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-hydroxy--methylbutyrate ingestion, PartII: effects on hematology, hepatic and renalfunction

PHILIP M. GALLAGHER, JOHN A. CARRITHERS, MICHAEL P. GODARD, KIMBERLEY E. SCHULZE,AND SCOTT W. TRAPPE

Human Performance Laboratory, Ball State University, Muncie, IN 47306

ABSTRACT

GALLAGHER, P. M, J. A. CARRITHERS, M. P. GODARD, K. E. SCHULZE, and S. W. TRAPPE. -hydroxyl--methylbutyrate

ingestion, Part II: effects on hematology, hepatic and renal function. Med. Sci. Sports Exerc., Vol. 32, No. 12, 2000, pp. 2116–2119.

Purpose: The purpose of this investigation was to examine the effects of differing amounts of -hydroxy--methylbutyrate (HMB),

0, 36, and 76 mg·kg-1·d-1, on hematology, hepatic and renal function during 8 wk of resistance training. Methods: Thirty-seven,

untrained collegiate males and were randomly assigned to one of the three groups, 0, 38, or 76 mg·kg-1·d-1. Resistance training consisted

of 10 exercises, performed 3 d·wk -1 for 8 wk at 80% of their 1-repetition maximum. Blood and urine was obtained before training, 48 h

after the initial session, 1 wk, 2 wk, 4 wk, and at 8 wk of resistance training. Blood was analyzed for glucose, blood urea nitrogen,

hemoglobin, hepatic enzymes, lipid profile, total leukocytes, and individual leukocytes. Urine was analyzed for pH, glucose, and protein

excretion. Results: The 38 mg·kg-1·d-1 group had a greater increase in basophils compared with 0 or 76 mg·kg-1·d-1 groups (P 0.05).

No difference occurred in any other blood and urine measurements. Conclusion: These data indicate that 8 wk of HMB supplemen-

tation (76 mg·kg-1·d-1) during resistance training had no adverse affects on hepatic enzyme function, lipid profile, renal function, or

the immune system. KEY WORDS: CHOLESTEROL, LEUKOCYTES, PLASMA ENZYMES, RESISTANCE TRAINING

-hydroxy--methylbutyrate (HMB) is a derivative of the

amino acid leucine and is metabolized from -ketoisocap-

roate (KIC), the keto acid of leucine, in the liver by KIC-

dioxygenase (12). HMB is present in both plant and animalfoods and is currently available as a nutritional supplement

(14). The theory behind HMB supplementation is that HMB

is metabolized to -hydroxy--methylglutaryl CoA (HMG-

CoA), which is used for cholesterol synthesis (8). HMG-

CoA can be a rate limiting substrate when cholesterol syn-

thesis 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 synthe-

sis 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). Extensive animal re-

search 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 chick-

ens (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). Previousstudies indicate that ingestion of 1.5–4 g·d-1 of HMB (man-

ufactures 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 hematologyand 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: -hydroxy--methylbutyrate ingestion, Part I:

effects on strength and fat free mass (3).

METHODS

Subjects. Thirty-seven healthy untrained male volun-

teers aged 18–29 completed the 8-wk supplementation and

resistance training program. Subject characteristics are re-

ported in Table 1 of the companion paper (3). Before any

0195-9131/00/3212-2116/$3.00/0

MEDICINE & SCIENCE IN SPORTS & EXERCISE®

Copyright © 2000 by the American College of Sports Medicine

Received for publication May 2000.

Accepted for publication May 2000.

2116



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 dis-

ease. All subjects gave written informed consent in accor-

dance with the University’s Institutional Review Board be-

fore participating in the study.

Experimental design. 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 corre-

spond to doses reported in previous studies (4,6,8) of ap-

proximately 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 partici-

pated in an 8-week resistance training protocol. Each train-

ing session was supervised and the subjects were required toattend the sessions three times per week. A detailed descrip-

tion of the resistance training program is illustrated in part

I (3).

Blood collection. Blood samples were taken from the

antecubital vein after an overnight fast for the measurement

of blood variables. Samples were collected before the ini-

tiation 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 con-

taining 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).

Urine collection. 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.

Blood analysis. The blood and urine samples for each

collection time point were placed in a specimen delivery kit

and shipped overnight to Labcorp (Roche Biomedical Lab-

oratories, Inc.) at 4°C for analysis. The blood was tested for

lactate dehydrogenase (LDH), alkaline phosphatase (ALP),

aspartate aminotransferase (SGOT), and alanine amino-

transferase (SGPT) on an Olympus AU5200 automated

chemistry analyzer (Melville, NY). Quantities of hemoglo-

bin, total white blood cells, polysinophils, lymphocytes,

monocytes, eosinophils, and basophils were determined us-

ing 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 triglyc-

erides (TRI). Total cholesterol and triglycerides were deter-

mined using an enzymatic colorimetric techniques. HDL

concentration was determined after precipitation with sul-

fated -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 reac-

tions, respectively.Urine analysis. Urine was analyzed for pH, glucose

using a hexokinase reaction, protein using a biuret assay,

and ketone levels using a nitroprusside reaction.

Statistical analysis. 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.

RESULTS

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.

Leukocyte levels. Pre- and post-supplementation

mean leukocyte levels are reported in Table 2. No differ-

ences were detected in any leukocyte variables at any time

point except for basophils. The 38 mg·kg·d-1

group exhibited a

TABLE 1. Pre- and post-supplementation plasma enzyme activity.

0 mgkg1d1 38 mgkg1d1 76 mgkg1d1

Alkaline phosphotase (IUL1)Pre 77.7 6.5 76.8 9.7 72.8 5.3Post 82.7 6.9 81.2 5.4 78.4 5.4

SGOT (IUL1)Pre 22.1 2.9 18.4 1.3 16.7 1.4Post 27.1 6.0 19.6 2.2 19.2 1.2

SGPT (IUL1)Pre 20.5 3.1 17.6 2.6 15.7 1.8Post 19.3 3.2 19.1 2.6 15.3 1.4

LDH (IUL1

)Pre 131.0 6.8 133.2 6.6 131.4 4.3Post 140.0 8.7 136.3 5.2 136.1 4.0

SGOT, aspartate aminotransferase; SGPT, alanine aminotransferase; LDH, lactate de-hydrogenase.Values are mean SE.

TABLE 2. Pre- and post-supplementation leukocyte levels; values are mean SE.


Total WBC (103L1)Pre 6.3 0.4 6.6 0.4 6.0 0.2Post 6.0 0.3 6.6 1.1 6.8 0.7

Polyosinophils(103L1)Pre 3.4 0.4 3.3 0.1 3.2 0.1Post 3.0 0.2 3.2 0.2 3.8 0.6

Lymphocytes (103L1)Pre 2.1 0.1 2.4 0.2 2.1 0.2

Post 2.2 0.1 2.5 0.1 2.1 0.2Monocytes (103L1)

Pre 0.5 0.0 0.6 0.0 0.6 0.1Post 0.5 0.1 0.5 0.0 0.6 0.1

Eosinophils (103L1)Pre 0.2 0.0 0.2 0.0 0.2 0.0Post 0.3 0.0 0.4 0.0 0.3 0.0

Basophils (103L1)Pre 0.08 0.01 0.07 0.01 0.09 0.02Post 0.08 0.02 0.11 0.02* 0.06 0.02

* Significantly greater increase from pre value (time).

HMB, HEMATOLOGY, AND HEPATIC AND RENAL FUNCTION Medicine & Science in Sport s & Exercise

2117



significant increase in basophils pre- to post-study (P 0.05).

However, all group mean leukocyte values, including ba-

sophils, were within normal limits at all time points throughout

the investigation.

Blood lipid profile. The blood lipid profile pre- andpost-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.

Blood chemistry. 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.

Urine analysis. No differences among the three groups

were observed in urine pH, or glucose, protein, and ketone

excretion levels pre- to post-supplementation and all urinedata 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.

DISCUSSION

The intent of this investigation was to examine the effects

of different amounts of HMB supplementation (0, 38, and76 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 phys-

iology 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 sub-

jects, one in the placebo group and the other in the 38

mg·kg·d-1, displayed consistently greater (0.5–1.1 10-

3·l-1) than normal levels (0.0 – 0.4 10-3·l-1) of eosino-

phils throughout the investigation. However, no differences

in group mean total white blood cells, polysinophils, lym-

phocytes, 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·l-1).

It has been suggested that HMB supplementation causes

an alteration in cholesterol synthesis via conversion to

HMG-CoA (8). -hydroxy--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 sup-

plementation causes a lowering of LDL cholesterol (8,10).

However, the current investigation does not support theprevious 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. Twosubjects, 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 pe-

riod; however, there was no consistent pattern to these

values.

Urine was analyzed to assess whether HMB supplemen-

tation 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.

TABLE 4. Pre- and post-supplementation blood urea nitrogen, blood glucose, andhemoglobin levels; values are mean SE.


Blood urea nitrogen (mgdL1)Pre 12.5 0.5 11.8 0.7 14.4 0.6Post 13.5 0.8 13.3 1.0 14.1 0.7

Glucose (mgdL1)Pre 97.8 2.7 97.2 1.5 90.8 1.9Post 92.3 3.1 95.5 4.8 90.4 2.5

Hemoglobin (mgdL1)Pre 15.1 0.2 15.8 0.2 14.9 0.3Post 15.3 0.2 15.6 0.2 15.1 0.2

TABLE 3. Pre- and post-supplementation plasma lipid profile.


Total cholesterol (mgdL1)Pre 160 7 173 5 168 13Post 154 8 160 9 172 12

HDL (mgdL1)Pre 47 3 43 2 45 3Post 50 3 42 1 46 4

LDL (mgdL1)Pre 92 7 106 4 98 21Post 82 8 96 8 98 8.5

VLDL (mgdL1

)Pre 19 2 22 2 18 3Post 21 4 20 2 18 2

Triglycerides (mgdL1)Pre 99 8.5 150 27 95 13Post 108 19 106 12 162 69

HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-densitylipoprotein.Values are mean SE.

2118 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org



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 hema-

tology, hepatic or renal function in young male adults.

This study was supported, in part, by a grant from MetabolicTechnologies, Inc. (Ames, IA) and Experimental and Applied Sci-

ence, Inc. (Golden, CO). Address of correspondence: Scott Trappe, Human Performance

Laboratory, Ball State, University, Muncie, IN 47306; E-mail:[email protected].

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