Serum Testosterone and Urinary Excretion of Steroid Hormone Metabolites After Administration of a High-Dose Zinc Supplement

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ORIGINAL ARTICLE Serum testosterone and urinary excretion of steroid hormone metabolites after administration of a high-dose zinc supplement

K Koehler1, MK Parr1, H Geyer1, J Mester2 and W Scha¨nzer1

1Institute of Biochemistry, German Research Centre of Elite Sport, German Sport University Cologne, Cologne, Germany and 2Institute of Training Science and Sport Informatics, German Research Centre of Elite Sport, German Sport University Cologne, Cologne, Germany

Objectives: To investigate whether the administration of the zinc-containing nutritional supplement ZMA causes an increase of serum testosterone levels, which is an often claimed effect in advertising for such products; to monitor the urinary excretion of testosterone and selected steroid hormone metabolites to detect potential changes in the excretion patterns of ZMA users. Subjects: Fourteen healthy, regularly exercising men aged 22–33 years with a baseline zinc intake between 11.9 and 23.2 mg dayÀ1 prior to the study. Results: Supplementation of ZMA significantly increased serum zinc (P ¼ 0.031) and urinary zinc excretion (P ¼ 0.035). Urinary pH (P ¼ 0.011) and urine flow (P ¼ 0.045) were also elevated in the subjects using ZMA. No significant changes in serum total and serum free testosterone were observed in response to ZMA use. Also, the urinary excretion pattern of testosterone metabolites was not significantly altered in ZMA users. Conclusions: The present data suggest that the use of ZMA has no significant effects regarding serum testosterone levels and the metabolism of testosterone in subjects who consume a zinc-sufficient diet. European Journal of Clinical Nutrition (2009) 63, 65–70; doi:10.1038/sj.ejcn.1602899; published online 19 September 2007

Keywords: zinc supplementation; athletes; serum testosterone; urinary steroid hormone metabolites; urinary zinc excretion

Introduction non-deficient athletes. Therefore, the advertised effects of nutritional supplements have to be evaluated critically based The ergogenic effect of numerous nutritional supplements upon independent trials. has been investigated to a great extent in the field of sports The interrelations of the trace element zinc and the male nutrition. Countless different supplements can be found on sexual hormone testosterone (T) have been known for many the market, which are advertised to be valuable for athletes. years. Dietary zinc deficiency has been found to cause Yet, only a few substances available have been scientifically hypogonadism and growth retardation for the first time in proven to improve athletic performance (Burke et al., 2000). the 1960s (Prasad et al., 1963). Reduced testosterone Even though supplementation may reverse negative synthesis due to impaired action of superordinate hormones effects of nutritional deficiencies (and consequently improve such as gonadotropin-releasing hormone, luteinizing athletic performance), this cannot be transferred directly to hormone and follicle-stimulating hormone (McClain et al., 1984), and altered enzymatic conversion of testosterone Correspondence: K Koehler, Institute of Biochemistry, German Research (Om and Chung, 1996) have been identified as the Centre of Elite Sport, German Sport University Cologne, Carl-Diem-Weg 6, main reasons for lower testosterone levels in zinc Cologne 50933, Germany. deficiency. E-mail: [email protected] Contributors: KK managed the realization of the study, sample and statistical In 2000, a placebo-controlled, double-blind study showed analysis and interpretation of the results and led the writing of the paper. MKP that the use of the nutritional supplement ZMA by initiated the trial, was in charge of the design and the implementation and semiprofessional athletes resulted in an increase of plasma contributed to the writing. HG, JM and WS instigated the study and assisted testosterone levels of approximately 30% and significantly with the interpretation of the data and the preparation of the manuscript. Received 23 November 2006; revised 10 April 2007; accepted 25 July 2007; improved muscle strength when compared to control published online 19 September 2007 athletes (Brilla and Conte, 2000). Changes in the excretion patterns of ZMA users K Koehler et al 66 Except for this trial, supplementation of zinc has only Table 1 Anthropometric data and baseline dietary zinc intake of the been reported to increase testosterone in pathological participants of the trial conditions linked to a low zinc status (Favier, 1992) and in Group N Age Weight Height Baseline elderly men (Haboubi et al., 1988; Prasad et al., 1996). (years) (kg) (cm) dietary zinc Therefore, the aim of the present study was to reinvestigate (mg dayÀ1) the claimed effect of the administration of ZMA on serum ZMA 7 27.074.2 83.679.2 184.477.9 18.073.4 testosterone levels in young, physically active, healthy men Placebo 7 27.472.7 78.176.1 188.077.7 16.373.4 in an independent placebo-controlled, double-blind trial. A further objective was to monitor the urinary excretion of Values are means7s.d. testosterone and selected steroid hormone metabolites to detect potential changes in the excretion patterns of ZMA users. investigators were unaware of the group assignments until the end of the study. In the course of the trial, the daily intake of zinc was Materials and methods determined on the base of diet history interviews, which were conducted with EBISpro software. This method has The nutritional supplement ZMA been validated and compared to other computerized methods The product ZMA (manufacturer: SNAC System Inc., (Landig et al., 1998). The interviews were performed by an Burlingame, CA, USA) was purchased from an Internet experienced nutritionist, and were designed to reflect the distributor of sport nutrition (Bodybuilding.com, 2005). habitual dietary intake of the subjects in the month before According to the manufacturer’s information, the supple- and during the trial period. ment contained zinc (30 mg per recommended dose of The daily zinc intakes of all subjects (range: 11.9–23.2 three capsules, present as monomethionine and aspartate), mg dayÀ1) were higher than the recommended daily allowance À1 magnesium (450 mg, as aspartate) and vitamin B6 (10.5 mg, of 11 mg day (Institute of Medicine, 2001), so all subjects as hydrochloride). Before the study, the zinc and magnesium were considered to be not zinc deficient. content of the supplement was analysed by a commercial laboratory using Inductively Coupled Plasma–Mass Spectro- metry after acid digestion. The concentrations of both Supplementation trial elements were similar to those labelled. According to their group assignment, all subjects ingested With respect to recent reports regarding contaminations either three capsules per day of ZMA or placebo for 56 days. (Geyer et al., 2004b) and adulterations (Geyer et al., 2004a) of Placebo capsules containing D-lactose monohydrate were nutritional supplements, prior to the trial the supplement indistinguishable from ZMA in size and colour. The partici- was confirmed to contain none of the following anabolic pants were asked to swallow the capsules with water between steroids: testosterone, 19-nortestosterone, prohormones of dinner and bedtime at minimum 1 h after the last food testosterone and 19-nortestosterone, tetrahydrogestrinone, intake. At least 2 weeks before and during the trial, all stanozolol, metandienone and trenbolone. The analysis of subjects had to refrain from using any nutritional supple- the supplement was performed according to a previously ment containing zinc. described method (Parr et al., 2004). For the detection of Blood and spot urine samples were collected the morning tetrahydrogestrinone, stanozolol, metandienone and tren- before the start of the supplementation (week 0). For the bolone, this method was adapted to liquid chromatographic- investigation of the time course of potential changes caused tandem mass spectrometric (LC/MS-MS) conditions, which by the administration of ZMA, blood and urine samples were are used for the detection of synthetic steroids in urine taken weekly within and at the end of the supplementation samples (Thevis et al., 2005). The limits of detection (signal/ period of 8 weeks (weeks 1–8). Samples were collected noise ratio X3) of the listed steroids were calculated to be in between 0900 and 1100 hours. To account for circadian the range of 0.2–5 ng gÀ1. rhythms of all measured parameters, the subjects reported to the laboratory at the same time of day every week. After centrifugation, serum was stored at –201C until analysis. Subjects Urine samples were also kept frozen until analysis. The placebo-controlled, double-blind trial was approved by the Local Ethics Committee of the German Sport University Cologne. All subjects gave their written consent prior to the Analytical methods trial. The study included 14 healthy male volunteers, who Serum and urinary zinc concentrations were assessed on a reported to exercise regularly on a recreational or semi- Perkin Elmer 2380 atomic absorption spectrometer (AAS). À1 competitive basis (2.5–10 h week ). After stratification by After dilution with 0.01 N nitric acid (serum: 1:10; urine: 1:2), weight (o80 and X80 kg), the subjects were randomly the samples were directly aspirated into the AAS system. The assigned to the study groups (Table 1). Subjects and urinary zinc excretion rate, which is the most commonly

European Journal of Clinical Nutrition Changes in the excretion patterns of ZMA users K Koehler et al 67 used measure in the evaluation of urinary zinc, was Parameters were presumed to be affected by the adminis- calculated by multiplying the urinary zinc concentration tration of ZMA if the estimate of ‘week Â group’ was with urine flow. significantly different from 0 (a ¼ 0.05). The mixed linear Serum total testosterone (total T) and serum sexual model also provided estimates for each point of time and hormone-binding globulin (SHBG) were both measured with each group as well as corresponding P-values, indicating commercially available immunoassays on a Modular Analy- significant differences from baseline values and/or from the tics E170 (Roche, Mannheim, Germany). Serum albumin was control group. determined with a ready-to-use kit using the bromcresol green method for the Modular/P-Modul system (Roche). Serum free testosterone was calculated from total T, SHBG Results and albumin according to a previously described method (Vermeulen et al., 1999). Serum concentrations Urinary creatinine was measured with an automatic Serum zinc was significantly raised following ZMA supple- enzymatic assay for the Modular/P-Modul system. Urinary- mentation but remained unchanged in the placebo group specific gravity was determined on a Paar DMA 38 Density (Figure 1, top). Even though serum zinc values differed from Meter and for pH analysis, a Consort C831 analyser was used. baseline concentrations at a significant level only after 5 and Urinary flow was calculated as the volume of the spot urine 6 weeks, the mixed linear model indicated a significantly over the time difference between the spot sample and the positive trend of 0.2970.13 nmol lÀ1 per week in the ZMA preceding urination, which the subjects were instructed to group (P ¼ 0.031). record to the minute. The time course of serum total T and free T is shown in For the determination of the urinary concentrations of Figure 2. Within each group, there were no statistically unconjugated and glucuronidated steroid hormones, a gas significant trends in the concentrations of total T (ZMA: chromatographic–mass spectrometric method routinely used P ¼ 0.42; placebo: P ¼ 0.69) or free T (ZMA: P ¼ 0.33; placebo: in doping analysis was utilized (Geyer et al., 1998). The P ¼ 0.56). following urinary steroids were quantified: T, epitestosterone (EpiT), 5a-dihydrotestosterone, 5a-androstane-3a,17b-diol (Adiol), 5b-androstane-3a,17b-diol (Bdiol), Urinary parameters androsterone, etiocholanolone, DHEA, 5b-pregnane-3a,20a- As illustrated in Figure 1, bottom, the urinary zinc excretion diol (Pdiol), 11b-hydroxy-androsterone, 11b-hydroxy-etiocho- rate was increased in the group supplementing ZMA. The lanolone, tetrahydrocortisol and allo-tetrahydrocortisol. Urinary concentrations were corrected to a mean specific gravity of 1.022 g mlÀ1 according to Equation (1).

À1 À1 ccorrected¼ crawÁð1:022À1ÞÁðspecific gravitysampleÀ1gml Þ ð1Þ

Additionally, the following ratios, which are usually recorded in routine doping analyses, were determined: T/EpiT, Adiol/Bdiol, androsterone/etiocholanolone, androsterone/T and androsterone/EpiT.

Statistical analysis Statistical analysis was performed using SAS software, version 9.1. Serum and urinary hormone concentrations were normalized by applying natural log transformation before statistical analysis. For determination of statistically significant changes of all measured parameters, a general mixed linear model for repeated measures was used (Littell et al., 1998). The assignment to each treatment group (‘group’) and the change within each treatment group over time (‘week- Figure 1 Serum zinc concentrations and urinary zinc excretion group’) were considered to be fixed effects. Subject variability rate. aSignificantly different from week 0, Po0.05; bsignificantly was deemed to be a random effect. Variances were assumed different from week 0, Po0.01; csignificantly different from placebo, to be different within each group. Po0.05; and dsignificantly different from placebo, Po0.01.

European Journal of Clinical Nutrition Changes in the excretion patterns of ZMA users K Koehler et al 68 Table 2 Mixed linear model estimates of the monitored urinary metabolites in each groupa

Group Baseline Week Â group P

Testosterone Placebo 4.0270.65 À0.02070.044 0.65 ZMA 3.6670.51 À0.01170.051 0.82

Epitestosterone Placebo 4.3870.27 À0.03070.034 0.38 ZMA 4.2470.16 À0.01870.029 0.53

5a-Dihydrotestosterone Placebo 1.4570.45 À0.03870.055 0.49 ZMA 1.2170.34 À0.00470.059 0.94

5a-Androstanediol Placebo 4.4370.25 À0.02170.030 0.49 ZMA 4.3970.19 À0.02170.036 0.56

5b-Androstanediol Placebo 4.9870.39 À0.02770.040 0.49 ZMA 4.7570.309 À0.01170.048 0.83

Figure 2 Serum total and serum free testosterone after use of ZMA Androsterone (grey) or placebo (white). Placebo 8.3770.23 À0.02070.030 0.50 ZMA 8.2170.15 À0.00370.027 0.92

Etiocholanolone mixed model showed a significant average increase of Placebo 8.0870.22 À0.02670.026 0.32 13.676.4 pmol minÀ1 per week (P ¼ 0.035), whereas only at ZMA 7.9770.17 À0.00970.031 0.77 weeks 4 and 8, the differences from baseline levels reached DHEA statistical significance. In the placebo group, the urinary zinc Placebo 4.5470.32 À0.05070.039 0.20 excretion rate was not altered (P ¼ 0.75). ZMA 4.2270.22 À0.00270.042 0.96 There were no significant differences in the urinary excretion of the monitored urinary steroid metabolites Pregnanediol Placebo 6.0470.27 À0.01670.035 0.64 (Table 2). Consequently, none of the ratios recorded in ZMA 6.0470.19 À0.03770.034 0.33 routine doping analysis was significantly altered by the use of ZMA (data not shown). 11-OH-androsterone 7 7 Supplementation with ZMA significantly elevated urinary Placebo 6.85 0.23 À0.022 0.034 0.42 ZMA 7.1270.16 À0.02770.029 0.39 pH by approximately 1 pH unit after 8 weeks (overall trend: P ¼ 0.011). Additionally, urine flow was almost doubled after 11-OH-etiocholanolone 8 weeks of ZMA use and the mixed model revealed a Placebo 5.2370.48 À0.04170.054 0.43 ZMA 5.4670.34 À0.03870.050 0.47 significantly positive trend (Figure 3; Table 3). In spite of the increase in urinary flow, there was neither a Tetrahydrocortisol significant change in urinary specific gravity nor in urinary Placebo 7.9070.26 À0.06370.041 0.13 creatinine concentration (Table 3). ZMA 7.8370.12 À0.04270.026 0.10

Allo-tetrahydrocortsiol Placebo 7.2770.19 À0.03870.049 0.44 ZMA 7.2570.19 À0.04970.036 0.17 Discussion Abbreviation: DHEA, dehydroepiandrosterone. aAll values have been logarithmized before statistical analysis. The present supplementation trial could not confirm the results of a previous study conducted by Brilla and Conte, who reported an increase of plasma testosterone levels by zinc levels before and after supplementation as well as about 30% after use of ZMA (Brilla and Conte, 2000). The baseline testosterone levels were similar in both trials. reasons for this can only be speculated on. It has to be noted Supplementation of ZMA caused a small but significant that the training level of the participants of the present trial increase in serum zinc and a more pronounced increase in seems somewhat lower than that of the previous study. urinary zinc. The fact that serum zinc was only mildly However, this is unlikely to have caused such a discrepancy increased after ZMA use agrees with the general observation between the study results, since serum (respectively plasma) that serum zinc concentrations are well regulated (Krebs,

European Journal of Clinical Nutrition Changes in the excretion patterns of ZMA users K Koehler et al 69 2000). The stronger increase of the urinary zinc excretion Consequently, urine markers of testosterone and its meta- rate indicates that renal zinc excretion substantially con- bolism were also not altered by the use of ZMA. tributed to zinc homeostasis in the subjects receiving ZMA. In contrast to the present study, previous trials which This gives additional support to the assumption that the reported an increase of testosterone levels after zinc basal zinc status of the participants of the study was good supplementation were conducted with zinc-deficient sub- prior to the start of the supplementation (Capel et al., 1982; jects: in pathological conditions, in which the zinc status is Verus and Samman, 1994). often poor, such as uraemia/haemodialysis (Antoniou et al., The high zinc dose of additional 30 mg dayÀ1 had no 1977; Mahajan et al., 1982), growth retardation (Ghavami- effects on serum testosterone concentrations in the subjects. Maibodi et al., 1983), sickle-cell anaemia (Prasad et al., 1981) or infertility (Netter et al., 1981; Favier, 1992), it was shown that administration of the trace element caused a reversal of previously lowered testosterone concentrations. Kilic et al. (2006) reported that supplementation of zinc reversed reduced serum testosterone levels caused by exhaustion exercise. In summary, it can be concluded that even if 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. However, it is noteworthy that the use of the supplement ZMA had significant effects on urinary pH and urine flow. There is only limited comparable data available on effects of supplementation of the major components of ZMA (zinc aspartate, zinc methionine and magnesium aspartate) on urinary pH and/or urine flow. Still, the increase in urinary pH after the use of ZMA stands in contrast to previous studies on the effects of magnesium-aspartate supplementation. Mu¨hlbauer et al. (1991) reported that magnesium-aspartate supplementation caused a mild decrease in urinary pH, whereas Classen et al. (1987) concluded that magnesium oxide but not magnesium-aspartate affects the acid–base Figure 3 Urine pH and urine flow in subjects using ZMA (grey) or a status as shown by an alkalization of the urine. So, further placebo (white). Significantly different from week 0, Po0.05; csignificantly different from placebo, Po0.05; and dsignificantly investigation is needed to clarify the effect of ZMA different from placebo, Po0.01. supplementation on urinary parameters of the acid–base

Table 3 General urinary parameters in each group

Week Urinary pH Urine flow (ml minÀ1) Specific gravity (g mlÀ1) Creatinine (g lÀ1)

Placebo ZMA Placebo ZMA Placebo ZMA Placebo ZMA

0 5.6470.27 6.0970.38 1.0370.25 1.0170.36 1.02270.004 1.02470.005 1.8970.43 1.9370.61 1 5.5970.20 5.9970.29 0.7970.34 1.1670.48 1.02370.003 1.02170.004 1.8370.30 1.8570.42 2 6.0570.35 6.2570.49 1.1570.18 0.9170.25 1.01970.002 1.02670.003a 1.0970.19b 2.0470.27a 3 5.2770.24 6.4870.33c 1.1470.14 1.0070.20 1.01970.002 1.02470.003 1.5270.29 1.6870.41 4 5.8170.47 6.3970.67 1.0770.25 1.1570.35 1.02070.002 1.02270.003 1.4170.21 1.4870.30 5 5.5270.28 7.0370.39c 1.3170.29 1.4370.41 1.01970.003 1.02170.004 1.3370.19 1.3170.27 6 5.5770.30 6.5570.43a 1.0970.32 1.0870.45 1.02270.002 1.02470.003 1.6270.26 1.9770.37 7 5.7170.35 6.7070.49 0.7970.31 1.2370.44 1.02570.002 1.02470.004 2.1470.35 1.9270.49 8 5.9070.36 7.1070.51a 1.4970.39 1.9670.55b 1.01970.003 1.01970.004 1.2570.21 1.3470.29 Week Â group 0.01170.033 0.12270.047 0.03170.032 0.08070.039 0.000070.0001 À0.000270.0002 À0.01270.038 À0.04470.035 P 0.74 0.011 0.33 0.047 0.98 0.38 0.75 0.22 aSignificantly different from placebo, Po0.05. bSignificantly different from week 0, Po0.05. cSignificantly different from placebo, Po0.01.

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