C-whey protein oxidation can be affected by other co-ingested nutrients like glucose.
Link to full text: https://www.mdpi.com/books/pdfview/book/1430
The amino acid profile of Rhynchophorus phoenicis, Zonocerus variegatus, Helix pomatia and Littorina littoria were determined using a Technicon sequential multi sample amino acid analyzer (TSM).The protein qualities of the samples were also estimated. The result of the study showed that R. phoenicis and Z. variegatus contain predominantly glutamic acid (13.71 and 12.96 g/100g protein) and aspartic acid (10.22 and 10.53 g/100g protein) respectively. The predominant amino acids in H.pomatia were arginine (12.25 g/100g) and lysine (12.03 g/100g), while in L. littoria, it was glutamic acid (9.54 g/100gprotein).The total essential amino acid content of L. littoria was low (29.29 g/100g) in comparison with 66.81, 54.70, and 50.38g/100g observed in H. pomatia, R. phoenicis and Z. variegatus respectively. The predicted protein efficiency ratio (P-PER) of the samples ranged from 2.12 in L. littoria to 3.58 in H. pomatia. The limiting amino acid for R. phoenicis and Z. variegatus were isoleucine and valine respectively, while for H. pomatia and L. littoria, it was methionine or cysteine and threonine respectively. The results revealed that the species of insects and mollusks studied contain nutritionally useful quantities of essential amino acids. Thus, they are very good sources of protein comparable to conventionally used protein foods.
Background
Sirtuin 1 (Sirt1) is suppressed in non‐alcoholic fatty liver disease (NAFLD), while its’ stimulation or overexpression results in reduced disease severity in pre‐clinical NAFLD models. Leucine allosterically activates Sirt1 and synergise with other Sirt/AMPK/NO pathway activators. We developed a triple combination of leucine, metformin and sildenafil (NS‐0200), which was effective in a mouse model of non‐alcoholic steatohepatitis (NASH).
Aim
To report the results from a Phase 2, randomised clinical trial of of NS‐0200 in 91 subjects with NAFLD (liver fat ≥15% by magnetic resonance imaging‐proton‐density fat fraction (MRI‐PDFF)).
Methods
Subjects were randomised to placebo, low‐dose (1.1 g leucine/0.5 g metformin/0.5 mg sildenafil) or high‐dose NS‐0200 (1.1 g leucine/0.5 g metformin/1.0 mg sildenafil) b.d. for 16 weeks; change in hepatic fat was assessed via MRI‐PDFF, and lipid metabolism was assessed via changes in the lipidomic signature. Seventy subjects completed the trial and met a priori compliance criteria. Analyses were conducted on the full cohort and on those with alanine aminotransferase (ALT) values above median (50 U/L; n = 35).
Results
In the full cohort, active treatments did not separate from placebo. High dose NS‐0200 reduced hepatic fat by 15.7% (relative change from baseline) in the high ALT group (P < 0.005) while low dose NS‐0200 and placebo did not significantly change hepatic fat. Lipidomic analysis showed dose‐responsive treatment effects in both overall and high ALT cohorts, with significant decreases in metabolically active lipids and up‐regulation of fatty acid oxidation.
Conclusion
These data support further evaluation of high‐dose NS‐0200 for treating NASH, especially in those with elevated ALT (NCT 02546609).
Branched-chain amino acids (BCAAs) appear to influence several synthetic and catabolic cellular signaling cascades leading to altered phenotypes in mammals. BCAAs are most notably known to increase protein synthesis through modulating protein translation, explaining their appeal to resistance and endurance athletes for muscle hypertrophy, expedited recovery, and preservation of lean body mass. In addition to anabolic effects, BCAAs may increase mitochondrial content in skeletal muscle and adipocytes, possibly enhancing oxidative capacity. However, elevated circulating BCAA levels have been correlated with severity of insulin resistance. It is hypothesized that elevated circulating BCAAs observed in insulin resistance may result from dysregulated BCAA degradation. This review summarizes original reports that investigated the ability of BCAAs to alter glucose uptake in consequential cell types and experimental models. The review also discusses the interplay of BCAAs with other metabolic factors, and the role of excess lipid (and possibly energy excess) in the dysregulation of BCAA catabolism. Lastly, this article provides a working hypothesis of the mechanism(s) by which lipids may contribute to altered BCAA catabolism which often accompanies metabolic disease.
Use of dietary supplements, especially among athletes is increasing. Among the amino acids, the branched-chain amino acids (BCAA-leucine, valine and isoleucine) are popular as dietary supplements especially among strength training athletes. In particular, leucine as a supplement is widely consumed because it has been implicated to be the key amino acid involved in stimulating muscle protein synthesis. This chapter will present current dietary leucine intakes, define the concept of safe upper limits (UL) and summarize a recent study in humans conducted to address leucine tolerability. In the 2005 Dietary References Intake (DRI) report, men 51 through 70 years of age had the highest intakes, at the 99th percentile for leucine, at 14,100 mg·d-1 (~ 201 mg·kg·d-1). In order to directly explore leucine tolerability in humans we proposed a novel model. We hypothesized that with increasing intakes of leucine above the estimated average requirement (EAR of 50 mg·kg-1·d-1) in adult men, the oxidation of leucine will increase and will reach a maximum, after which the leucine oxidation will achieve a plateau. This ‘metabolic limit’ to oxidize leucine may be used as a marker of an intake after which increasing intakes may result in increasing risk of adverse effects. Five healthy young men participated in the study. Each subject participated in a dose-escalation study design, where graded stepwise increases in leucine intake (150, 250, 500, 750, 1000 and 1250 mg·kg-1·d-1 corresponding to the EAR, EARx3, x5, x10, x15, x20 and x25) were provided on each study day. Oxidation of L-[1-13C]leucine to 13CO2 in breath (F13CO2) was measured on each study day. With increasing intakes of leucine, a dose-response in leucine oxidative capacity was observed, with a breakpoint estimated at 550 mg·kg-1·d-1 or 39 g·d-1. Simultaneous and significant increases in blood ammonia concentrations, plasma leucine concentrations and urinary leucine excretion were observed with leucine intakes higher than 500 mg·kg-1·d-1. These results taken together with the recent animal data suggest that under acute dietary conditions, as a cautious estimate, intakes greater than 500 mg leucine·kg-1·d-1 may potentially increase the risk of adverse events, and could be proposed as the UL for leucine in healthy adults.
Substantial evidence has been accumulated suggesting that branched-chain amino acid (BCAA) supplementation or BCAA-rich diets have a positive effect on the regulation of body weight, muscle protein synthesis, glucose homeostasis, the ageing process and extend healthspan. Despite these beneficial effects, epidemiological studies have shown that BCAA plasma concentrations and BCAA metabolism are altered in several metabolic disorders, including type 2 diabetes mellitus and cardiovascular diseases. In this review article, we present an overview of the current literature on the different effects of BCAAs in health and disease. We also highlight the results showing the most promising therapeutic effects of dietary BCAA supplementation and discuss how BCAAs can trigger different and even opposite effects, depending on the catabolic and anabolic states of the organisms. Moreover, we consider the effects of BCAAs when metabolism is abnormal, in the presence of a mixture of different anabolic and catabolic signals. These unique pharmacodynamic properties may partially explain some of the markedly different effects found in BCAA supplementation studies. To predict accurately these effects, the overall catabolic/anabolic status of patients should be carefully considered. In wider terms, a correct modulation of metabolic disorders would make nutraceutical interventions with BCAAs more effective.
Linked articles:
This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.
Background:
We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD(+), thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction.
Methods:
Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies.
Results:
Leu (0.5mmol/L)+Met (50-100μmol/L) synergistically activated Sirt1 (p<0.001) at low (≤100μmol/L) NAD(+) levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met-Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~50%, p<0.002) evident within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~25%, while addition of Leu+Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p=0.023), restored normal glucose utilization and increased fat oxidation ~two-fold (p<0.005), while metformin exerted no independent effect at any concentration (0.1-0.5mmol/L).
Conclusion:
Thus, Leu and Met synergize to enable Sirt1 activation at low NAD(+) concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.
Protein consumption as well as the effects of isolated amino acids have long been studied for their possible ergogenic effects on skeletal muscle mass and strength. This chapter discusses in detail the role of specific amino acids, namely the branched-chain amino acids (BCAAs), especially leucine, regarding their metabolism, the molecular pathways they activate in skeletal muscle and their physiological effects. Studies regarding the effects of protein and amino acids supplementation on gain of muscle mass and strength are discussed with an emphasis on the activation of key proteins (such as mTOR and p70S6k) that, once phosphorylated, are able to stimulate the initiation of protein synthesis. The effects of different sources of protein, dose and timing and their physiological effects are also discussed. The last section gives practical recommendations regarding amino acid and protein supplementation, their safety, as well as the possible side effects when amino acids are given under specific metabolic conditions.
Cobalamin C (cblC) deficiency impairs the biosynthesis of 5'-deoxyadenosyl-adenosyl- and methyl-cobalamin, resulting in methylmalonic acidemia combined with hyperhomocysteinemia and hypomethioninemia. However, some patients with cblC deficiency are treated with medical foods, devoid of methionine and high in leucine content, that are formulated for patients with isolated propionate oxidative defects. We examined the effects of imbalanced branched-chain amino acid intake on growth outcomes in cblC-deficient patients.
Dietary intake was correlated with biochemical, anthropometric, and body composition measurements and other disease parameters in a cohort of 28 patients with early-onset cblC deficiency.
Protein-restricted diets were followed by 21% of the patients, whereas 32% received medical foods. Patients on protein-restricted diets had lower height-for-age z-score (P = 0.034), whereas patients consuming medical foods had lower head circumference Z-scores (P = 0.037), plasma methionine concentrations (P = 0.001), and predicted methionine influx through the blood-brain barrier Z-score (-1.29 vs. -0.0617; P = 0.007). The combination of age at diagnosis, a history of seizures, and the leucine-to-valine dietary intake ratio best predicted head circumference Z-score based on multiple regression modeling (R(2) = 0.945).
Patients with cblC deficiency treated with medical foods designed for isolated methylmalonic acidemia are at risk for iatrogenic methionine deficiency that could adversely affect brain growth and development.Genet Med advance online publication 13 August 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.107.
The aging process is a continuum throughout life and often associated with deterioration of body function as well as accumulation of chronic disabilities and of disease. The impact of nutritional status on morbidity and mortality is unquestioned. Malnutrition increases the risk for frailty and nutritional deficits can influence immune status, response to medical treatments and recovery from acute illnesses, including surgery. Health-promoting interventions implemented individually, such as exercise programs, preventive home visits, comprehensive geriatric evaluation and management, and attention to adequate nutrition with or without nutritional supplements, have been shown in separate studies to be both feasible and effective in reducing age-related deterioration. Protein and its constituent amino acids (AA) are key components of any healthy diet. Sarcopenia, the slow but progressive loss of lean muscle mass associated with advancing age, has been the focus of many studies but there is no clear-cut answer to the question of how to restrain the process. The more general question of how the requirements for protein and specific AA change with age continues to be investigated. A shift towards studying the efficacy and safety of specific AA or combination of AA that may sustain and/or enhance physiologic processes, ranging from specific tissue metabolism to overall function (e.g. exercise performance, immune function, cognition, and chronic disease development) has occurred. This review focuses on recent studies examining the use of specific AA or mixtures as supplements in the elderly and whether/how AA may assist in the maintenance of health and independence.
Leucine has been reported to be an important regulator of protein metabolism. We investigated the effect of intravenous infusion of l-leucine versus saline on amino acid metabolism in eight healthy human subjects. Plasma concentrations of amino acids were measured and protein turnover was estimated using l-(1-13C)lysine and l-(3,3,3-2H3)leucine as tracers. Glucose kinetics were measured using d-(6,6-2H2)glucose as a tracer. Leucine infusion increased the plasma leucine concentration from 103 ± 8 to 377 ± 35 μmol/L (P < .01). Plasma concentrations of essential amino acids, including threonine, methionine, isoleucine, valine, tyrosine, and phenylalanine were significantly decreased by leucine infusion. Leucine infusion did not change lysine flux significantly (108 ± 4 during saline v 101 ± 4 μmol/kg/h−1 during leucine infusion), but decreased lysine oxidation (13.2 ± 0.9 v 10.7 ± 1 μmol/kg/h, P < .05) and endogenous leucine flux (from 128 ± 4 to 113 ± 7 μmol/kg/h, P < .05) when plasma (2H3) ketoisocaproate (KIC) was used for calculation. During leucine infusion, the (2H3) KIC to (2H3) leucine plasma enrichment ratio increased from 0.76 ± 0.02 to 0.88 ± 0.01 (P < .001), while estimation of leucine flux using plasma (2H3) leucine showed no change in endogenous leucine flux. Leucine infusion decreased hepatic glucose production and metabolic clearance of glucose, but did not change plasma concentrations of glucose, insulin, C-peptide, glucagon, epinephrine, norepinephrine, or free fatty acids. We conclude that leucine spares glucose and lysine catabolism and decreases plasma concentrations of essential amino acids. This study also demonstrated that the ratio of plasma KIC enrichment to leucine enrichment does not remain constant in all study conditions.
The purpose of this study was to determine whether a practical leucine-protein, high-carbohydrate postexercise feeding regimen could improve recovery, as measured by subsequent cycling performance and mechanistic markers, relative to control feeding. In a crossover, 10 male cyclists performed 2- to 2.5-h interval training bouts on 3 consecutive evenings, ingesting either leucine-protein, high-carbohydrate nutrition (0.1/0.4/1.2/0.2 g·kg–1·h–1; leucine, protein, carbohydrate, fat, respectively) or isocaloric control (0.06/1.6/0.2 g·kg–1·h–1; protein, carbohydrate, fat, respectively) nutrition for 1.5 h postexercise. Throughout the experimental period diet was controlled, energy and macronutrient intake balanced, and protein intake clamped at 1.6 g·kg–1·day–1. The alternate supplement was provided the next morning, thereby isolating the postexercise nutrition effect. Following 39 h of recovery, cyclists performed a repeat-sprint performance test. Postexercise leucine-protein ingestion improved mean sprint power by 2.5% (99% confidence limit, ±2.6%; p = 0.013) and reduced perceived overall tiredness during the sprints by 13% (90% confidence limit, ±9.2%), but perceptions of leg tiredness and soreness were unaffected. Before exercise, creatine-kinase concentration was lowered by 19% (90% confidence limits, ±18%), but lactate dehydrogenase and pressure-pain threshold were unaltered. There was a small reduction in anger (25% ± 18%), but other moods were unchanged. Plasma leucine (3-fold) and essential amino acid (47%) concentrations were elevated postexercise. Net nitrogen balance trended mildly negative in both conditions (mean ± SD: leucine-protein, –20 ± 46 mg·kg–1 per 24 h; control, –25 ± 36 mg·kg–1 per 24 h). The ingestion of a leucine-protein supplement along with other high-carbohydrate food following intense training on consecutive days enhances subsequent high-intensity endurance performance and may attenuate muscle membrane disruption in well-trained male cyclists.
To investigate the effects of daily oral L-leucine ingestion on strength, bone mineral-free lean tissue mass (LTM) and fat mass (FM) of free living humans during a 12-wk resistance-training program.
Twenty-six initially untrained men (n = 13 per group) ingested either 4 g/d of L-leucine (leucine group: age 28.5 ± 8.2 y, body mass index 24.9 ± 4.2 kg/m2) or a corresponding amount of lactose (placebo group: age 28.2 ± 7.3 y, body mass index 24.9 ± 4.2 kg/m2). All participants trained under supervision twice per week following a prescribed resistance training program using eight standard exercise machines. Testing took place at baseline and at the end of the supplementation period. Strength on each exercise was assessed by five repetition maximum (5-RM), and body composition was assessed by dual energy X-ray absorptiometry (DXA).
The leucine group demonstrated significantly higher gains in total 5-RM strength (sum of 5-RM in eight exercises) and 5-RM strength in five out of the eight exercises (P < .05). The percentage total 5-RM strength gains were 40.8% (± 7.8) and 31.0% (± 4.6) for the leucine and placebo groups respectively. Significant differences did not exist between groups in either total percentage LTM gains or total percentage FM losses (LTM: 2.9% ± 2.5 vs 2.0% ± 2.1, FM: 1.6% ± 15.6 vs 1.1% ± 7.6).
These results suggest that 4 g/d of L-leucine supplementation may be used as a nutritional supplement to enhance strength performance during a 12-week resistance training program of initially untrained male participants.
Our laboratory is interested in the metabolic effects of ingested proteins. As part of this research, we currently are investigating the metabolic effects of ingested individual amino acids. The objective of the current study was to determine whether leucine stimulates insulin and/or glucagon secretion and whether, when it is ingested with glucose, it modifies the glucose, insulin, or glucagon response. Thirteen healthy subjects (6 men and 7 women) were studied on 4 different occasions. Subjects were admitted to the special diagnostic and treatment unit after a 12-hour fast. They received test meals at 8:00 am. On the first occasion, they received water only. Thereafter, they received 25 g glucose or 1 mmol/kg lean body mass leucine or 1 mmol/kg lean body mass leucine plus 25 g glucose in random order. Serum leucine, glucose, insulin, glucagon, and alpha-amino nitrogen concentrations were measured at various times during a 2.5-hour period after ingestion of the test meal. The amount of leucine provided was equivalent to that present in a high-protein meal, that is, that approximately present in a 350-g steak. After leucine ingestion, the leucine concentration increased 7-fold; and the alpha-amino nitrogen concentration increased by 16%. Ingested leucine did not affect the serum glucose concentration. When leucine was ingested with glucose, it reduced the 2.5-hour glucose area response by 50%. Leucine, when ingested alone, increased the serum insulin area response modestly. However, it increased the insulin area response to glucose by an additional 66%; that is, it almost doubled the response. Ingested leucine stimulated an increase in glucagon. Ingested glucose decreased it. When ingested together, the net effect was essentially no change in glucagon area. In summary, leucine at a dose equivalent to that present in a high-protein meal, had little effect on serum glucose or insulin concentrations but did increase the glucagon concentration. When leucine was ingested with glucose, it attenuated the serum glucose response and strongly stimulated additional insulin secretion. Leucine also attenuated the decrease in glucagon expected when glucose alone is ingested. The data suggest that a rise in glucose concentration is necessary for leucine to stimulate significant insulin secretion. This in turn reduces the glucose response to ingested glucose.
The definition of amino acid requirements for neonates receiving total parenteral nutrition (TPN) is critical for the further improvement of this nutritional regimen. In the present study we investigated the kinetics and requirements of phenylalanine and tyrosine in neonatal piglets receiving TPN. Twenty-four 3-d-old male Yorkshire piglets were fitted with external jugular and femoral catheters and maintained on identical TPN formulations for 5 d. Total amino acid, phenylalanine, tyrosine, and energy intakes were 15, 0.61, and 0.51 g. kg-1 . d-1 and 1.1 MJ . kg-1 . d-1, respectively. On day 5, piglets (three per level) were randomly assigned to receive one of eight phenylalanine intakes: 0.2. 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 1.2 g. kg-1 . d-1. On day 6, phenylalanine kinetics were measured during a 4-h primed, continuous infusion of L-[1-14C]phenylalanine. Plasma phenylalanine and phenylalanine oxidation were statistically similar for the three lowest phenylalanine intakes and increased thereafter. Crossover regression analysis yielded estimates for the mean requirement and safe phenylalanine intake of 0.45 and 0.48 g . kg-1 . d-1, respectively (equivalent to 30 and 32 mg/g amino acids, respectively), in the presence of excess tyrosine. An inability of piglets to maintain a linear oxidative response at phenylalanine intakes > 0.8 g . kg-1 . d-1 (equivalent to 53 mg/g amino acids) was found. These data represent the first direct estimates of phenylalanine requirements in neonates receiving TPN and demonstrate the use of oxidation techniques for the estimation of amino acid requirements during parenteral nutrition.
We report on research progress on two approaches that may be useful in determining the upper adequacy range for macronutrients such as amino acids. One approach was to attempt to identify "toxic metabolites" that were responsible for toxicity or biomarkers for the toxicity of excessive intake of an amino acid in rats. We found that there was hepatic toxicity that was specifically associated with L-cystine excess, but not with L-cysteine excess. We analyzed urine samples from rats fed basal diets or L-cystine or L-cysteine excess diets and identified 25 peaks from gas chromatography mass spectrometry analysis that were specific for L-cystine excess and also correlated with toxicity markers. Another approach was to try to identify "metabolic limits" by measuring CO(2) arising from amino acid excess. Uniformly (13)C labeled L-leucine was used as tracer, in diets with added L-leucine fed to rats, and (13)CO(2) arising from its metabolism was collected over 24 h and the fraction of the ingested L-leucine that was exhaled as CO(2) was calculated. The fractional exhalation of (13)CO(2) increased with increasing L-leucine dose, but showed an inflexion point at approximately 8.9 g/kg body weight, after which it reached a plateau. This suggested that >8.9 g/kg BW, the catabolism of L-leucine changed and this approximately coincided with the dose above which a statistically significant decrease in body weight was seen.
Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day.
Branched chain amino acids (BCAA), particularly leucine, have been suggested to be ergogenic for both endurance and strength/power performance. This study investigated the effects of dietary leucine supplementation on the exercise performance of outrigger canoeists. Thirteen (ten female, three male) competitive outrigger canoeists [aged 31.6 (2.2) year, VO(2max) 47.1 (2.0) ml kg(-1) min(-1)] underwent testing before and after 6-week supplementation with either capsulated L: -leucine (45 mg kg(-1) d(-1); n = 6) or placebo (cornflour; n = 7). Testing included anthropometry, 10 s upper body power and work and a row to exhaustion at 70-75% maximal aerobic power where perceived exertion (RPE), heart rate (HR) and plasma BCAA and tryptophan concentrations were assessed. Leucine supplementation resulted in significant increases in plasma leucine and total BCAA concentrations. Upper body power and work significantly increased in both groups after supplementation but power was significantly greater after leucine supplementation compared to the placebo [6.7 (0.7) v. 6.0 (0.7) W kg(-1)]. Rowing time significantly increased [77.6 (6.3)-88.3 (7.3) min] and average RPE significantly decreased [14.5 (1.5)-12.9 (1.4)] with leucine supplementation while these variables were unchanged with the placebo. Leucine supplementation had no effect on the plasma tryptophan to BCAA ratio, HR or anthropometric variables. Six weeks' dietary leucine supplementation significantly improved endurance performance and upper body power in outrigger canoeists without significant change in the plasma ratio of tryptophan to BCAA.
Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day.
Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasma [1-13Clleucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.
Preface.Vectors of Random Variables.Multivariate Normal Distribution.Linear Regression: Estimation and Distribution Theory.Hypothesis Testing.Confidence Intervals and Regions.Straight-Line Regression.Polynomial Regression.Analysis of Variance.Departures from Underlying Assumptions.Departures from Assumptions: Diagnosis and Remedies.Computational Algorithms for Fitting a Regression.Prediction and Model Selection.Appendix A. Some Matrix Algebra.Appendix B. Orthogonal Projections.Appendix C. Tables.Outline Solutions to Selected Exercises.References.Index.
The purpose of this study was to investigate the ability of whey-protein and leucine supplementation to enhance physical and cognitive performance and body composition. Thirty moderately fit participants completed a modified Air Force fitness test, a computer-based cognition test, and a dual-energy X-ray-absorptiometry scan for body composition before and after supplementing their daily diet for 8 wk with either 19.7 g of whey protein and 6.2 g leucine (WPL) or a calorie-equivalent placebo (P). Bench-press performance increased significantly from Week 1 to Week 8 in the WPL group, whereas the increase in the P group was not significant. Push-up performance increased significantly for WPL, and P showed a nonsignificant increase. Total mass, fat-free mass, and lean body mass all increased significantly in the WPL group but showed no change in the P group. No differences were observed within or between groups for crunches, chin-ups, 3-mile-run time, or cognition. The authors conclude that supplementing with whey protein and leucine may provide an advantage to people whose performance benefits from increased upper body strength and/or lean body mass.
The authors examined the effect of branched-chain amino acid (BCAA) supplementation on squat-exercise-induced delayed-onset muscle soreness (DOMS) using 12 young, healthy, untrained female participants. The experiment was conducted with a crossover double-blind design. In the morning on the exercise-session day, the participants ingested either BCAA (isoleucine:leucine:valine = 1:2.3:1.2) or dextrin at 100 mg/kg body weight before the squat exercise, which consisted of 7 sets of 20 squats/set with 3-min intervals between sets. DOMS showed a peak on Days 2 and 3 in both trials, but the level of soreness was significantly lower in the BCAA trial than in the placebo. Leg-muscle force during maximal voluntary isometric contractions was measured 2 d after exercise (Day 3), and the BCAA supplementation suppressed the muscle-force decrease (to ~80% of the value recorded under the control conditions) observed in the placebo trial. Plasma BCAA concentrations, which decreased after exercise in the placebo trial, were markedly elevated during the 2 hr postexercise in the BCAA trial. Serum myoglobin concentration was increased by exercise in the placebo but not in the BCAA trial. The concentration of plasma elastase as an index of neutrophil activation appeared to increase after the squat exercise in both trials, but the change in the elastase level was significant only in the placebo trial. These results suggest that muscle damage may be suppressed by BCAA supplementation.
The existing data on the safe upper limits of amino acid intake in humans is essentially observational; how much do individuals ingest and what side effects do they have? There are numerous studies in humans comparing the effects of high doses of amino acids given as protein bound vs. as free amino acids. These studies have shown that protein-bound amino acids have much less effect on plasma levels of the test amino acid, because protein intake stimulates protein synthesis as another sink for the increased amino acid intake. In practice, the highest amino acid intakes occur with free amino acid supplements that may be ingested by athletes who believe that the amino acids will benefit them in training and/or performance. Previously, in a piglet study, we were able to define the point at which maximal phenylalanine oxidation occurred, above which plasma phenylalanine concentration and body balance rose exponentially. We regard this value of maximal disposal (oxidation) of an amino acid as one metabolic marker of the upper limit of intake. Recently, others have demonstrated a similar maximal oxidation rate for leucine in rats. Based on these experimental data and the paucity of published human data in controlled experiments, we think that a systematic approach needs to be undertaken to define the maximal oxidation rate for all dietary indispensable amino acids and other amino acids that may be ingested in excess by humans. We believe that this will provide a rational basis to begin to define the upper limits of tolerance for dietary amino acids. However, some amino acids, such as threonine and methionine, will be more difficult to study, because they have more than 1 route of disposal or very complex metabolic regulation, in which case defining their upper limits will be more multifaceted.
Four healthy young men were studied to explore the effects of dietary leucine intake on plasma levels of leucine,isoleucine and valine. Leucine-, valine-, leucine-valine-, and leucine-isoleucine-valine-free amino acid mixtures were studied during four-day experimental diet periods. The effects of giving amino acid mixtures with high-carbohydrate, low-fat or low-carbohydrate, high-fat diets were also studied. Deficient leucine intake increased plasma levels of valine and isoleucine, but a valine-free diet did not affect plasma levels of the other branched-chain amino acids. Leucine influence was evident during the postprandial and fasted phases. Valine and isoleucine levels were less markedly reduced when diets devoid of these amino acids were also leucine-free, as compared with a diet providing adequate leucine. Changes in the major dietary energy source failed to influence the qualitative effects of dietary leucine adequacy on the branched-chain amino acid levels in plasma. The results suggest that leucine facilitates both tissue uptake of branched-chain amino acids and their intracellular metabolism.
A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.
Estimates of substrate oxidation obtained from appearance of 13C or 14C from tracers in breath must be corrected for retention of labeled carbon in the body. We aimed to determine the effect of a defined experimental diet and metabolic status on recovery of infused Na [13C]bicarbonate in breath. Six healthy male subjects consumed an experimental diet for 7 days before receiving a continuous infusion of formula without tracer on day 8 and received either an intragastric (ig) or intravenous (iv) infusion of Na [13C]bicarbonate on day 9 or 11 during a 4-h postabsorptive (PA), 4-h continuously fed period. A trend toward increasing PA breath enrichment during the first 7 diet days approached statistical significance (P = 0.051), whereas breath enrichments measured 3 h postbreakfast were consistently higher than PA values throughout and did not change over the 7-day period. Breath enrichments during a 4-h continuous ig infusion of formula without tracer on day 8 rose 2.0 +/- 0.5 atom percent excess (APE).10(-3) above base line (P less than 0.001, ANOVA). In the tracer studies, breath enrichments were similar for the ig and iv routes of tracer infusion. For the ig infusion the fraction of infused Na [13C]bicarbonate recovered in breath as 13CO2 was 0.74 +/- 0.02 for the PA period and 0.79 +/- 0.02 for the fed period. For the iv infusion the fraction recovered was 0.70 +/- 0.04 for the PA period and 0.82 +/- 0.03 for the fed period. Fractional recoveries were not significantly different for ig and iv routes of administration but were different for PA and fed periods (P less than 0.0001, 2-way ANOVA). The fractional recoveries for the fed period obtained here were similar to the value 0.81 reported in a number of other studies. Recovery of tracer in breath increased linearly with O2 uptake and CO2 production, suggesting that factors affecting respiratory gas exchange may alter recovery. We conclude that the primary factor determining label recovery is the immediate and recent nutritional status of the host.
Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.
Refined methods for separating PTC-amino acids on reverse phase columns may pose a challenge to traditional ion exchange techniques.
The response of rat skeletal muscle branched-chain alpha-keto acid dehydrogenase to administration of branched-chain amino acids in vivo was determined using a soluble preparation of the enzyme. After detergent extraction of the complex in the presence of kinase and phosphatase inhibitors, initial in vivo activity was typically 1 nmol X min-1 X g muscle-1, with 0.1 mM alpha-[1-14C]ketoisocaproate as substrate. Total activity of the dephosphorylated complex, measured after preincubation with 15 mM Mg2+, typically reached a maximum of 29 nmol X min-1 X g-1. Thus in overnight-fasted rats the complex was 2-3% active. Initial activity increased three- to fivefold after leucine or isoleucine (at higher concentrations) but not valine administration in vivo. After intravenous leucine injection (0.25 mmol/kg) initial muscle enzyme activity increased rapidly and subsequently decreased, paralleling plasma leucine concentrations, while plasma valine and isoleucine decreased. In conclusion, muscle branched-chain alpha-keto acid dehydrogenase complex is rapidly activated when circulating leucine is increased to concentrations that may occur after meals. During hyperleucinemia accelerated valine and isoleucine degradation by muscle may account for the observed "antagonism" among the branched-chain amino acids.
A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.
The short-term effects of feeding rats high levels of L-leucine or L-isoleucine on valine metabolism in vivo have been investigated. Consumption of a low-protein diet containing an additional 5% of leucine resulted in depression within one hour of the plasma concentrations of isoleucine, valine, alpha-keto-beta-methylvalerate, and alpha-ketoisovalerate. Concurrently with these changes in blood branched-chain amino acids and branched-chain ketoacids was a rapid increase (51%) in whole-body L-[1-14C]-valine oxidation. Studies with intragastrically administered leucine solutions indicated that the depressions in blood concentrations of valine occurred over the same time period as the stimulation in valine oxidation. In contrast, consumption of a low-protein diet containing an additional 5% of isoleucine had no significant effect on the plasma concentrations of leucine, valine, and alpha-ketoisocaproate; a significant (P less than 0.01) depression in the plasma concentration of alpha-ketoisovalerate was observed three hours after the diet containing excess isoleucine had been consumed. In contrast to the results obtained with excess leucine, consumption of excess isoleucine had no significant effect on the rate of valine oxidation in vivo. As part of an effort to explain the leucine-induced depletion of plasma valine and stimulation of valine oxidation, liver and muscle branched-chain aminotransferase and liver branched-chain ketoacid dehydrogenase activities were measured. Consumption of excess leucine had no significant effect on either muscle or liver aminotransferase activities, but was associated with a greater than two-fold increase in hepatic dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.
1., Intravenous infusions of l-valine (600 μmol/min), l-isoleucine (150 μmol/min), l-leucine (300 μmol/min) and a mixture of the three branched-chain amino acids (70% l-leucine, 20% l-valine, 10% l-isoleucine; 270 μmol/min) were given to four groups of healthy volunteer subjects. Whole-blood concentrations of amino acids and glucose and serum insulin were measured before and during the infusions.
2. Valine and isoleucine infusions resulted in twelve- and six-fold increases in the respective amino acid. During valine infusion, tyrosine was the only amino acid for which a decrease in concentration was seen (25%, P < 0.05). With isoleucine administration, no significant changes were found. In contrast, leucine infusion (during which the leucine concentration rose about sixfold) was accompanied by significant decreases in tyrosine (35%), phenylalanine (35%), methionine (50%), valine (40%) and isoleucine (55%). The arterial glucose concentration fell slightly (5%) and the insulin concentration increased 20% during leucine infusion.
3. Infusion of the mixture of the three branched-chain amino acids resulted in marked decreases in tyrosine (50%), phenylalanine (50%) and methionine (35%). The decreased amino acid levels remained low for 2 h after the end of the infusion.
4. The present findings demonstrate that intravenous infusion of leucine (not infusion of valine or isoleucine) results in marked reductions in the concentrations of the aromatic amino acids and methionine. Infusion of a mixture of the three branched-chain amino acids gives results similar to those obtained with leucine infusion alone. Thus a mixed branched-chain amino acid solution with leucine as its main constituent seems to be the best alternative in the treatment of patients with hepatic cirrhosis and encephalopathy.
Study of the amino acid metabolism of vulnerable groups, such as pregnant women, children and patients, is needed. Our existing protocol is preceded by 2 d of adaptation to a low 13C formula diet at a protein intake of 1 g. kg-1. d-1 to minimize variations in breath 13CO2 enrichment and protein metabolism. To expand on our potential study populations, a less invasive protocol needs to be developed. We have already established that a stable background 13CO2 enrichment can be achieved on the study day without prior adaptation to the low 13C formula. Therefore, this study investigates phenylalanine kinetics in response to variations in prior protein intake. Healthy adult subjects were each fed nutritionally adequate mixed diets containing 0.8, 1.4 and 2.0 g protein. kg-1. d-1 for 2 d. On d 3, subjects consumed an amino acid-based formula diet containing the equivalent of 1 g protein. kg-1. d-1 hourly for 10 h and primed hourly oral doses of L-[1-13C]phenylalanine for the final 6 h. Phenylalanine kinetics were calculated from plasma-free phenylalanine enrichment and breath 13CO2 excretion. A significant quadratic response of prior protein intake on phenylalanine flux (P = 0.012) and oxidation (P = 0.009) was identified, such that both variables were lower following adaptation to a protein intake of 1.4 g. kg-1. d-1. We conclude that variations in protein intake, between 0.8 and 2.0 g. kg-1. d-1, prior to the study day may affect amino acid kinetics and; therefore, it is prudent to continue to control protein intake prior to an amino acid kinetics study.
Leucine, isoleucine and valine, the branched-chain amino acids (BCAA), make up about one-third of muscle protein. Of these, leucine has been the most thoroughly investigated because its oxidation rate is higher than that of isoleucine or valine. Leucine also stimulates protein synthesis in muscle and is closely associated with the release of gluconeogenic precursors, such as alanine, from muscle. Significant decreases in plasma or serum levels of leucine occur following aerobic (11 to 33%), anaerobic lactic (5 to 8%) and strength exercise (30%) sessions. In skeletal muscle, there is a decrease in leucine level and a reduction in glycogen stores during exhaustive aerobic exercise. Basal fasting serum leucine levels decrease by 20% during 5 weeks of speed and strength training in power-trained athletes on a daily protein intake of 1.26 g/kg bodyweight.
The leucine content of protein is assumed to vary between 5 and 10%. There are suggestions that the current recommended dietary intake of leucine be increased from 14 mg/kg bodyweight/day to a minimum of 45 mg/kg bodyweight/day for sedentary individuals, and more for those participating in intensive training in order to optimise rates of whole body protein synthesis.
Consumption of BCAA(30 to 35% leucine) before or during endurance exercise may prevent or decrease the net rate of protein degradation, may improve both mental and physical performance and may have a sparing effect on muscle glycogen degradation and depletion of muscle glycogen stores.However, leucine supplementation (200 mg/kg bodyweight) 50 minutes before anaerobic running exercise had no effect on performance. During 5 weeks of strength and speed training, leucine supplementation of 50 mg/kg bodyweight/day, supplementary to a daily protein intake of 1.26 g/kg bodyweight/day, appeared to prevent the decrease in the serum leucine levels in power-trained athletes.
According to 1 study, dietary supplementation of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) 3 g/day to humans undertaking intensive resistance training exercise resulted in an increased deposition of fat-free mass and an accompanying increase in strength.Muscle proteolysiswas also decreased with HMB, accompanied by lower plasma levels of enzymes indicating muscle damage and an average 50% decrease in plasma essential amino acid levels. Furthermore, BCAA supplementation (76% leucine) in combination with moderate energy restriction has been shown to induce significant and preferential losses of visceral adipose tissue and to allow maintenance of a high level of performance.
Caution must be paid when interpreting the limited number of studies in this area since, in many studies, leucine has been supplemented as part of a mixture of BCAA. Consequently, further research into the effects of leucine supplementation alone is needed.
Previous recommendations for branched-chain amino acids (BCAA), based on nitrogen balance studies, were found to be low in a series of stable isotope-labeled amino acid studies. The BCAA requirement was increased in the new dietary reference intake (DRI) report on the basis of a series of stable isotope studies examining the requirement of leucine and valine individually, but not isoleucine. To reduce the possibility of interactions among these amino acids and imbalances in the mixture affecting the estimate of requirements, we decided to determine the requirement for the total BCAA of young healthy adult men, receiving a mixture of BCAA based on the proportion of these amino acids in egg protein, by use of indicator amino acid oxidation. Seven men were assigned to receive nine graded intakes of a BCAA mixture in random order: 34, 50, 66, 80, 100, 120, 140, 160 and 180 mg/(kg. d). The rate of release of (13)CO(2) from the oxidation of L-[1-(13)C]phenylalanine (F (13)CO(2)) was measured and a two-phase linear regression crossover model was applied to determine total BCAA requirement. The mean requirement and population-safe level (upper limit of 95% confidence interval) of the total BCAA were 144 and 210 mg/(kg. d), respectively. Based on the balance of BCAA in egg protein, our estimate for the mean leucine requirement is 55 mg/(kg. d), which is substantially higher than the 34 mg/(kg. d) recommended by the DRI.
The proportions of amino acids in diets typical of human populations usually differ from the proportions in which they are required, although adverse effects due to such differences are not common. However, there is little systematic information about the adverse effects and the pathophysiological mechanisms of excessive intakes of single or mixtures of amino acids in human subjects. To promote the safe and effective application of amino acids in clinical nutrition and for health promotion it is necessary to establish a sound scientific basis for evaluating their efficacy and safety under various conditions of use. Hence, a series of Amino Acid Assessment Workshops (AAAW) are being organized to bring together experts in amino acid nutrition, metabolism, cell and molecular biology, toxicology and regulation/policy with the eventual purpose of establishing a paradigm for the characterization of risks associated with the ingestion of specific intakes of amino acids by humans. In this introductory paper I summarize the major issues arising at the 1st AAAW, held in Tokyo June, 2001, and provide an introductory context to the present, 2nd AAAW.
This study described the effect of leucine supplementation on serum amino acid concentration during two different exercise sessions in competitive male power athletes. The subjects performed a strength exercise session (SES; n = 16; 26 +/- 4 years) or a maximal anaerobic running exercise session (MARE; n = 12; 27 +/- 5 years) until exhaustion twice at a 7-day interval. The randomized subjects consumed drinks containing leucine (100 mg x kg/body weight before and during SES or 200 mg x kg/body weight before MARE) or placebo. Blood specimens taken 10 min before (B) and after (A) the sessions were analyzed for serum amino acids. In SES the concentration of leucine was distinctly higher in the leucine supplemented group than in the placebo group in both B (p < 0.001) and A (p < 0.001) samples. The leucine concentration decreased in placebo but not in the leucine supplemented group following the exercise session. Isoleucine (p = 0.017) and valine (p = 0.006) concentration decreased more in the leucine supplemented group than in placebo in A samples. In MARE the concentration of leucine was higher in the leucine supplemented group than in placebo in both B (p < 0.001) and A (p < 0.001) samples and increased (p < 0.001) in the supplemented group following the session. Isoleucine (p = 0.020) and valine (p = 0.006) concentration decreased in the supplemented group in A samples. There were no differences in a counter movement jump after SES or in the running performance in MARE between the leucine supplemented group and placebo. These findings indicate that consuming leucine before or before and during exercise sessions results in changes in blood amino acid concentration. However, the supplementation does not affect an acute physical performance.
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).
Daily requirements for protein are set by the amount of amino acids that is irreversibly lost in a given day. Different agencies have set requirement levels for daily protein intakes for the general population; however, the question of whether strength-trained athletes require more protein than the general population is one that is difficult to answer. At a cellular level, an increased requirement for protein in strength-trained athletes might arise due to the extra protein required to support muscle protein accretion through elevated protein synthesis. Alternatively, an increased requirement for protein may come about in this group of athletes due to increased catabolic loss of amino acids associated with strength-training activities. A review of studies that have examined the protein requirements of strength-trained athletes, using nitrogen balance methodology, has shown a modest increase in requirements in this group. At the same time, several studies have shown that strength training, consistent with the anabolic stimulus for protein synthesis it provides, actually increases the efficiency of use of protein, which reduces dietary protein requirements. Various studies have shown that strength-trained athletes habitually consume protein intakes higher than required. A positive energy balance is required for anabolism, so a requirement for "extra" protein over and above normal values also appears not to be a critical issue for competitive athletes because most would have to be in positive energy balance to compete effectively. At present there is no evidence to suggest that supplements are required for optimal muscle growth or strength gain. Strength-trained athletes should consume protein consistent with general population guidelines, or 12% to 15% of energy from protein.
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.
Some athletes can have quite high intakes of branched-chain amino acids (BCAAs) because of their high energy and protein intakes and also because they consume protein supplements, solutions of protein hydrolysates, and free amino acids. The requirement for protein may actually be higher in endurance athletes than in sedentary individuals because some amino acids, including the BCAAs, are oxidized in increased amounts during exercise compared with rest, and they must therefore be replenished by the diet. In the late 1970s, BCAAs were suggested to be the third fuel for skeletal muscle after carbohydrate and fat. However, the majority of later studies, using various exercise and treatment designs and several forms of administration of BCAAs (infusion, oral, and with and without carbohydrates), have failed to find a performance-enhancing effect. No valid scientific evidence supports the commercial claims that orally ingested BCAAs have an anticatabolic effect during and after exercise in humans or that BCAA supplements may accelerate the repair of muscle damage after exercise. The recommended protein intakes for athletes (1.2 to 1.8 g . kg body mass(-1) . d(-1)) do not seem to be harmful. Acute intakes of BCAA supplements of about 10-30 g/d seem to be without ill effect. However, the suggested reasons for taking such supplements have not received much support from well-controlled scientific studies.
Amino acids (AAs), especially BCAAs, play pivotal roles in hormonal secretion and action as well as in intracellular signaling. There is emerging data showing that BCAAs regulate gene transcription and translation. Signaling proteins such as the mammalian target of rapamycin act as sensors of BCAAs, especially leucine, to modulate anabolic action. AAs stimulate protein synthesis and inhibit protein breakdown in skeletal muscle and liver. The specific role of BCAAs in regulating synthesis and breakdown of individual protein or proteins with common function or functions remains to be defined. Future studies should also focus on potential adverse effects of BCAAs on insulin sensitivity, renal function, and tumor growth. It also remains to be determined whether potential adverse effects of BCAA supplementation is similar in people of different age groups.