Effect of Zinc and Selenium Supplementation on Serum Testosterone and Plasma Lactate in Cyclist After an Exhaustive Exercise Bout
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- Shafiei Neek, L., Gaeini, A.A. & Choobineh, S. Biol Trace Elem Res (2011) 144: 454. doi:10.1007/s12011-011-9138-2
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Zinc and selenium are essential minerals and have roles for more than 300 metabolic reactions in the body. The purpose of this study was to investigate how exhaustive exercise affects testosterone levels and plasma lactate in cyclists who were supplemented with oral zinc and selenium for 4 weeks. For this reason, 32 male road cyclists were selected equally to four groups: PL group, placebo; Zn group, zinc supplement (30 mg/day); Se group, selenium supplement (200 μg/day); and Zn–Se group, zinc–selenium supplement. After treatment, free, total testosterone, and lactate levels of subjects were determined before and after exhaustive exercise. Resting total, free testosterone, and lactate levels did not differ significantly between groups, and were increased by exercise (P > 0.05). Serum total testosterone levels in Zn group were higher than in Se group after exercise (P < 0.05). Serum-free testosterone levels in the Zn group were higher than the other groups (P < 0.05).There was an insignificant difference between levels of lactate in the four groups after exercise (P > 0.05). The results showed that 4-week simultaneous and separately zinc and selenium supplementation had no significant effect on resting testosterone and lactate levels of subjects who consume a zinc and selenium sufficient diet. It might be possible that the effect of zinc supplementation on free testosterone depends on exercise.
KeywordsExhaustion exerciseLactateRoad cyclistsTestosteroneSelenium supplementationZinc supplementation
Many researchers concentrate on the relation between exercise and antioxidant supplementation; therefore, it can be stated that there is an increasing interest about evaluation of the ergogenic effects of minerals such as zinc and selenium on exercise.
Zinc is the fourth most abundant intercellular metal, and a biologically essential trace metal is found in over 200 enzymes and proteins . Since zinc is necessary for the activity of a number of enzymes in the energy metabolism, low muscle zinc levels may lead to a reduction in endurance capacity . Zinc plays a key role in reproductive physiology [3, 4]. There was a positive relation between zinc and testosterone . Lactic dehydrogenase is a Zn-containing enzyme. Adequate muscle zinc concentration may facilitate the reduction of lactic acid to pyruvate through the action of lactic dehydrogenase in exercising skeletal muscle and, therefore, may decrease muscle fatigue . Kaya et al.  reported that zinc supplementation leads to a significant increase in testosterone levels and a significant decrease in lactate levels in response to exhaustive exercise.
Also, selenium, which is commonly found in nature, is an essential trace element required for the normal development of human and animal organisms [8, 9]. The increase in oxidative stress caused by exercise and the recognition of the stimulation of antioxidant activity by selenium inevitably entails a relation between selenium and exercise . Selenium is also needed for normal testosterone metabolism and testicular morphology, which may explain the presence of several other selenoproteins in the male gonads . Akil et al.  reported that the increase in free radical production and lactate levels due to acute swimming exercise in rats might be offset by selenium supplementation.
A relationship between exercise and testosterone, which has important effects on energy metabolism, seems inevitable. From a large number of studies exploring the relationship between exercise and testosterone, no definite conclusion can be drawn [12–14]. Bosco et al.  reported that short-term strenuous exercise increased total and free testosterone levels by 12% and 13%, respectively. But Kilic et al.  reported that exhaustive exercise decreased thyroid hormone and testosterone concentrations in elite athletes.
Studies on the relation between selenium and exercise and zinc and exercise mainly focus on the antioxidant role of selenium and the distribution of zinc in the body on response to exercise. There is limited information about the effect of zinc and selenium supplementation especially around their relationship with testosterone hormone and exercise. Accordingly, this topic needs to be studied. The purpose of this study was to examine how exhaustion exercise affects testosterone and lactate levels in athletes who consume a zinc and selenium sufficient diet, and without any lack of selenium and zinc, who are supplemented oral zinc and selenium for 4 weeks.
Materials and Methods
Anthropometric data and baseline zinc and selenium intake of the cyclist
Aerobic power (Watt)
Baseline dietary zinc (mg d−1)
Baseline dietary selenium (mg d−1)
176.87 ± 8.02
66.15 ± 6.4
21.12 ± 0.98
291.5 ± 44.56
15.67 ± 0.89
64.05 ± 15.02
176.87 ± 8.2
68.11 ± 8.66
21.75 ± 2.5
299.88 ± 30.17
16.40 ± 1.02
59.98 ± 13.29
177.75 ± 4.23
61.66 ± 4.75
20.5 ± 1.26
304.06 ± 31.99
14.98 ± 1.08
56.87 ± 14.67
174.81 ± 4.3
63.17 ± 8.45
21.57 ± 2.1
296.12 ± 41.54
16.23 ± 0.45
62.56 ± 12.34
None of the subjects had ingested zinc and selenium or any other dietary supplements, for a minimum of 2 weeks before the initiation and during the study. The subjects were assigned base on body composition, and they were equally divided into four groups by using a double-blind design.
(a) Placebo (PL, 30 mg of dextrose; n = 8), (b) zinc (Zn, 30 mg zinc sulfate), (c) selenium (Se, 200 μg sodium selenite), (d) zinc–selenium (Zn–Se, 30 mg zinc sulfate–200 μg selenium selenite).
According to their group assignment, all subjects ingested one capsule of zinc, selenium, zinc–selenium, and placebo each day for 4 weeks.
Diets' Zinc and Selenium Assay
In the course of trial, the daily intake of zinc and selenium was determined on the base of diet history interviews (24-h recall and food frequency questionnaire), which were conducted with Dorosty Food Processor (version 2.1). The interviews were performed by an experimental nutritionist, and were designed to reflect the habitual dietary intake of the subjects in the month before and during the trial period. The daily zinc and selenium intake of all subjects (16/34 ± 1/23 mg d−1 and 64/45 ± 19/03 mg d−1, respectively) were higher than the recommended daily allowance of 11 mg d−1 and 55 μg d−1 , respectively for zinc and selenium intake, so all subjects were considered to be not zinc and selenium deficient; therefore, the usage of supplements was more than their daily needs.
One week before the actual experiment started, subjects had to perform a graded exercise test in order to determine their maximal power output (Wmax) . For 3 days before the experiments, the subjects were obliged to abstain from strenuous exercise. No caffeine, only tea was permitted during the 48 h before the experiment. On the experimental days, subjects reported to the NOPA.I.R.Iran after an overnight fast. Exercise test started with a 10-min warm-up period at a workload of 50% Wmax. Thereafter, the subjects were instructed to cycle 2-min block periods at alternating workloads of 90% and 50% of Wmax, respectively. This was continued until the subjects were no longer able to complete the 2 min at 90% Wmax. That moment was defined as the time at which the subject was unable to maintain cycling speed at 60 revolutions per minute. At that moment, the high-intensity block was reduced to 80% Wmax. Again, the subjects had to cycle until they were unable to complete a 2-min block at 80% Wmax, after which the high-intensity block was reduced to 70% Wmax. The subjects were allowed to stop when pedaling speed could not be maintained at 70% Wmax. Water was provided ad libitum during the exercise protocol . To remain blood samples were taken from all subjects pre and post exercise.
Blood samples collected from the subjects were centrifuged for 10 min at 3,000 rpm and were kept frozen at −20°C until analysis. Free and total testosterone analyses were conducted on the serum samples. Lactate, zinc, and selenium were assessed on the plasma samples.
Zinc and Selenium Analysis
Plasma zinc and selenium concentrations were assessed on a Younglin AAS 8020 atomic absorption spectrometer (AAS). Values were expressed as micrograms per deciliter.
Lactate analyses were carried out in the separated plasma samples according to colorimetric method (using Roche Diagnostic lactate kits) in a Cobas Integra 400 autoanalyzer. Plasma lactate levels (read at 552 nm wavelength) are expressed as milligrams per deciliter.
Free Testosterone Measurements
Serum-free testosterone analyses were done in Elisa test kit (LDN Company) by enzyme-linked immunosorbent assay method; the results are expressed as picogram per milliliter.
Total Testosterone Measurements
Serum total testosterone was measured with chemiluminescence immunoassay on a LIAISON (Diasorin) analyzer. The results are expressed as nanograms per milliliter.
Statistical analysis was performed with SPSS version 16 program (SPSS Inc., Chicago, IL, USA). Statistical evaluation was done by Kolmogrov–Smirnov test at first to examine the normal distribution and Leven's test for homogeneity. One-way ANOVA was performed in order to compare between groups in anthropometric data and aerobic power. Two-way ANOVA was also performed to assess differences between intra and intergroups followed by Bonferroni's test for multiple comparisons. All results were shown as means±SD in all statistical comparisons P < 0/05 was used as the criterion for statistical significance.
Considering that the results of this study about two different sides of the nutrition–exercise effects (Zn, Se, and Zn–Se groups) and exercise effects (PL group) are comparable with the related obtained results, therefore, the results of this research will be discussed in these two fields separately.
In results of our study, resting free and total testosterone levels measured were significantly lower than those measured after an exhaustive exercise bout. This result reveals that there is a positive correlation between testosterone and exhaustive exercise.
In fact, results of studies examining the relation between exercise and testosterone illustrates that there is no agreement on this topic. In other words, there are contradictions about the cause and effect of physical exercise and the amount of testosterone in different researches [7, 16, 20–22]. Besides the studies reporting that free and total testosterone levels did not change with exercise [23–25], there are also those noting that free and total testosterone levels significantly decreased with exercise [26–28]. The increase we obtained in free and total testosterone levels immediately after exhaustion is in contrast with the findings of these researchers. Ransen et al.  showed that there was an important increase in levels of testosterone, epinephrine, norepinephine, ACTH, cortisol, and growth hormone of the subjects following hight-intensity endurance exercise on a cycle ergometer. The results of this study are consistent with our findings.
The mechanisms responsible for the increase in serum testosterone levels during exercise are controversial, and may include decreased testosterone clearance, hemoconcentration, increased SHBG, catecholamine, precursor molecule DHEA sulfate, and testosterone production due to stimulation by factors other than LH and the slight decrease in progesterone [30–35].
Probably, the reason of contradiction around this issue is about the difference between physical fitness of the subjects, exercise intensity, type and duration, and the work load. Jezova et al.  reported that both plasma testosterone and catecholamine responses to physical effort depend more on work intensity than on work duration or total work output.
Zinc enhances human chorionic gonadotropin-induced production of cAMP and consequently testosterone in rat testes . Additionally, zinc may increase the conversion of androstenedione to testosterone in the periphery tissue . Zinc also interferes with the metabolism of testosterone by decreasing its hepatic clearance and reducing hepatic 5 alpha-reductase activities . The findings of our study demonstrated that resting free and total testosterone levels after a 4-week zinc supplementation had no variation relative to PL group. Exercise increased total testosterone levels in the Zn group than Se group, and also free testosterone levels in Zn group than other groups. Kilic et al.  showed that 4-week zinc supplementation increased resting levels of both thyroid hormones and testosterone concentrations, and exhaustion exercise led to a significant inhibition of those hormones, but that 4-week zinc supplementation prevented this inhibition in wrestlers. Similarly, Kilic  reported mentioned results about sedentary males in response to fatiguing bicycle exercise. Kilic concluded that “administration of a physiologic dose of zinc can be beneficial to performance.” Contradictory, Koehler et al.  noted that zinc supplementation may reverse lowered testosterone levels and restore disturbed testosterone metabolism in cases of mild or severe zinc deficiency; it is not capable of further increasing serum testosterone when sufficient zinc is provided by the regular diet.
The disparity in results in the aforementioned studies with our findings might be due to variations in the consumed dose of zinc supplement, status of zinc in subjects, amount of zinc intake on diet, as well as the improper time duration for the effect of supplementation does in our study.
Previous studies demonstrated that significant decrease in polymorphonuclear leukocyte and lymphocyte activity, and high-density lipoprotein cholesterol after 6 weeks of 150 mg Zn per day [41, 42]. Similarly, that consumption of zinc supplements in excess of 50 mg/day have been linked to impair copper absorption . For these reasons, use of Zn supplements should be limited to those containing no more than 30 mg/d . Therefore, in the present study, the considered supplementation of consumed dose 30 mg/d was performed by its practical order.
In the Leydig cells, glutathione peroxidise (se-dependent) has been localized immunocytochemically in the cytoplasm in close relationship to the smooth endoplasmic reticulum, and it is possible that the metabolic pathway of testosterone biosynthesis requires protection against peroxidation and is thus affected by a decrease in the activity of this enzyme . In our study, 4-week selenium supplementation had no effect on resting levels of total and free testosterone and plasma lactate in cyclist. Probably, the normal status of selenium in cyclists and the enough amount of intake selenium from diet can be the other reasons of the results about the selenium group.
It is known that plasma lactate concentration increases together with increased exercise . This study showed that 4-week zinc and selenium supplementations had no effect on plasma lactate in pre and post-exhaustive exercise. There are only a limited number of studies about how selenium, which is known to reduce oxidative damage in exercise, affects glucose metabolism, lactate levels, and tiredness in physical activity . Akil et al.  investigated the effects of selenium on lipid peroxidation and lactate levels in rats subjected to acute swimming exercise. He reported that the increase in free radical production and lactate levels due to acute swimming exercise in rats might be offset by selenium supplementation. Selenium supplementation may be important in that it supports the antioxidant system in physical activity. Baltaci et al.  demonstrated that 4-week zinc deficiency increased plasma lactate in rats after acute swimming and zinc supplementation (3 mg/kg weight) has the opposite effect.
In summary, even though supplementation may reverse negative effects of nutritional deficiencies (and consequently improve athletic performance), this cannot be transferred directly to non-deficient athletes. In most cases, if energy intakes are sufficient, the mineral needs of athletes are analogous to healthy individuals. Some athletes, however, may have greater requirements as a consequence of disproportionate losses of I nutrients in sweat and urine. For these athletes, supplementation may need to be considered on an individual basis to maintain normal body stores, not for ergogenic purposes. A systematic approach to the study of minerals and exercise performance is needed. This approach needs to use the same protocol to evaluate whether minerals can be effective ergogenic aids. It would require longer supplementation periods, control of exercise settings, multi-center trials, men and women participants, elite and recreational athletes, and precise measures of mineral status.