β-hydroxy-β-methylbutyrate (HMB) is a derivative of the amino acid leucine and is metabolized from α-ketoisocaproate (KIC), the keto acid of leucine, in the liver by KIC-dioxygenase (12). HMB is present in both plant and animal foods and is currently available as a nutritional supplement (14). The theory behind HMB supplementation is that HMB is metabolized to β-hydroxy-β-methylglutaryl CoA (HMG-CoA), which is used for cholesterol synthesis (8). HMG-CoA can be a rate limiting substrate when cholesterol synthesis is in great demand, such as during periods of rapid cell growth or membrane repair (8). Thus, HMB may provide the necessary amount of HMG-CoA for cholesterol synthesis and subsequent membrane production during periods of high muscular stress. Preliminary studies indicate that HMB ingestion may enhance strength gains, decrease low-density lipoprotein (LDL) concentrations, decrease muscle damage, and increase lean body mass during periods of intense resistance training (4–6,8,10,13).
E-tensive animal research has been conducted analyzing the safety of HMB supplementation. No adverse effects have been reported in animals consuming between 8 and 5000 mg·kg·d-1 for a period of 1–16 wk (7,8,10). Furthermore, HMB has been shown to increase immune function in sheep (8) and chickens (9), decrease total subcutaneous fat in cattle, and lower LDL levels in chickens (9). Several studies have examined the safety of HMB consumption in humans (6,8). Previous studies indicate that ingestion of 1.5–4 g·d-1 of HMB (manufactures recommended dose of HMB in humans is 3 g·d-1 or approximately 38 mg·kg·d-1) for up to 7 wk elicits no negative effects (8). In addition, HMB has been shown to increase strength gains and fat free mass, and lower LDL levels in humans (6,8). Use of HMB has increased in recent years and numerous consumers are ingesting more than the recommended dose (11). However, no studies have been published investigating the safety of higher doses of HMB in humans (i.e., > 4 g·d-1). Therefore, the purpose of this investigation was to compare different amounts of HMB supplementation (0, 38, and 76 mg·kg·d-1) on hematology and hepatic and renal function during 8-wk of resistance training. This study was performed in conjunction with an investigation of HMB effects on muscle strength and body composition: β-hydroxy-β-methylbutyrate ingestion, Part I: effects on strength and fat free mass (3).
Thirty-seven healthy untrained male volunteers aged 18–29 completed the 8-wk supplementation and resistance training program. Subject characteristics are reported in Table 1 of the companion paper (3). Before any testing, the subjects were interviewed by a member of the investigating team, and potential subjects were excluded from the study if they had engaged in resistance training within the last 3 months or were taking any nutritional supplements. Subjects were also excluded if they smoked or had a history of metabolic, cardiac, and/or pulmonary disease. All subjects gave written informed consent in accordance with the University’s Institutional Review Board before participating in the study.
The subjects were matched based on their body weights and randomly assigned, in a double-blind order, to one of three groups: placebo, 38 mg·kg-1·d-1, or 76 mg·kg-1·d-1 of HMB. These doses correspond to doses reported in previous studies (4,6,8) of approximately 0, 3 or 6 g·d-1, respectively. Further description of the supplementation protocol is provided in part I (3).
Resistance training protocol.
The subjects participated in an 8-week resistance training protocol. Each training session was supervised and the subjects were required to attend the sessions three times per week. A detailed description of the resistance training program is illustrated in part I (3).
Blood samples were taken from the antecubital vein after an overnight fast for the measurement of blood variables. Samples were collected before the initiation of the training program and 48 h after the first, 3rd (1 wk), 6th (2 wk), 12th (4 wk), and 24th (8 wk) lifting session. Approximately 4 mL of blood was collected in a vial containing SST® gel and clot activator (Vacutainer, Franklin Lakes, NJ) and then centrifuged for 15 min. An additional 3 mL of blood was collected in a vial containing 0.057 mL of 0.34 molar EDTA (Vacutainer, Franklin Lakes, NJ).
Approximately 5 mL of urine was collected in a urine collection vial from the subjects at the same time that the blood was drawn (within 5 min) and immediately placed in a 4°C environment.
The blood and urine samples for each collection time point were placed in a specimen delivery kit and shipped overnight to Labcorp (Roche Biomedical Laboratories, Inc.) at 4°C for analysis. The blood was tested for lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (SGOT), and alanine aminotransferase (SGPT) on an Olympus AU5200 automated chemistry analyzer (Melville, NY). Quantities of hemoglobin, total white blood cells, polysinophils, lymphocytes, monocytes, eosinophils, and basophils were determined using a Coulter counter (Miami, FL). A lipid profile was also performed to examine the levels of total cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and triglycerides (TRI). Total cholesterol and triglycerides were determined using an enzymatic colorimetric techniques. HDL concentration was determined after precipitation with sulfated α-cyclodextrin in alkaline magnesium chloride. LDL and VLDL concentrations were calculated based on the Friedewald equation (2). In addition, the blood was also analyzed for blood urea nitrogen (BUN) and glucose using urease with glucose dehydrogenase and hexokinase reactions, respectively.
Urine was analyzed for pH, glucose using a hexokinase reaction, protein using a biuret assay, and ketone levels using a nitroprusside reaction.
The data were analyzed using the SPSS for windows statistical program (v. 8.0.0). A general linear model with repeated measures was performed on all variables with the within and between subject factors being time and group (0 mg·kg-1·d-1, 38 mg·kg-1·d-1, or 76 mg·kg-1·d-1), respectively. The alpha level was set at P < 0.05. Values found to be significantly different were compared using a Bonferroni post hoc test. All data are presented as mean ± standard error.
Blood enzyme activity.
Group mean pre- and post-supplementation values for the plasma enzymes are reported in Table 1. Normal values were observed for LDH, ALP, SGOT, and SGPT. No differences were exhibited among the three groups at any point during the investigation.
Pre- and post-supplementation mean leukocyte levels are reported in Table 2. No differences were detected in any leukocyte variables at any time point except for basophils. The 38 mg·kg·d-1 group exhibited a significant increase in basophils pre- to post-study (P < 0.05). However, all group mean leukocyte values, including basophils, were within normal limits at all time points throughout the investigation.
Blood lipid profile.
The blood lipid profile pre- and post-supplementation is reported in Table 3. All blood lipid variables were normal and no differences were noted in lipid levels at any time point among the three groups.
No differences were observed in blood glucose, hemoglobin, or BUN levels pre- to post-supplementation among the three groups (Table 4). All mean values were within normal limits and no differences were observed at any time point throughout the investigation.
No differences among the three groups were observed in urine pH, or glucose, protein, and ketone excretion levels pre- to post-supplementation and all urine data were within normal limits. The mean pre- and post-supplementation urine pH values were 6.2 ± 0.4 and 5.9 ± 0.3 for the placebo group, 5.2 ± 0.2 and 5.6 ± 0.3 for the 38 mg·kg·d-1 group, and 5.6 ± 0.2 and 5.5 ± 0.3 for the 76 mg·kg·d-1 group. Throughout the study the urine glucose, protein, and ketone assessments were negative for all but a few individuals with no established patterns.
The intent of this investigation was to examine the effects of different amounts of HMB supplementation (0, 38, and 76 mg·kg·d-1) on hematology and hepatic and renal function during an 8-wk resistance training study. To our knowledge, this is the first study investigating whether adverse effects occur during HMB ingestion greater than 38 mg·kg·d-1 in humans. No alterations in blood and urine markers of physiology were observed. The present study demonstrates that short-term oral ingestion of HMB, up to 76 mg·kg·d-1, does not alter hematology hepatic or renal function.
Similar to previous investigations (8), no harmful effects of HMB supplementation were observed in any of the subject’s plasma enzyme markers (Table 1). There were no differences in LDH, ALP, SGOT, or SGPT over time or among the placebo and HMB supplemented groups.
The mean leukocyte values were within normal limits throughout the supplementation period (Table 2). Two subjects, one in the placebo group and the other in the 38 mg·kg·d-1, displayed consistently greater (0.5–1.1 × 10-3·μl-1) than normal levels (0.0–0.4 × 10-3·μl-1) of eosinophils throughout the investigation. However, no differences in group mean total white blood cells, polysinophils, lymphocytes, monocytes, or eosinophils were displayed during the entire the investigation. Although the 38 mg·kg·d-1 group exhibited a significant increase in basophils pre- to post-supplementation (P < 0.05), the values were still within normal limits (0.0–0.2 × 10-3·μl-1).
It has been suggested that HMB supplementation causes an alteration in cholesterol synthesis via conversion to HMG-CoA (8). β-hydroxy-β-methylglutaric acid has been shown to produce an inhibition of liver cholesterol synthesis (1). Thus, the changes in cholesterol synthesis could elicit an alteration in blood cholesterol levels. Previous research in both humans and animals has demonstrated that HMB supplementation causes a lowering of LDL cholesterol (8,10). However, the current investigation does not support the previous findings as no differences were observed in any lipoprotein cholesterol values over time or among groups. One explanation may be that the intensity and quantity of exercise elicited a greater demand of HMB (and HMG-CoA) then previous studies, thus attenuating the inhibition of liver cholesterol synthesis. It could be suggested that the ingested HMB was excreted in the urine; however, it has been shown that less HMB is excreted than consumed (see part I (3)).
Similar to previous investigations, no differences were observed in blood glucose, hemoglobin, or BUN levels among the placebo and HMB supplemented groups. Two subjects, one in the placebo group and the other ingesting 38 mg·kg-1·d-1 HMB, displayed slightly higher (111–163 mg·dL-1) than normal (65–109 mg·dL-1) glucose values before and sporadically throughout the supplementation period; however, there was no consistent pattern to these values.
Urine was analyzed to assess whether HMB supplementation has any adverse effects on renal function. No changes were observed in excretion of glucose, proteins, ketones, or hydrogen ions in any of the subjects. Based on the excretion levels of these compounds, it can be assumed that HMB does not adversely affect the kidney.
In summary, all of the examined variables were within normal limits and no differences were observed among the three groups in any variables, except for basophils. Thus, based on the results of present investigation, it appears that short-term (8-wk) HMB supplementation (up to 76 mg·kg-1·d-1) is safe and does not alter or adversely affect hematology, hepatic or renal function in young male adults.
This study was supported, in part, by a grant from Metabolic Technologies, Inc. (Ames, IA) and Experimental and Applied Science, Inc. (Golden, CO).
Address of correspondence: Scott Trappe, Human Performance Laboratory, Ball State, University, Muncie, IN 47306; E-mail: [email protected]
1. Beg, Z. H., and P. J. Lupien. The protective action of hepatic cholesterol synthesis by 3-hydroxy-3-methylglutaric acid. Biochim. Biophys. Acta 260: 439–448, 1972.
2. Friedewald, W. T., R. J. Levy, and D. S. Fredrickson. Estimation of the concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin. Chem. 18: 499–509, 1972.
3. Gallagher, P. M., J. A. Carrithers, M. P. Godard, K. E. Schulze, and S. W. Trappe. β-hydroxy-β-methylbutyrate ingestion part I. Effects on strength and fat free mass. Med. Sci. Sports Exerc.
32: \b\b\b–\b\b\b, 2000.
4. Nissen, S., L. Panton, R. Wilhelm , and J C. Fuller, Jr. Effects of β-hydroxy β-methylbutyrate (HMB) supplementation on strength and body composition of trained and untrained males undergoing intense resistance training
. FASEB J.
10: A287, 1996.
5. Nissen, S., L. Panton, J. Fuller, Jr., D. Rice, and R. Sharp. Effects of feeding β-hydroxy β-methylbutyrate (HMB) on body composition and strength of women. FASEB J. 11: A150, 1997.
6. Nissen, S., R. Sharp, M. Ray,et al. Effects of leucine metabolite β-hydroxy β-methylbutyrate on muscle metabolism during resistance-exercise training. J. Appl. Physiol. 81: 2095–2104. , 1996.
7. Nissen, S., D. Morrical, and J. C. Fuller, Jr. The effects of the leucine catabolite β-hydroxy- β-methylbuyrate on the growth and health of growing lambs. J. Anim. Sci. 77: 243, 1994.
8. Nissen, S. L., and N. N. Abumrad. Nutritional role of the leucine metabolite β-hydroxy β-methylbutyrate (HMB). Nutr. Biochem. 8: 300–311, 1997.
9. Nonnecke, B. J., S. T. Franklin, and S. L. Nissen. Leucine and its catabolites alter mitogen-stimulated DNA synthesis by bovine lymphocytes. J. Nutr. 121: 1665–1672, 1991.
10. Ostaszewksi, P. K. G., B. Balasinska, W. Barej, and S. Nissen. Effects of 3-hydroxy 3-methyl butyrate and 2-oxoisocaporate on body composition and cholesterol metabolism in rabbits. VII Symposium on Protein Metabolism and Nutrition,
Vale de Santarim. 162, 1995.
11. Phillips, B. Sports Supplement Review. Golden, CO: Mile High, 1997.
12. Sabourin, P. J., and L. L. Bieber. Formation of β-hydroxy-isovalerate from a-ketoisocaproate by a soluble preparation from rat liver. Dev. Biochem. 18: 149–154, 1981.
13. Vuckovich, M. D., N. D. Stubbs, R. M. Bohlken,et al. The effects of dietary β-hydroxy β-methylbutyrate (HMB) on strength gains and body composition changes in older adults. FASEB J. 12: A652, 1998.
14. Zhang, Z., C. Coates, J. Rathmacher, T. D. Faidley, J. Sultan, and S. Nissen. Occurrence of β-hydroxy- β-methylbutyrate in foods and feeds. FASEB J. 8: A464, 1993.