INTRODUCTION

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PARTICIPATION IN INTENSE, excessive, and/or unaccustomed exercise typically causes delayed-onset muscle membrane damage. If the exercise involves a large eccentric component, such as with downhill running, damage is generally more severe. The high mechanical forces produced during exercise can cause disruption and/or degradation of structural proteins in the muscle fibers as well as in the connective tissue (26). Damage to the muscle typically leads to diminished physical performance (2, 10). Therefore, lessening the amount of muscle damage and subsequent protein breakdown typically associated with high-intensity, long-duration exercise (4, 8, 14) would be beneficial.

Research has shown that the leucine metabolite, -hydroxy--methylbutyrate (HMB) may play a role in inhibiting muscle damage and protein breakdown (16, 17). Studies examining the effects of HMB supplementation on strength and body composition have demonstrated that, in combination with a resistance training program, HMB supplementation resulted in increased muscular strength and lean mass and tended to decrease fat mass (16, 17) as well as indicators of muscle damage (16). Research has also shown that HMB supplementation in trained cyclists resulted in a significantly greater time to reach peak oxygen uptake, which suggests that HMB supplementation may have positive effects on performance (27).

Whereas it has been shown that muscle damage and subsequent protein breakdown are typically followed by impaired muscle function (2, 4, 8, 10, 14), the present study extends those findings by testing the hypothesis that HMB supplementation can prevent or slow the muscle damage associated with intense distance running. Therefore, the purpose of this study was to examine the effects of HMB supplementation on muscle damage after a prolonged run.

	METHODS

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Eight male and eight female volunteers, 20-50 yr of age, were recruited for this study. The purpose and the procedures were explained to each subject, and they signed a consent form that outlined the experimental design and potential discomforts. The study was approved by the Iowa State University's Human Subject's Review Committee.

Only subjects running at least 48 km/wk were selected to participate. A training log assessed the subject's compliance with the mileage requirement and was checked periodically throughout the study by the investigators.

Data collection occurred over a 10-wk period before and after a prolonged run (Table 1). Pre- and postrun measurements included maximal oxygen consumption (O_2 max), body composition, plasma creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity. After 6 wk of supplementation, subjects participated in a prolonged run. The race took place on a 20-km collegiate cross-country course with ~4,800 m (2,400 m of incline and 2,400 m of decline) of hills. Subjects repeated a 10-km loop two times and had access to water at stations located throughout the course. The temperature and humidity at the time of the run were ~66°F and 90%, respectively.

Blood samples were collected from an antecubital vein from each subject after a 12-h fast. Samples were drawn before the subjects began supplementation, 4 wk after supplementation (Pre), immediately after completion of the prolonged run (Post), and every day for 4 days after the prolonged run (1d Post-4d Post). Serum samples were analyzed for CPK and LDH activity by a commercial laboratory (Lab Corp, Kansas City, MO). In addition, plasma was collected and frozen and was later analyzed for HMB by gas chromatography-mass spectrometry methodology (18).

Estimates of lean body mass and fat mass were determined by using the seven-site skinfold method (11, 12).

Subjects were paired according to their best 2-mile run time and past running experience. Treatments, either 3.0 g of HMB or a placebo (rice maltodextrin) supplement, were randomly assigned in a double-blind fashion. Subjects were instructed to take four capsules three times per day with their meals. Supplements were taken for ~6 wk before and continued for 4 days after completion of the prolonged run. All subjects were given a log to record the days and the times they took their supplements to verify compliance in taking the supplement. At the end of the study, the subjects were informed of their treatment assignments.

Statistical analysis. Values are presented as least squares means ± SE. Data were analyzed by using the general linear model procedures of SAS (23). CPK and LDH activities were analyzed by using a repeated-measures analysis of covariance. Because the amount of mileage run per week and gender may affect CPK and LDH levels, the prerun CPK and LDH levels were used as covariates. In addition, fat-free mass (FFM) was used as a covariate to adjust the CPK and LDH values. Main effects were considered significant at P0.05. Independent two-tailed t-tests were used to evaluate descriptive data between the two subject groups (HMB vs. placebo).

	RESULTS

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Thirteen subjects, five men and eight women, were able to finish the entire study. Complete data were obtained on only five of the original eight subjects in the placebo-supplemented group because two had to withdraw from the study due to personal reasons before the date of the prolonged run, and one dropped out during the prolonged run (20-km course) and did not complete the postrun evaluation. All of the subjects initially assigned to the HMB-supplemented group completed the study. Descriptive characteristics of the subjects are presented in Table 2. The two groups did not differ on variables of height, body composition, or O_2 max at any time. There was, however, a trend (P0.10) for a significant difference in body mass between the two groups. However, there was no difference between treatments in the percentage of FFM (83 ±2.2 and 84 ±1.9% for placebo and HMB, respectively). Therefore, the proportion FFM to extracellular water should be equivalent and should not bias the CPK and LDH data. Although the HMB-supplemented group was significantly older (P0.05) than the placebo-supplemented group, this did not affect the subjects' ability to perform aerobically, as evidenced by the similarity in O_2 max between groups. Average weekly training distance, although slightly lower than the 48 km/wk study requirement, was also not significantly different between groups.

The HMB-supplemented group had significantly (treatment main effect, P  0.05) higher levels of HMB in their blood after beginning supplementation. This finding confirms that subjects complied with the supplementation requirements (Table 3).

Ten of the 13 subjects completed the entire prolonged run. The 3 subjects that did not run the same distance as the other 10 were kept in the study because the prolonged run of ~15 km was intense enough to elicit muscle damage. The relative increase in CPK was similar to their treatment cohorts. There was no difference in run time between groups in those subjects completing the entire 20-km course (111.8 ± 7.0 and 115.7 ± 15.7 min for the placebo- and HMB-supplemented groups, respectively).

CPK and LDH. Mean serum CPK levels before and after the prolonged run are presented in Fig. 1A. Serum CPK activity was elevated in both groups immediately after and for 4 days after the prolonged run. Approximately 24 h postrun, CPK activity reached peak levels of 341 ± 68 U/l (unadjusted for prerace values) for the placebo-supplemented group and 282 ± 49 U/l (unadjusted) for the HMB-supplemented group. Analysis of covariance using the prerun CPK level as the covariate showed a significant main effect of group (HMB and placebo) on the postrun CPK response (P = 0.05). Specifically, the HMB group experienced a significantly lower CPK response compared with the placebo group over the 4 days after the prolonged run. Analysis of covariance using the prerun CPK level and FFM as the covariates statistically demonstrated a similar treatment response as CPK levels tended to be lower after the prolonged run in HMB-supplemented subjects (treatment main effect, P = 0.13; 126, 158, 258, 182, 159, and 158 U/l for Pre, Post, and 1d-4d Post prolonged run, respectively) compared with placebo supplemented subjects (110, 177, 379, 270, 235, and 177 U/l for Pre, Post, and 1d-4d Post prolonged run, respectively). A similar response was seen when the statistical model accounted for the gender effect (treatment main effect, P = 0.12, Table 4).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   A: creatine phosphokinase (CPK) activity before and after a prolonged run in placebo- and -hydroxy--methylbutyrate (HMB)-supplemented subjects. Values are least squares means ± SE, adjusted to the prerace values. Pre, 4 wk after supplementation; Post, immediately after completion of the prolonged run; 1d Post-4d Post, days 1-4 after the prolonged run, respectively. Main effects: treatment, P = 0.05; time, P = 0.77; time × Pre, P = 0.05; treatment × time, P = 0.23. B: lactate dehydrogenase (LDH) activity before and after a prolonged run in placebo- and HMB-supplemented subjects. Values are least squares means ± SE, adjusted to the prerace values. Main effects: treatment, P = 0.003; time, P = 0.0208; time × Pre, P = 0.21; treatment × time, P = 0.57.

	DISCUSSION

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Research has shown that repetitive, intense exercise, especially that which involves a large eccentric component, is typically associated with disruption and damage to skeletal muscle connective and/or contractile tissue (1, 4, 7, 9). Because injury to skeletal muscle is often characterized by impaired muscle function (5, 6, 7, 24), attempts have been made to find mechanisms to reduce exercise-induced muscle protein breakdown and stimulate protein synthesis (16, 17). Armstrong (3) reported, in the middle to late 1980s, that there was no direct evidence that any medical or drug treatment could either prevent exercise-induced muscle damage or hasten recovery of the injured muscles after exercise. Nissen et al. (16), however, has recently demonstrated that supplementation of 1.5 or 3.0 g HMB/day can reduce exercise-induced muscle damage and subsequent proteolysis sustained as a result of intense resistance training. The present study was conducted to determine whether HMB supplementation would have similar effects after intense endurance exercise. The results indicated that HMB supplementation lessens muscle damage, as judged by CPK and LDH responses.

One of the major findings in this study was that HMB supplementation, compared with supplementation with a placebo, resulted in smaller increases in CPK and LDH levels after the prolonged run. The placebo-supplemented subjects compared with the HMB-supplemented subjects exhibited a significantly higher increase in CPK activity and LDH activity after the prolonged run, despite the fact that there were no differences in run performance between the groups. Because both groups performed equally well in the prolonged run, the larger increases in enzyme activity in the placebo-supplemented group suggest that they sustained more muscle damage as a result of the run. These results are consistent with those found by Nissen et al. (17) in a previous study involving resistance training. This study showed that CPK levels in the subjects who were not supplemented with HMB increased to 15,868 U/ml after 1 wk of training, whereas subjects taking 1.5 or 3.0 g of HMB had lower CPK levels (15,355 and 7,859 U/ml, respectively). Although the differences in CPK activity between the groups were not significantly different after the first week of the study, researchers noted that HMB supplementation did significantly decrease CPK activity in a dose-responsive manner by the third week (17). In addition, Nissen et al. demonstrated that HMB supplementation also tended to decrease plasma LDH activity in a dose-responsive manner during the second and third week of the study.

Muscular injury causes enzymes such as CPK and LDH to be released into the bloodstream, and thus they are generally considered valid indicators of the degree of muscular injury (13, 19). However, Kuipers (15) has noted that CPK activity does not necessarily reflect the amount of structural damage done to the muscle. Because plasma CPK responses after a similar bout of exercise may differ between individuals, some researchers have concluded that fiber damage is not necessarily reflected in proportional increases in CPK activity (15).

The purpose for measuring CPK activity was to use it as a measure for determining whether HMB supplementation helps to attenuate muscle damage and subsequent proteolysis. CPK activity was elevated in this study but not to the extreme levels reported in marathon runners (19, 21, 25). When examining the difference demonstrated in this study after a prolonged run, two conclusions regarding the variations in the CPK response between the two groups can be made. The first is that the differences may be attributed to supplementation with HMB. Because HMB supplementation has been instrumental in decreasing muscle damage associated with resistance training, it is likely that it also helps to decrease muscle damage caused by intense endurance exercise. It has been demonstrated that HMB is metabolized to HMG-CoA in the cytosol of the muscle cell and serves as a precursor for de novo cholesterol synthesis (16). This is important because the muscle cell relies on de novo synthesis of cholesterol. It has been hypothesized that the de novo synthesized cholesterol is used to support the cell membrane integrity and help prevent muscle damage or used to regenerate damaged muscle cell membranes. Therefore, HMB may help prevent muscle damage or help regenerate a damaged muscle more quickly, which is evident by less CPK leaking from the cell membrane. HMB may also help prevent muscle proteolysis by an alternative mechanism (16).

The second possible explanation for the differences seen in CPK levels may be attributed to differences in lean body mass between the two groups due to gender. The placebo-supplemented group tended (P  0.10) to weigh more than the HMB-supplemented group. Therefore, the differences in CPK activity may be simply due to the fact that one group tended to have more muscle mass than the other. However, the percentage of FFM was not different. CPK activity is expressed as units per liter and will only be affected by LBM if the percentage of FFM were different, which was not the case. Research by Rogers et al. (20) has shown that, after a marathon, men had significantly greater mean serum total CPK activity than women. The gender effect was attributed to a larger muscle mass in men. If this assumption is true, then the reason that the placebo-supplemented group had higher levels of CPK compared with the HMB-supplemented group could simply be due to the fact that they tended to have a greater amount of muscle mass. However, the statistical model corrected for the gender differences in CPK and LDH observed before the prolonged run. In addition, when these data are also corrected for the differences in LBM, a similar treatment response is demonstrated for CPK activity (treatment main effect, P = 0.13) and LDH activity (treatment main effect, P = 0.01).

Although LDH is often used as an indicator for muscle damage, previous research on the LDH response after exercise has shown conflicting results. For instance, Rose et al. (21) and Nuviala et al. (19) have shown significant increases in LDH activity immediately after a marathon, whereas Schwane et al. (23) have reported no significant changes in LDH activity after 45 min of downhill running. The results obtained in the present study are similar to those reported by Rose et al. (21) and Nuviala et al. (19), in that there was a significant increase in LDH activity after a long run. The fact that the placebo-supplemented subjects exhibited higher LDH activity after the prolonged run compared with the HMB-supplemented subjects suggests that they would tend to sustain more muscle damage as a result of the run. This finding further supports the hypothesis that HMB helps to prevent against muscle injury.

Conclusions. Dietary supplementation of 3.0 g HMB/day in individuals undergoing intense endurance exercise resulted in decreased CPK and LDH responses after a prolonged run. These findings are consistent with the hypothesis that HMB-supplemented subjects experienced less muscle damage or that the HMB-supplemented group could have sustained a similar amount of muscle damage as the placebo-supplemented group but recovered at a faster rate.