Effects of acute androstenedione supplementation on testosterone levels in older men

Abstract

The purpose of the study was to examine the effects of acute androstenedione supplementation on hormone levels in older men at rest and during exercise. Men (n = 11) between the ages of 58 and 69 were divided into an experimental (n = 6; 62.33 ± 2.57 y) and control (n = 5; 60.2 ± 1.02 y) groups. Each participant received an oral 300 mg dose of either androstenedione (experimental) or a cellulose placebo (control) for 7 d. Pre- and post-supplementation participants completed two separate, 20-min strength tasks consisting of leg extension and leg curls at different percentages of their 10-RM. Researchers collected blood samples pre-, during, and post-exercise. Blood samples were analyzed for testosterone, androstenedione, and estradiol levels. The researchers found a significant difference between pre- (4.36 ± 56 ng/mL) and post- (5.51 ± 0.35 ng/mL) testosterone levels, as well as pre- (0.88 ± 0.20) and post- (7.46 ± 1.25) androstenedione levels, but no significant differences between pre- and post-estradiol levels for either group. It appears that short-term androstenedione supplementation augmented acute testosterone responses to resistance exercise in older men. However, further study of this supplement is needed to determine any potential it may have in mitigating andropause.

• Aerobic exercise
• aging
• muscle strength
• resistance exercise
• sex hormones

Introduction

The progressive loss of muscle mass and functionality that can occur with aging [1–8] has been defined as sarcopenia. This loss of muscle function with sarcopenia is based upon the decreased capacity of the muscles to generate force [9]. To illustrate, muscle strength levels necessary for functional activities appear to be well-maintained for most men until the fifth decade of life, but researchers have documented declines up to 15% per decade that occur in the sixth and seventh decades of life, after which the strength losses increase to 30% per decade [10–14]. The onset of sarcopenia may be the result of many factors, including: reduced activation of motor units, reduced contractile properties within the muscle, lesser physical activity, inadequate protein intake, decreased neuromuscular coordination, and decreased circulating hormones [8].

It has been suggested that aging men experience andropause, a hormonal decline similar to the hormonal changes women experience during menopause [15], and this hormonal decline partially explains the functional decrements seen in sarcopenia. This similarity between the sexes is based on the commonalities between estrogens and testosterone in regulating secondary sex characteristics. The impact of testosterone starts at puberty, greatly enhancing protein synthesis through a direct interaction with its cytoplasmic receptor that stimulates RNA synthesis and promotes muscle growth [16–18]. The anabolic effects of testosterone (along with growth hormone) then work to maintain muscle mass throughout much of the lifespan, particularly among those who regularly engage in resistance training. Craig and coworkers [19,20] are among the researchers who have previously established the influence of resistance training on the release of testosterone and growth hormone [20,21], investigating a sample of male subjects (6 young, 23 ± 1 yrs and 9 elderly, 63 ± 1 yrs) who underwent a 12-week supervised progressive resistance weight lifting program. The data presented in the first paper [19] indicated that both the young and elderly subjects showed significant increase (p < 0.05) in lean body mass and strength measures, whereas the data presented in the second paper [20] indicated that while strength training can induce growth hormone and testosterone release, regardless of age, the elderly response was well below that of the young. These results were supported by Häkkinen et al. [21], who measured growth hormone and testosterone responses to an acute bout of heavy resistance training. They found that the hormonal responses of the elderly (70 ± 3.7 yrs) were below those of the young (26.5 ± 4.8 yrs) subjects they tested.

Other lines of research also suggest that hormonal levels likely play an important role in age-related decreases in muscle strength and size. For example, Urban and colleagues [22] examined the effects of testosterone supplementation in six men greater than 65 years of age, and found that exogenous testosterone administration increased muscle strength and protein synthesis in their subjects. Similarly it has been shown that the suppression of androgen expression in men leads to reduced muscle strength and functional performance [23].

Physicians and other licensed health-care professionals are interested in pharmaceutical and dietary supplements that may be used clinically to counteract the effects of sarcopenia, thus improving quality of life and exercise capacity in of older patients [24] who experience age-associated declines in testosterone levels. One such strategy is to add androstenedione to dietary supplements. Androstenedione is a precursor hormone to testosterone, and therefore in theory should increase testosterone levels. The German patent for androstenedione claims that the ingestion of androstenedione by young healthy subjects increases serum testosterone levels by as much as 237% within 15 min, followed by a secondary increase of 48–97% within 3 to 4 d [25]. These increases in serum testosterone concentrations represent profound responses to this dietary supplementation, but it has been listed on the World Anti-Doping banned list [26] and thus is unlikely to have acceptable uses for young athletes.

However, androstenedione supplementation may afford quality of life and functional benefits to older men experiencing the debilitating effects of andropause. A thorough review of scientific databases reveals that androstenedione supplementation among aging males has received little attention, and that the studies completed provide little or no agreement on its short-term or long-term effects. More specifically, existing work suggests that androstenedione supplementation may have an effect under certain conditions. Androstenedione supplementation appears to be effective in younger participants [27–29], but when older participants were treated, the results are mixed [24,29,30]. A study by Wallace et al. [31] found no significant differences in lean body mass, strength, or serum testosterone levels after 12 weeks of supplementation of either androstenedione or dehydroepiandrosterone in middle-aged men. Alternatively, in a study with 55 adult men, Brown et al. [24] found that supplementation of 100 mg of androstenedione three times per day did not raise total testosterone levels but did cause an increase in free testosterone, androstenedione, estrogen, and dihydrotestosterone. Finally, Leder et al. [29] found significant increases in both serum testosterone and estradiol when participants were given 300 mg/day of androstenedione for 7 d.

The overall interpretation of these results indicates that age and dosage appear to influence the net effectiveness of androstenedione supplementation, and as is the case with other medications and/or dietary supplements, these findings may have tangible ramifications when applied to clinical populations. In the studies that used either higher levels of androstenedione [29] or lower doses given at higher frequency [24], the researchers found changes in hormone levels in the older participants. However, no study has been completed using solely older participants and resistance exercise. The current study aimed to fill this gap in the literature, addressing the lack of research on older participants by examining the potential of androstenedione supplementation to enhance testosterone levels in this age group. Therefore, the purpose of this investigation was to determine the short-term physiological responses of androstenedione supplementation on resting and exercise testosterone levels in physically active men older than 50 years of age.

Methods

Experimental approach

To examine the effects of androstenedione supplementation on resting and exercising testosterone levels in older men, participants (n = 11) between the ages of 58 and 69 were divided into two groups. The experimental (EXP) group consisted of 6 men (mean age ± SE, 62.33 ± 2.57) taking 300 mg of androstenedione supplement and a control (CON) group that consisted of 5 men (mean age ± SE, 60.2 ± 1.02) taking the 300 mg of a cellulose placebo. The study duration was 7 d. The participants were blinded to the supplement assigned to them, as were the fitness professionals who administered the testing. Participants in both groups had been participating in a university adult fitness program (UAFP) in the Midwestern United States, for at least one year, incorporating both aerobic and resistance training into their workouts. Testosterone, estradiol, and androstenedione were analyzed via hormone assay pre- and post-supplementation.

Participants

Fourteen healthy male participants 58–69 years of age were recruited from the UAFP for this one-week study. In order to account for the influence of exercise habits on hormonal levels, only subjects who regularly participated in an exercise regimen were included in the study population. Participants were excluded if they were currently taking androgenic/anabolic supplements, diagnosed with either pancreatic or prostate cancer, or had any contraindications for high intensity exercise; these exclusion criteria greatly reduced the potential sample size for the present study. All participants were currently training utilizing aerobic, anaerobic, or both types of exercise in the fitness center (FC) of the UAFP.

Fourteen participants were originally recruited for this study, but only 11 completed all of the required testing procedures. Reasons for withdrawal from this study included: concern over medical implications related to the androstenedione supplementation (one participant) and scheduling difficulties (two participants).

The 11 individuals who met the inclusion criteria were asked to participate in the study, and they had the procedures and associated risks of the study explained to them. An institutionally approved informed consent form was read by each of the participants and signed by both the participant and the principal investigator prior to the preliminary testing. All participants stated they would strictly adhere to the pretest protocol. The study was approved by the university Institutional Review Board.

Pretesting measures

The participants reported to the university human performance laboratory (HPL) at 6:00 am, where their height and weight were determined on a stadiometer and calibrated scale. The participants warmed up with 5 min of unloaded cycling at a self-selected pace and they completed a warm-up exercise set on both the leg extension (LE) and leg curl (LC) machines to minimize risk of injury during the 10 repetition maximum (10-RM) test. The machine order was controlled for accurate exercise measurements. For the 10-RM test, each participant chose a weight (50% of subjective perceived maximum) arbitrarily to begin the test. Participants performed 10 repetitions of each weight with the resistance being increased accordingly based on the ease/difficulty of the previous lift until the heaviest weight was found that each participant was able to lift 10 times with proper form. A minimum of 2-min rest was allowed between attempts. The maximum weight each participant could lift 10 times for each exercise was recorded as his 10-RM.

The 11 participants were randomly placed into either an EXP (n = 6) or CON (n = 5) group following pretesting. The EXP group received 300 milligrams per day of androstenedione and the CON group received 300 milligrams of a cellulose placebo, for 7 d following the first testing session. The study utilized a double-blind technique to control for researcher bias during the study protocol.

Testing sessions

One week after the pretesting session, the participants reported to the HPL at 6:00 am, at which time the researchers prepped them for the blood draws as described below, and then took them to the FC for their first testing session. Participants reported to the HPL at the same time for all subsequent testing days. Participants refrained from strenuous activity for 24 h prior to the testing procedure. Upon arrival at the HPL, the participants assumed a supine position and a researcher inserted a 21-G Teflon catheter into the participant’s antecubital vein and attached it to a three-way stopcock to allow for blood draws. The catheter and the stopcock were kept patent with saline. Researchers drew 5 mL of blood (Sample 1) after the participant was in the supine position for approximately 15 min; this sample served to establish a baseline measurement. Once the catheter was secured with elastic tape, the researchers flushed the site with saline and covered it with sterile gauze, which was secured with elastic tape. After this, the participant reported to the FC for the supervised exercise session. The participant warmed up with 5 min of unloaded cycling at a self-selected pace. He then performed one set of 10 repetitions for LE and LC at 30% of his 10-RM, as a light warm-up to help prevent injury during the exercise stimulus.

After a 2-min rest, each participant performed three sets (total) of 10 repetitions on the LE and LC machines, the order of which was assigned using a randomized format. Each participant started the exercise stimulus with either the LE extension or LC, completing sets at 50%, 70%, and 90% of his 10-RM with 1-min rest between sets. At the conclusion of this exercise bout, the participants moved to a weight bench where the researchers drew another 5 mL of blood (Sample 2). After a 2-min rest, the participant repeated the protocol on the second exercise (i.e. leg curl or leg extension), and gave a third 5 mL blood sample immediately following this exercise stimulus (Sample 3); researchers drew additional samples at 5, 10, 15, and 20 min post-exercise (Samples 4–7). The blood draw sequence is shown in Figure 1. Then, using a counterbalanced design that reversed the order of the LE and LC exercise bouts completed by each participant during the initial exercise stimulus session, this protocol was repeated after 7 d of supplementation in order to determine the short-term effects of androstenedione supplementation on hormone levels.

Figure 1. The blood draw sequence.

Supplementation

As indicated, the participants were divided into EXP and CON groups and ingested one 300 mg capsule of either androstenedione (EXP group) or cellulose placebo (CON group) in a double-blind fashion at the same time each morning with breakfast. The supplementation lasted for exactly 7 d, beginning on the first day after their initial testing session and concluding on the day of their last testing session. During the course of the supplementation, participants were asked to continue their normal exercise routine with exception of the 24-h period prior to each testing session. Participants were also asked to continue their normal diet during the study, but with the aid of a 1-d dietary recall, participants were asked to repeat their pretest diet for the 24-h period prior to the post-testing session in order to attempt to control for dietary variations upon hormone levels.

Hormone analysis

Researchers analyzed androstenedione, estradiol, and total testosterone using radioimmunoassay kits (Diagnostic Systems Labs, Inc., Webster, TX). Blood samples were collected into a test tube and cooled on ice until the researchers centrifuged them for 20 min at 1169 g. The resulting serum was then aliquoted and frozen at −20 °C until analysis. The intra-assay coefficients of variation for androstenedione, estradiol, and testosterone were 5.8%, 6.9%, and 5.7%, respectively.

Statistical analysis

Dependent measures were checked for normality using Shapiro–Wilk tests. The pre- to post-supplement subject descriptive data (age, weight, height, and body mass index [BMI]) and plasma androstenedione measurements were statistically compared using an independent Student’s t-test. The workout loads were examined for reporting purposes, and pre- to post-supplementation measurements for testosterone and estradiol were analyzed using a two-way repeated measures ANOVA (Group × Time (Pre, Post) × Sample (1–7)), with any differences compared post-hoc with a Scheffe F-test. Modern statistical software packages were used to perform the analysis (G*Power 3 & SPSS 17.0, Chicago, IL) and statistical significance was set a priori at alpha < 0.05. All data are presented as mean ± SE.

Results

Shapiro–Wilk test results suggested that all data sets were normally distributed (p > 0.05). Participant characteristics are presented in Table 1. The participants in the EXP group were 52–69 years of age with a range in BMI of 23.96 to 32.51 (kg/m²). The subjects in the CON group were 58–63 years of age with a range in BMI of 23.53 to 30.38 (kg/m²). The 10-RM for the LE and LC for the EXP and CON groups are located in Table 2. The weights used by the EXP and CON groups for the 50%, 70%, and 90% of the 10-RM for the LE exercise were: 21.97 ± 1.82 versus 20.91 ± 2.31 kg (50%), 31.06 ± 1.82 versus 27.27 ± 3.21 kg (70%), and 37.88 ± 2.79 versus 34.55 ± 4.22 kg (90%). The weights used by the EXP and CON groups for the LC exercise were: 26.51 ± 2.17 versus 26.36 ± 4.41 kg (50%), 37.88 ± 3.46 versus 35.45 ± 5.45 (70%), and 47.73 ± 4.35 versus 44.55 ± 7.44 kg (90%). There were no significant differences between groups for performance on LE and LC exercises.

Table 1. Characteristics of the EXP and CON groups (means ± SE).

Characteristics	EXP (n = 6)	CON (n = 5)
Age (yrs.)	62.33 ± 2.57	60.2 ± 1.02
Height (em.)	179.49 ± 0.03	177.80 ± 0.02
Weight (kg)	85.31 ± 3.56	80.59 ± 3.09
BMI (kg/m^2)	26.51 ± 1.36	25.55 ± 1.27

Table 2. Ten-repetition maximum (10-RM) and 50%, 70%, and 90% of 10-RM means for the EXP and CON groups (means ± SE).

Lift	EXP (n = 6)	CON (n = 5)
10-RM Leg extension (kg)	43.18 ± 3.05	38.18 ± 4.90
10-RM Leg curl (kg)	52.27 ± 4.35	50.0 ± 8.00
50% Leg extension (kg)	21.97 ± 1.82	20.91 ± 2.31
50% Leg curl (kg)	26.51 ± 2.17	26.36 ± 4.41
70% Leg extension (kg)	31.06 ± 1.82	27.27 ± 3.21
70% Leg curl (kg)	37.88 ± 3.46	35.45 ± 5.45
90% Leg extension (kg)	37,88 ± 2.79	34.55 ± 4.22
90% Leg curl (kg)	47.73 ± 4.35	44.55 ± 7.44

Androstenedione

The pre- and post-supplemental data for the androstenedione assay are presented in Table 3. The analysis of the androstenedione assays revealed that the EXP group had a significant increase in androstenedione as compared to the CON group.

Table 3. Pre- to post-supplementation (between group) comparison of hormone levels for the EXP and CON groups (means ± SE).

	EXP (n = 6)		CON (n = 5)
	Pre	Post	Pre	Post
Testosterone (ng/mL)	4.36 ± 0.56	5.51 ± 0.35*	3.89 ± 0.57	3.82 ± 0.35
Estradiol (pg/mL)	15.95 ± 3.13	20.63 ± 2.67	14.71 ± 3.43	14.48 ± 2.92
Androstenedione (ng/mL)	0.88 ± 0.20	7.46 ± 1.25*	0.96 ± 0.20	0.95 ± 0.26

*p < 0.05

Testosterone

The pre- and post-supplemental data for the testosterone assay are presented in Table 3. The analysis via repeated measures ANOVA revealed a significant Group × Time (pre–post) interaction (F_1,8=6.626, p = 0.033, η_p² = 0.453) with the CON group having a mean change less than that of the EXP group. The analysis revealed no significant main or interaction effect (p > 0.05) for the Samples (1–7) during the data collection. The testosterone exercise data (Figure 2) showed that blood testosterone values remained relatively constant during the exercise session then declined slightly for both groups for 20-min post-exercise. These data also show that there was a significant increase pre- to post-supplementation in testosterone in the EXP group compared to the CON group.

Figure 2. Serum testosterone (ng/mL) post-leg extension, leg curl and during recovery by group (placebo versus androstenedione).

Estradiol

The pre- to post-supplement data for the estradiol assay are presented in Table 3. The analysis of the estradiol assays revealed that the EXP group had an increase in estradiol level, although not significantly (F_{1, 8}=3.927, p = 0.079, η_p² = 0.304). The estradiol exercise data (Figure 3) showed that estradiol declined slightly for both groups throughout the exercise session and increased slightly post-exercise, peaking around 15 min then declining toward 20 min post-exercise.

Figure 3. Serum estradiol (ng/mL) post-leg extension, leg curl and during recovery by group (placebo versus androstenedione).

Discussion

The major finding of this study was that seven consecutive days of androstenedione supplementation significantly increased the participant’s serum levels of testosterone. The novelty of this work is the demonstration of significant increase of testosterone level following androstenedione supplementation in older male participants. The results contrast with previous literature stating that androstenedione supplementation had no effect in young participants and inconsistent results were found in the older men [24,25,27,30].

Only one previous study [29] has noted a significant increase in blood levels of testosterone with androstenedione supplementation. Differences in participant age and testosterone level likely help to explain why most previous research has not consistently demonstrated significantly increased basal testosterone levels with androstenedione supplementation. Research has demonstrated that as men age, basal testosterone levels begin to decline [15,19–21]. The results from the Massachusetts Male Aging study showed total testosterone levels of 5.8 ng/mL for subjects in their 40s declined to 4.6 ng/mL by age 60, suggesting that total testosterone decreased by 0.8% per year, whereas both free and albumin bound testosterone declined by 2% per year [32]. The mean age for the participants in the present study was 62.33 ± 2.565 years, and basal mean testosterone levels were 4.645 ± 0.514 ng/mL, which is similar to the data reported by Feldman et al. [32], and suggests that at the outset of the current study the sample of participants had testosterone levels typical of men in their 60s. This suggests that the effectiveness of androstenedione supplementation is influenced by baseline testosterone levels, and may help to explain the current results, which showed testosterone levels increased from measures typical of men in their 60s to measures more typical of men in their 40s after androstenedione supplementation. Blood levels of testosterone are controlled by a feedback mechanism that shuts down production when testosterone reaches its basal set point, with any excess testosterone being taken up by fat cells and converted into estradiol. At the onset of this study, our EXP group had testosterone levels typical of males in their 60s and at the end of 7 d of supplementation their testosterone levels were typical of males in their 40s. The rise in estradiol of our EXP group suggests that any testosterone over the normal basal level was likely converted to estradiol.

The feedback control of basal testosterone levels was also demonstrated in earlier studies in which the participants who were too young to have diminished basal testosterone level were not affected by androstenedione supplementation. A study by King et al. [28] reported that blood estradiol increased by 41% for the androstenedione group who took 300 mg/day for 8 weeks. Leder and colleagues [29] reported an estradiol concentration increase of 125% in area under the curve (AUC) in participants who were supplemented for 300 mg/d of androstenedione. The study by Rasmussen et al. [33] reported an increase in estradiol of 70% from the basal levels. The study by Brown and colleagues [24] reported a 75% increase in estradiol for their 50-year-old subjects. The androstenedione group in the present study showed only a statistically non-significant 23% increase in estradiol; this change in estradiol occurred concomitantly with a significant increase in the participants’ testosterone level to measures comparable to normal for 40-year-old men.

The influence of fat cells on estradiol may be accelerated by aging, as evidenced by the tendency of males to accumulate body fat as they age. The fat cells of the body have the ability to convert excess testosterone into estradiol, which may help to explain why androstenedione supplementation appears to be ineffective in younger participants. In other words, androstenedione supplementation may provide little boost to young men who regularly exercise, as they tend to have abundant baseline testosterone levels and possess relatively lesser percentages of body fat. Conversely, older men who exercise tend to have lesser testosterone and more abundant body fat, so their endocrine systems may be able to use the exogenous androstenedione in ways that young men cannot. Thus, given the interactions between hormonal levels, body fat, exercise habits, and aging, the present findings suggest further study is needed on androstenedione supplementation on men in the early stages of andropause who regularly exercise.

Another reason why the data from this study do not agree with prior research could be related to the dose of androstenedione supplementation used. The majority of the previous studies in this area used supplementation dosages of 100–200 mg of androstenedione. The study by Wallace et al. [31] showed a basal testosterone mean of 6.08 ng/mL for their participants and a mean age of 48.1 ± 3.9 years. The age of their participants may lead one to believe that these researchers possibly could have seen a significant increase in blood testosterone levels. However, the participants ingested only 100 mg of androstenedione for a period of twelve weeks. This supplementation dose coincides with the study by Rasmussen et al. [33], who studied six participants who had a mean age of 32 ± 4 years. These researchers did not report mean basal testosterone levels, making the interpretation less apparent. Further, a dose of 100 mg of androstenedione has been shown to be ineffective for raising blood levels of testosterone [33].

In contrast, the report by Leder and colleagues [29] showed that while a supplement of 100 mg of androstenedione per day for a period of 7 d was insufficient to raise blood testosterone levels, 300 mg doses did significantly raise testosterone levels. Their participants had a mean age of 31.5 years and mean basal testosterone levels of 4.93 ng/mL. They put participants through two levels of androstenedione supplementation, 100 mg and 300 mg per day for a period of 7 d. The results reported by Leder and colleagues [29] most closely resemble that of the present study in the respect that the participants in the 300 mg group had a significant increase in mean blood testosterone, rising to 8.72 ng/mL (p < 0.001), whereas the 100 mg group did not (p = 0.48). Their conclusion was that 300 mg of androstenedione per day was substantial enough to raise basal levels of blood testosterone, while 100 mg per day was not.

While the information provided via the data from this study adds to the literature, it is not without limitations. The small sample size does impact the interpretation of the results, however, it should be noted that there is a limited pool of healthy males in the age group who are not on medications, or who do not have contraindications for high intensity exercise. Additionally, although base line assessments of testosterone were not measured 7 of the 14 participants demonstrated low levels of testosterone ∼3 ng/mL as assessed during the course of the study. Even with the small sample size in the present study, the acute physiological effect of a 300 mg dose of androstenedione supplementation in older men is apparent. The results of this study indicate that acute androstenedione supplementation can raise testosterone levels in older men who regularly exercise. While assessing the clinical relevance of androstenedione supplementation is beyond the aims of the present study, the rise in basal levels of testosterone could have major health implications for older males, in that testosterone is responsible for assisting males to retain skeletal muscle mass through the lifespan. However, future research using longer study periods are needed to examine the chronic effects of androstenedione supplementation on testosterone levels in older men. Clinical studies utilizing a dose-response protocol to identify the level of androstenedione that results in an optimal level of testosterone without a significant increase in estradiol would be valuable for licensed health-care providers who care for older men. The benefits of additional research would be to identify strategies to offset the age-related decline in muscle mass without deleterious side effects.

Declaration of interest

The authors do not declare any conflict of interest in having performed the research associated with this paper.