Leucine is an essential branched-chain amino acid present in a number of protein-containing foods (1). Leucine metabolites, such as α-hydroxyisocaproic acid (α-HICA) and β-hydroxy-β-methylbutyrate (HMB), have been proposed to promote changes in body composition, performance, and reduce indirect markers of muscle damage (2,3). A product of leucine metabolism, α-HICA (leucic acid) is formed in many human tissues (4). Research on α-HICA is scarce, with only one study (3) in male soccer players. Based on this single investigation (3), it has been suggested that α-HICA may exert an anticatabolic action.
Supplementation with the leucine metabolite HMB in the calcium form, HMB-Ca, has shown positive effects on performance and body composition (5–8), whereas other studies report no effect (9–11). The suggestions have been that the most effective use of HMB would be in older adults, untrained individuals, and injured or energy-restricted athletes (12). A free acid (FA) form of HMB (HMB-FA) has a higher bioavailability when comparing with HMB-Ca (13); however, there is no difference between HMB-Ca and HMB-FA in their ability to enhance muscle protein synthesis in young men (14). A recent systematic review suggested that HMB-FA, in conjunction with resistance training, may attenuate markers of muscle damage, augment acute endocrine responses, and enhance training-induced increases in muscle mass and strength (2). However, the reported effects on muscle mass and strength in that review are strongly driven by two research studies (2), which contained extraordinary results (15,16) that have been questioned due to extensive limitations (17). Thus, it is unclear at this time as to the true efficacy of HMB-FA in promoting changes in body composition.
Meta-analyses indicate small effects of HMB on markers of muscle damage, endocrine responses, and training-induced chances in muscle mass and strength (2), whereas other analyses do not support this conclusion (18,19). To our knowledge, there has been no randomized trial to investigate the effects of commercially available forms of these compounds on muscle thickness (MT) and selected performance outcomes. This is, in our view, exceptionally important because consumers are taking these supplements direct “from the shelf” often based on claims stemming from research. Thus, we undertook a pragmatic controlled, double-blind randomized trial to compare commercially available supplements, HMB-FA, α-HICA, and HMB-Ca, on resistance training-induced changes in MT and performance. Our working hypothesis was that so long as participants adhered to a diet containing adequate energy and dietary protein that there would be no differences between those receiving the leucine metabolites—HMB-FA, α-HICA and HMB-Ca—when compared with a placebo-consuming group.
Ethics and general study design
This investigation was approved by the Faculty of Human Kinetics Institutional Review Board (approval number 15/2017) and conformed to all standards of human research set out in the declaration of Helsinki. The trial was registered at clincicaltrials.gov as NCT03511092. Before engaging in any of the study procedures, the purpose and design of the study, the data collection methodologies, and all potential risks and benefits were explained to potential research participants. All participants gave their verbal and written informed consent before enrolling. Fifty-three men were recruited according to the eligibility criteria with 40 completing the investigation. Participants were between the ages of 18 and 45 yr and were recruited from social networks and local gyms. Participants were randomly assigned to one of the following four groups: α-HICA, HMB-FA, HMB-Ca, placebo (PLA). For details, please refer to the Consolidated Standards of Reporting Trials (CONSORT) flow diagram (Fig. 1).
All supplements were generously donated directly from shelf-stock from a local supplement store. All brands presented a certificate of analysis regarding the content of each supplements. Third party testing (Labs-Mart Inc. Edmonton, AB) of the same supplements (reports 70496-1, 70496-2, and 70496-3) for α-HICA, HMB-FA, and HMB-Ca (using HPLC-UV) indicated the supplements contained 97% ± 2.9% of the α-HICA, 95% ± 2.3% of the HMB-FA, and 96% ± 2.1% of the HMB-Ca reported. Placebo consisted of magnesium stearate. Assignment was according to a randomly generated list and was blocked in varying block sizes with participants matched for grip strength, age, and dual-energy x-ray absorptiometry (DXA)-measured fat-free mass (FFM). Thus, at baseline, there were no statistically significant differences between groups for handgrip strength, age, or FFM (Table 1). Participants received packaged supplements and instructions in a double-blinded fashion. Supplementation compliance was assessed by having the participants hand their empty supplement packets back to the researchers at the end of each 2-wk block of training. The training protocol consisted on whole body hypertrophy-type resistance training routine for intermediate-trained individuals and consisted of three training sessions per week for 8 wk.
Evaluations of strength, skeletal muscle and performance took place at baseline and weeks 4 and 8. In addition, each subject had a blood sample collected in the same time in the morning by the same technician and were repeated at the end of week 4 and 8 of the study. All evaluations of the eligible participants and blood samples collection were performed early in the morning after a 12-h fast and without prior exercise, or consumption of alcohol, or caffeine/stimulant beverages. Muscle strength and power were assessed in a fed state after the ingestion of a meal replacement bar (Matrix Bar, Olimp Labs, Pustynia, Poland: comprised of 26.2 g protein, 20.5 g carbohydrate, and 8 g fat.
Participants were healthy men between 18 and 45 yr old, currently engaged in resistance training for at least 1 yr and at least three times per week. Sample size was calculated from previous studies (please refer to statistics section for more details). Participants taking any type of medication or supplements aimed at enhancing body composition or performance before the research, were excluded (only protein supplements and multivitamins were allowed). All participants were nonsmokers and free from clinical conditions that might have compromised their tolerance of the supplements, training program, or influence body composition and performance. Participants with more than 25% body fat were also excluded.
Body composition was determined at baseline by DXA and MT by ultrasonography. Participants underwent a whole-body DXA scan according to the procedures recommended by the manufacturer on a Hologic Explorer-W, fan-beam densitometer (Hologic, Waltham, MA). The equipment measures the attenuation of X-rays pulsed between 70 and 140 kV synchronously with the line frequency for each pixel of the scanned image. A step phantom with six fields of acrylic and aluminum of varying thickness and known absorptive properties was used as an external standard calibrator for the analysis of different tissue components. The same technician positioned the patient, performed the scan, and executed the analyses (software QDR for Windows version 12.4; Hologic) according to the operator’s manual using the standard analysis protocol. The coefficients of variation in our laboratory, based on 10 young active adults (five males and five females), is 1.6% for bone mineral content, 1.7% for FM, and 0.8% for FFM (20).
Muscle thickness of the vastus lateralis (VL) and the rectus femoris (RF) was assessed at rest using B-mode ultrasound imaging with a 9-cm-long 10-MHz linear-array transducer (model EUB-7500; Hitachi Medical Corporation, Tokyo, Japan). Longitudinal and transversal scans were taken from the muscles mid-belly corresponding to 39% (VL) and 56% (RF) of the distance from the proximal edge of the patella to the anterior superior iliac spine, according to Blazevich et al. (21). Participants were positioned in a seated position with their knee flexed at 10° (0° being full extension), participant’s legs were supported during the scan and their muscles relaxed. To ensure that repeated scans (weeks 4 and 8) were taken from the same site, scanning locations were mapped with a malleable transparent plastic sheet at the baseline measurement, along with other distinguishing surface landmarks (e.g., border of patella, tattoos, scars, moles). We defined MT as the perpendicular distance between the subcutaneous adipose tissue-muscle interface and intermuscular interface, and quantified three times from the ultrasound scans using the image analysis software, IMAGEJ 1.42q (National Institutes of Health, Bethesda, MD). Averaged values were considered for further analysis. All measures were collected and digitally analyzed by the same operator with an intra-rater coefficient of variation of 0.5% for VL and 0.6% for RF.
Blood samples were collected by standard procedures into ethylenediaminetetraacetic acid tubes, centrifuged at 500g at 4°C for 15 min, and plasma was frozen at −80°C. Plasma samples were then analyzed for growth hormone (GH), total testosterone, insulin-like growth factor 1 (IGF-1) and cortisol at the Core Laboratory of McMaster University Medical Centre using solid-phase, two site chemiluminescence immunometric assays (Immulite; Intermedico, Holliston, MA). All intra-assay coefficients of variation for these markers were below 5% and all assays included external and internal standards and daily quality controls. Plasma creatine kinase (CK) was determined by a kit according to the manufacturers specifications (Sigma Aldrich, St. Louis, MO). The intra-assay coefficient of variation for CK was below 8%. Hemoglobin was analyzed by photometry (Beckman, DU68) by the Drabkin method and hematocrit by capillary microcentrifugation (Sigma Aldrich). Concentrations of hormones and metabolites were corrected for plasma volume variation with hemoglobin concentration and hematocrit.
Muscle strength and power
Muscle strength was assessed at study entry by evaluating grip strength and one repetition maximum (1RM) of the back squat and bench press at baseline and at the end of weeks 4 and 8. Maximal isometric forearm strength was determined using a handgrip dynamometer (Jamar, Sammons Preston, Inc, Bolingbrook, IL). The evaluation of maximum strength during the protocol was obtained from 1RM testing of the back squat and bench press exercises on a Multipower machine (model-M953; Technogym, Cesena, Italy). The determination of 1RM from these exercises was conducted according to the National Strength and Conditioning Association (NSCA) guidelines and supervised by an NSCA-certified strength and conditioning specialist.
Muscle power was assessed with a supramaximal cycling test (Wingate) and a countermovement jump (CMJ). During the Wingate test (using a cycle ergometer—Monark ergomedic 894 E; Monark Exercise AB, Vansbro, Sweden), volunteers were instructed to cycle against a predetermined resistance (7.5% body weight) as fast as possible for 30 s (22). Peak power and average power were calculated. Countermovement jump was assessed on a contact platform controlled by an open-source hardware and software model (Chronojump, Barcelona, Spain), which computed and stored flight time with a temporal resolution of 1 ms. The best attempt out of 3 was considered for analysis (23).
Supplementation and diet control
Each participant received a commercial form of either α-HICA (HICA, Onsalesit, SA, Funchal, Portugal), HMB-FA (Beta-TOR, Body Attack, Hamburg, Germany), HMB-Ca (HMB Mega Caps 1250, Olimp Labs, Pustynia, Poland), or placebo (magnesium stearate; EightJuice, Seixal, Portugal). Participants were only aware that these were leucine derivatives and that a placebo group existed. Compounds were distributed in a double-blind manner. The investigator responsible for the sample randomization and compound distribution was not directly involved in participants’ eligibility interview or data collection. Individuals ingested supplements or placebo three times daily, alongside with meals or prior to training, according with previous research: 3 × 500 mg for α-HICA (3) and 3 × 1 g for HMB-FA (16), HMB-Ca (24), or placebo.
Participants were individually instructed by licensed and trained dieticians to consume sufficient energy and protein to allow for training-induced gains of lean mass. Participants self-reported 3-d dietary intake through dietary logs at the beginning, fourth and last week of the study. Food logs were analyzed by Food Processor 10.12 (ESHA Research Inc., Salem, OR) for energy and macronutrients. Before the beginning of the study, if participants were not ingesting at least 1.6 g·kg−1·d−1 protein per body weight (25) and at least 45 kcal·kg−1 of FFM a day (26), a registered dietitian would provide counseling on foods to consume to adjust the protein and/or energy intake (Table 2). Eleven participants required protein adjustments, which were normalized to ≈2.2 g·kg−1·d−1 protein per body weight (using protein supplements or food, depending on individual’s convenience). This consequently raised daily mean protein intakes into a high-protein approach in all groups. These strategies were used to assure that participants were in an estimated positive energy balance and that protein ingestion allowed for muscle hypertrophy development.
Training and exercise protocols
The resistance training protocol was designed according to the guidelines for hypertrophy type of resistance training for intermediate-trained individuals and consisted of three sessions per week during an 8-wk period, with a minimum of 48-h interval between sessions (27). The following exercises were performed in the described order during each resistance training session: barbell back squat, deadlift, machine leg extension, barbell flat bench press, dumbbell military press, lat pull-down, seated cable row. During the first 3 wk, participants performed three (weeks 1 and 2) or four (week 3) sets of 12 repetitions with 60-s interval between sets and exercises. In weeks 4, 5, and 6, participants performed three (week 4) or four (weeks 5 and 6) sets of 10 repetitions with 90-s interval between sets and exercises. During the last 2 wk (weeks 7 and 8), participants performed four sets of eight repetitions with 120-s interval between sets and exercises.
Each repetition was performed in a controlled manner for 2 s during the eccentric phase and 1 s in the concentric phase. Given that athletes had a resistance training background of at least 1 yr, sets were carried to the point of concentric muscle failure while maintaining proper exercise form. All exercises were performed according to the NSCA guidelines and under the direct 1:1 supervision of an experienced strength and conditioning coach.
Sample size was calculated through an a priori power analysis (G*Power Version 126.96.36.199, Heinrich Heine Universitat Dusseldorf, Germany), based on FFM changes from previous studies (15) and power of 0.80 and alpha of 0.05. Statistical analysis was performed using IBM SPSS statistics version 22.0 (IBM, Chicago, IL). Normality of the distribution of variables was tested by Shapiro–Wilk test. Baseline characteristics between groups and delta from baseline to week 8 for MT of both VL and RF were analyzed by a one-way ANOVA, since normality was observed. Time and time–group interactions were evaluated by repeated-measures ANOVA. The equality of the matrix of variance and sphericity were explored with the Levene F test and Mauchly’s test, respectively. Overall significance level for α was set at P ≤ 0.05.
According to food records, there were no differences between groups at baseline and no differences occurred from baseline to the end of the study in dietary intake. Participants were compliant with the supplementation, taking 84% ± 1% of supplements with no adverse effects being reported at the end of the 4th or 8th week of the study. Participants completed 94% ± 5% (α-HICA, 96% ± 4%; HMB-FA, 92% ± 5%; HMB-Ca: 96% ± 5%; PLA, 95% ± 6%) of the prescribed training sessions during the study.
Muscle thickness increased for VL by 8% (95% confidence interval [CI], 2.5–13.6; α-HICA), 6% (95% CI, 3.5–8.6; HMB-FA), 9% (95% CI, 1.7–15.8; HMB-Ca), and 8% (95% CI, 2.17–13.1; PLA), and for RF by 3% (95% CI, −3.1 to 8.9; α-HICA), 4% (95% CI, 1.7–6.1; HMB-FA), 6% (95% CI, 1.0–10.3; HMB-Ca), and 8% (95% CI, 3.2–13.4; PLA), from baseline to week 4 (VL and RF: P < 0.001) and from week 4 to week 8 (VL: P = 0.009; RF: P = 0.018), with no difference between groups regarding delta changes from baseline to week 8 (Fig. 2).
Plasma CK, IGF-1, total cortisol, and GH increased from baseline to week 4 (P < 0.001) with no differences from week 4 to week 8 (Fig. 3). There was no change in total testosterone.
Muscle strength and power
A time effect was found for Wingate peak power (P = 0.018), CMJ height (P = 0.028), CMJ power (P = 0.006), and 1RM back squat and bench press (P < 0.001). Wingate peak power decreased from baseline to week 4 (P = 0.007) returning to baseline values at the end of week 8; both CMJ height and power increased from baseline to week 8 (CMJ height: P = 0.043; CMJ power: P = 0.007); and 1RM back squat and bench press increased from baseline to week 4 (P < 0.001) and remained elevated at week 8 in both exercises (P < 0.001 and P = 0.001, respectively) (Table 3). There were no differences in these outcomes by group.
Our study is the first to directly compare the efficacy off-the-shelf commercially available forms of the leucine metabolites α-HICA, HMB-Ca, HMB-FA versus a placebo on resistance training-induced adaptations in performance and MT in young men consuming a balanced diet with sufficient protein intake. No differences were observed between supplements and placebo in anthropometric measures, MT, blood markers, or muscle strength and power. We observed training-induced effects for all performance-related variables, including MT, that was comparable to investigations of similar duration and with a similar group of trainees (10,11,28,29).
Our results of a lack of effect of HMB-Ca are in broad agreement with a variety of other investigations in which HMB-Ca supplementation did not influence resistance training-induced outcomes (11,29–31). We lack robust data for a comparison of an effect of α-HICA (3); however, our data provide no support for the concept that α-HICA is anabolic over and above normal protein and energy intake. Limited data exist on HMB-FA, but our data are in direct and sharp contrast to the few studies that have used this supplement (15,16).
We failed to reproduce the results of Kraemer et al. (24) in recreationally active participants who reported an increase in FFM in young men taking the same dose of HMB-Ca of ~9 kg in 12 wk as well as substantially greater increases in CMJ power and 1RM for squat and bench press. However, the supplement used by Kraemer et al. included other dietary ingredients (glutamine, arginine, and taurine) which would preclude direct attribution of any changes seen specifically to HMB-Ca; however, we can find no compelling evidence why these other metabolites would be anabolic (32,33). We also note that there are no meaningful differences in muscle protein synthesis when leucine and HMB in either calcium or FA form are compared (14,34). An obvious difference between our work and that of Kraemer et al. (24) is the training program; however, recent evidence (35) shows that when leucine and HMB-Ca were compared, using the identical training program as that of Kraemer et al., that there were no differences between groups. Moreover, in this work (35) gains in FFM were in-line with recent systematic reviews (25). Meta-analyses show that HMB-Ca modestly augments training-induced increases in FFM in untrained participants and to some extent in elderly populations (6,19) or older persons in bed rest (5). Regarding HMB-FA, our findings are in stark contrast with previously reported results by Wilson et al. (16) and Lowery et al. (15). However, these studies have been subject to considerable scientific scrutiny and have inherent methodological limitations (17).
Insofar as protein intake is concerned, our study has displayed a mean intake 3.1 ± 0.5 g·kg−1·d−1 protein per body weight which we believe is the highest intake reported with leucine metabolites to date. Other previous studies with HMB-Ca, which are in broad agreement with our results, reported intakes ranging from 1.9 to 2.4 g·kg−1·d−1 protein per body weight (29,36,37). These amounts are deemed sufficient according to the latest body of evidence (25). Our higher-protein intake is the result of the dietary intervention correcting intakes below 1.6 to 2.2 g·kg−1·d−1 protein per body weight as suggested by Morton et al. (25). Studies reporting extraordinary body composition outcomes with HMB, have failed to provide absolute energy and protein intakes (15,16,24). Thus, whether they were consuming sufficient protein and in an estimated positive energy balance cannot be determined, which poses a serious limitation.
As far as α-HICA is concerned, our findings are in contrast to the FFM increase reported by Mero et al. (3). The participants in this investigation were younger soccer players with no experience in resistance training. Whether α-HICA can improve training-induced changes in body composition or recovery in different populations requires further clarification. Nonetheless, our data provide no support for the contention that α-HICA is anabolic.
Changes in blood markers did not reveal any effect of the leucine metabolites or training protocol on total testosterone. These results do not align with those of some previous investigations (24,38) and are in agreement with others (15,19). The current body of evidence, in addition to our findings, does not support the concept that HMB can increase testosterone levels. As would be expected, training raised CK, cortisol, IGF-1, and GH. Unlike previous investigations, our findings did not support any reduction in CK activity (2). Although circulating CK is a weak proxy marker of muscle damage, it is often used (39). Based on our results, we did not see that either form of HMB or α-HICA was able to prevent exercise-induced muscle damage when compared to placebo. It has been questioned, however, whether compounds that are proposed to suppress damage and proteolysis would be effective treatment tools as protein removal is needed to promote clearance of damaged proteins (40).
Cortisol was also elevated during our training protocol, with no effect of any supplement when compared with placebo. Supplementation with HMB has been touted to suppress cortisol elevation to resistance training (16,24), whereas others failed to detect significant differences (6,38). We observed a small but significant rise in GH over time, but with no effect of any supplement. Some reported elevations for GH with HMB (24,41), whereas others reported no change with similar protocols (6). In our study, we did not detect a change in IGF-1 with any supplement which is in line with some studies (42) but not with others (41,43). Although IGF-1, GH, and testosterone are commonly described as anabolic hormones, current evidence suggests that acute elevations of these hormones does not correlate with resistance training-induced changes in FFM, or strength (44).
None of the leucine metabolites we studied augmented training-induced changes in any index of performance or MT. Our participants were, according to diet records, in a positive energy balance and consuming a high-protein diet. Our results are, at least from a mechanistic perspective, not surprising. It is known that leucine and HMB-FA exert similar effects on protein turnover in humans (14). In addition, there is no apparent anabolic advantage of HMB-FA over HMB-Ca in terms of stimulating protein synthesis (34), despite an apparent difference in bioavailability (34). An important question is how much more efficacious a metabolite of leucine could be than leucine itself in stimulating anabolism and/or suppressing catabolism to affect hypertrophy? We propose that it is unlikely that a metabolite of the same compound would be that much more effective that it could account for the type of growth many have reported (15,16). This bears particular consideration because leucine and its metabolites share the same canonical signaling mechanisms leading to stimulation of anabolism and suppression of catabolism (14).
Some of the strengths of the following pragmatic trial are the applicability of this research. Although most research uses supplements directly supplied from manufacturers, this was not the case in our trial because all supplements were obtained directly from a supplement store, with no interference from manufacturers. Here, we present a tightly controlled pragmatic randomized, double-blinded controlled trial where three commercially available supplements were directly evaluated and compared with placebo. We propose that our findings present a relevant contribution to the current body of evidence regarding these compounds and that our results are noteworthy.
Some weaknesses of our trial include the fact that supplements were presented in capsules and tablets that did not allow for a truly equivalent placebo form. Another limitation is related with the fact that α-HICA was administered in a different fashion than previous research by Mero et al. (3). However, it is unlikely that timing of ingestion of the α-HICA might have influenced results, because different timing protocols have been used with leucine derivatives, and no differences were reported (11,28).
We conclude that when consuming a high-protein diet and in an estimated positive energy balance, none of the investigated leucine metabolites resulted in an ergogenic effect on any outcome variable, in young moderately trained men. Our trial represents a true pragmatic trial with supplements that were commercially available as they would be to consumers. Our findings do not support the use of leucine metabolites as supplementation strategy to augment training-induced gains in performance or body composition in young men.
The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. S. M. P. reports having received funding, honoraria, and travel expenses from the US National Dairy Council, research funding from Pepsico, and research funding from the Dairy Farmers of Canada. This study was financed by the Interdisciplinary Center for the Study of Human Performance (CIPER). All supplements were freely donated by Body Temple, Lda. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
F. J. T. withholds a position as technical manager for Body Temple, Lda a company that sells HMB-Ca and HMB-FA.
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