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Journal of the American College of Nutrition, Vol. 22, No. 5, 363-371 (2003)
Published by the American College of Nutrition

Original Research

Ingestion of a Dietary Supplement Containing Dehydroepiandrosterone (DHEA) and Androstenedione Has Minimal Effect on Immune Function in Middle-Aged Men

Marian L. Kohut, PhD, James R. Thompson, MS, Jeff Campbell, BA, Greg A. Brown, MS, Matthew D. Vukovich, PhD, Dave A. Jackson, MS and Doug S. King, PhD

Department of Health and Human Performance, Iowa State University, Ames, Iowa

Address correspondence to: Marian L. Kohut, Ph.D., Department of Health and Human Performance, Iowa State University, 235 Forker Building, Ames IA 50011. E-mail: mkohut{at}iastate.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: This study investigated the effects of four weeks of intake of a supplement containing dehydroepiandrosterone (DHEA), androstenedione and herbal extracts on immune function in middle-aged men.

Design: Subjects consumed either an oral placebo or an oral supplement for four weeks. The supplement contained a total daily dose of 150 mg DHEA, 300 mg androstenedione, 750 mg Tribulus terrestris, 625 mg chrysin, 300 mg indole-3-carbinol and 540 mg saw palmetto.

Measurements: Peripheral blood mononuclear cells were used to assess phytohemagglutinin(PHA)-induced lymphocyte proliferation and cytokine production. The cytokines measured were interleukin (IL)-2, IL-4, IL-10, IL-1ß, and interferon (IFN)-{gamma}. Serum free testosterone, androstenedione, estradiol, dihydrotestosterone (DHT) were also measured.

Results: The supplement significantly increased serum levels of androstenedione, free testosterone, estradiol and DHT during week 1 to week 4. Supplement intake did not affect LPS or ConA proliferation and had minimal effect on PHA-induced proliferation. LPS-induced production of IL-1beta, and PHA-induced IL-2, IL-4, IL-10, or IFN-gamma production was not altered by the supplement. The addition of the same supplement, DHEA or androstenedione alone to lymphocyte cultures in vitro did not alter lymphocyte proliferation, IL-2, IL-10, or IFN-{gamma}, but did increase IL-4. In addition, serum HDL-C concentration significantly declined.

Conclusion: These findings suggest that, although chronic intake of a complex dietary supplement containing DHEA, androstenedione and herbal extracts increases serum androgen levels, it has minimal effect on immune function in middle-aged men.

Key words: aging, cytokines, lymphocyte, hormones, androstenedione, DHEA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The plasma concentration of several androgenic steroid hormones declines with advancing age [1]; therefore, the older adult population may consume supplements containing androgenic steroids in an effort to reverse age-associated changes such as the decrease in immune function. In addition, strength/power athletes may use these substances in an attempt to enhance performance. Various combinations of herbal extracts have been added to the androgenic steroid-containing supplements that are sold over the counter, in an attempt to maximize the "androgenic effect" of these dietary supplements. Although nutritional supplements containing different combinations of these androgenic steroid hormones are sold, the potential effects of these supplements on immune function have not been characterized.

Immune function may decline with age, and the results from several studies suggest that supplementation with the steroid dehydroepiandrosterone (DHEA) may modify the age-related changes of immune response. It is well documented that the production of the cytokine interleukin-2 (IL-2) declines with age [2]. It has been suggested that a dysregulation in cytokine balance (reduced T helper type 1 (Th1) cytokines, increased T helper type 2 (Th2) cytokines) may also accompany the aging process [2–5]. IL-2 and interferon-{gamma} (IFN-{gamma}) are Th1 cytokines that drive cell-mediated immune responses, whereas Th2 cytokines promote humoral-mediated immunity. The age-related dysregulation in Th1/Th2 cytokine balance may contribute to an impaired ability to defend against infection [3]. Studies using animal models have demonstrated that one of the steroids contained in the over-the-counter "androgenic dietary supplements," dehydroepiandrosterone (DHEA), may reverse the age-related decline of immune function and provide protection from infection [6]. Aged mice treated with DHEA exhibit enhanced interleukin (IL)-2 and interferon (IFN)-{gamma} production with no effect on IL-4 or IL-10, perhaps reversing the age-associated change in Th1/Th2 cytokine balance [4,5].

In addition to its potential effect on cytokines, other studies have shown that the same component, DHEA, contained in "androgenic dietary supplements" has a protective effect against infection with numerous pathogens (M. tuberculosis, West Nile virus, Herpes simplex type, Coxsackie virus) in mice [7–9]. However, it is important to note that lifelong treatment with DHEA did not reverse immunosenescence or enhance resistance to infection in rodents [10]. Fewer human studies exist, and there is limited evidence suggesting that DHEA can reverse immunosenescence. In several trials, oral DHEA administration did not significantly improve the immune response to influenza immunization [11–13]. The effect of "androgenic dietary supplements" on overall immune function is not well studied in humans. Based on two reports of healthy, older adults, some aspects of immune function may be enhanced by DHEA treatment, whereas others show no effect or are inhibited [14,15]. The effect of a complex "androgenic dietary supplement" on immune responsiveness has not been studied in humans to our knowledge.

Other weak androgenic steroids include dihydrotestosterone (DHT), androstenedione, androstenediol and androstenetriol. These steroids may also enhance immunity and protect against infection [16–19]. DHT is the most potent androgen [20] and binds to the androgen receptor more tightly than any other androgen [21]. Some investigators have demonstrated that the beneficial effect of these other steroid hormones, in terms of immune function, may be greater than DHEA [17–19]. However, the immunoenhancing effect of these steroids or combination of steroids has only been observed in animal models, and, to our knowledge, these have not been tested in humans.

The metabolic fate of ingested DHEA and androstenedione is not clear. DHEA may be converted to androstenedione or androstenediol, and either of these steroids may undergo further conversion to testosterone, a more potent androgen. Testosterone can undergo further conversion to DHT by the enzyme 5-alpha reductase or may undergo aromatization resulting in estrogen formation. In order to maximize the "androgenic" effect of these steroids and reduce the possibility of steroid conversion to DHT or estrogens, several herbal extracts have been added to nutritional supplements. These extracts include saw palmetto extract, which may inhibit 5-alpha reductase, preventing conversion to DHT [22], indole-3-carbinol and chrysin, which prevent aromatization of androgens [23,24], and Tribulus terrestris, suggested to increase serum lutenizing hormone, although this claim has not been verified in a controlled trial. These herbal extracts are not known to exhibit significant immunomodulatory properties. The purpose of this study was to investigate the immunological effects of four weeks of oral ingestion of a supplement containing DHEA, androstenedione, saw palmetto, Tribulus terrestris, chrysin and indole-3-carbinol in middle-aged men. Serum levels of several steroid hormones were also monitored. In addition, blood samples from middle-aged men were collected to assess the in vitro effects of the supplement and its androgenic components.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects
Sixteen healthy men between the ages of 50 and 59 not taking medications or dietary supplements were recruited for this double-blind study. Written informed consent was obtained from each participant, and the protocol was approved by the Iowa State University Human Subjects Review Board. Each subject completed a medical history, to eliminate subjects with known chronic disease, and a dietary evaluation. Subjects were randomly assigned to consume either a rice flour oral placebo (n = 8) or an oral dose of the dietary supplement (n = 8) for 28 days. The supplement contained 150 mg DHEA, 300 mg androstenedione, 750 mg Tribulus terrestris, 625 mg chrysin, 300 mg indole-3-carbinol and 540 mg saw palmetto. The supplement was consumed in three equal doses taken before 0900, at 1500 and before bedtime. The supplement was supplied by Experimental and Applied Sciences, Golden, CO, and content of the supplement was verified by HPLC at an independent laboratory (Integrated Biomolecule, Tuscon, AZ). Subjects were asked to maintain normal diet and activity patterns throughout the study. Subjects recorded dietary intake and activity for the two days prior to each blood draw. Diet analysis was performed with Nutritionist 4 software. Six additional young male subjects (ages 21 to 32) were included only as an age comparison group. These young subjects also completed a medical history and a dietary evaluation to eliminate any subjects with known chronic disease and/or consuming dietary supplements. The young subjects did not consume oral placebo or dietary supplement. Instead, this group was included only to document the age-related changes of immune function.

Blood Collection and Peripheral Blood Mononuclear Cell (PBMC) Isolation
Fasting blood samples were collected from the older subjects between 6:30 and 8:00 am prior to supplementation and once per week during supplementation. Blood was collected between 6:30 and 8:00 am from the young subjects at week 0, week 1 and week 4. PBMC were isolated by density gradient centrifugation (Ficoll-Paque Plus, Amershem Pharmacia Biotech, NJ). Cells were washed two times with sterile Hank’s Balanced Salt Solution (HBSS; Sigma Chemical Co. St. Louis, MO), suspended in RPMI (Life Technologies, Rockville, MD) supplemented with 10 mM Hepes (Sigma Chemical Co.), 50 U/mL penicillin, 50 mg/mL streptomycin (both from Life Technologies) and 5% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), and counted using a hemocytometer. All cells were then frozen in RPMI media containing 30% FBS and dimethylsulfoxide (DMSO) and stored in liquid nitrogen until later analysis. All cell samples were thawed on the same day to perform the mitogen-stimulated proliferation and cytokine production assays to limit inter-assay variability. After thawing, the cells were washed twice in supplemented RPMI and adjusted to the appropriate concentration. Cell viability was >=85% after thawing, and cell counts were adjusted based on the number of live cells.

Peripheral Blood Mononuclear Cell (PBMC) Proliferation
PBMC proliferation was measured using a colorimetric assay described by Mosmann (J Immunol Methods 65:55–63, 1983). Cells were plated at 1 x 105 cells per well in 96-well, flat bottom tissue culture plates (Corning, Corning, NY), containing a final concentration of 5 µg/mL phytohemagglutinin (PHA; Sigma Chemical Co.) or media alone in a total volume of 100 µL. Plates were incubated at 37°C in 5% CO2 for 72 hours. During the last four hours of incubation, 10 µL of 5.0 mg/mL 3-(4,5-dimethylthiozol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co.), in PBS was added to each well. Then, 100 µL of 0.04N HCl in isopropanol was added to dissolve the colored precipitate. Absorbance was read at a dual wavelength of 570 and 630 nm using an automated plate reader (BioRad Instrument Co., Richmond, CA).

Cytokine Production: IL-1ß, IL-2, IL-4, IL-10, IL-12 and IFN-{gamma}
Cytokine concentration was measured in supernatant 24 or 48 hours after the addition of LPS or PHA. Control wells received media without mitogens. IFN-{gamma} and IL-2 was measured after 24 hours in culture with PHA (5 µg/mL). IL-4 and IL-10 was measured after 48 hours in culture with PHA (5 µg/mL). IL-1ß was measured after 24 hours in culture with LPS, whereas IL-12 was measured after 48 hours in culture with LPS (100 ng/mL). A standard ELISA kit specific for each of the six cytokines to be assayed was used (OptEIA Sets, PharMingen, San Diego, CA).

In Vitro Effects of DHEA, Androstenedione and Supplement
Blood samples were collected from five men (40 to 58 years of age), and PBMC were isolated as described above with the exception that cells were not frozen and thawed. Instead, all blood was collected, cells were isolated and cultured on the same day. Cells were counted and adjusted to 1 x 106 cells/mL. 100 µL of cells were added to 96 well plates for the mitogen-induced proliferation assay and cytokine production. The proliferation assay was performed as described above. Both the proliferation assay and the cytokine assay used mitogen and supplement components at the following doses. Wells contained one of the following: PHA (5 µg/mL), LPS (100 ng/mL) or media plus DHEA (10-5–10-9 M), androstenedione (10-5–10-9 M), complete dietary supplement with DHEA and androstenedione concentrations at 10-5–10-9M, or media alone. The steroids were first dissolved in ethanol and then diluted to the appropriate concentrations. The media control wells contained the same final concentration of ethanol as the wells containing steroid (0.00001–0.1% ethanol). Plates were incubated for 72 hours at 37°C in 5% CO2, and proliferation was assessed using MTT as described above. In the cytokine analyses, cells were incubated 37°C in 5% CO2 for 24 to 48 hours, and the supernatant was collected from the appropriate wells according to the following time points. IL-2 and IFN{gamma} were measured after 24 hours incubation with PHA ± supplement components, IL-4 and IL-10 were measured after 48 hours incubation with PHA ± supplement components, and IL-1 was measured after 24 hours incubation with LPS ± supplement components. A standard ELISA kit specific for each of the cytokines to be assayed was used (OptEIA Sets, PharMingen, San Diego, CA).

Serum Hormones and Blood Chemistry
The serum concentrations of androstenedione, estradiol, free testosterone and total testosterone from the older subjects were measured with commercial radioimmunoassay kits (Diagnostic Products Corp. Los Angeles, CA and Diagnostic Systems Laboratory, Webster, TX). An ELISA kit was used to measure serum level of dihydrotestosterone (Immuno-Biological Laboratories, Hamburg, Germany). A commercial laboratory performed blood chemistry analysis (Quest Diagnostics, Wood Dale, IL).

Statistics
Statistical analyses were performed to determine the effect of supplement using a 2 factor mixed ANOVA (time x supplement). A 2 factor mixed ANOVA (time x age) was used to test the effect of age. A one-way ANOVA was used to test the potential effects of the supplement components added to cells in vitro. All analyses were performed with SPSS software (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
Diet/Activity
There was no difference between the placebo and supplement groups with respect to any of the nutrients analyzed with the Nutritionist-4 software. Also, activity patterns were not different between the two groups.

PHA-Induced Proliferation
With respect to PHA-induced proliferation, there were main effects of supplement treatment and time (Fig. 1). PHA-induced proliferation did not differ between placebo and supplement groups at baseline (week 0) or at week 4, but was greater in subjects consuming the supplement at week 1 (p = 0.023) (Fig. 1). A significant main effect of age was observed (young > old, p < 0.001), and a significant age x time interaction (p = 0.036) was observed, suggesting that PHA proliferation did not change over time in young subjects.



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  		Fig. 1. PHA-induced proliferation in PBMC from middle-aged subjects consuming either placebo or supplement and young subjects that did not consume placebo or supplement. Results are expressed as mean ± SEM. Asterisk indicates significant differences (p < 0.05) between placebo and supplement.

 
Cytokine Production
The production of IFN-{gamma} and IL-2 (Th1 cytokines) was not altered by consumption of the dietary supplement at any time point throughout the four-week period (Fig. 2). However, a significant effect of age was observed for IL-2 (young > old, p < 0.001) and IFN-{gamma} (young > old, p = 0.001). Dietary supplementation also did not have an effect on the production of IL-4 and IL-10 (Th2 cytokines) at week 1 or at week 4 (Fig. 3). A significant main effect of age was observed with respect to IL-4 (young < old, p = 0.025); however, we did not observe an effect of age on IL-10 production. Finally, the production of the pro-inflammatory cytokine IL-1ß was not altered by the supplement (Fig. 4).



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  		Fig. 2. PHA-induced production of the Th1 cytokines, IL-2 and IFN-{gamma} in supernatant from PBMC cultured with PHA at 5 µg/mL. PBMC were obtained from middle-aged subjects consuming either placebo or supplement, or from young subjects consuming no supplement at the time points indicated. Results are expressed as mean ± SEM.

 


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  		Fig. 3. PHA-induced production of the Th2 cytokines, IL-4 and IL-10 in supernatant from PBMC cultured with PHA at 5 µg/mL. PBMC were obtained from middle-aged subjects consuming either placebo or supplement, or from young subjects that did not consume placebo or supplement at the time points indicated. Results are expressed as mean ± SEM.

 


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  		Fig. 4. LPS-induced production of IL-1ß in supernatant from PBMC culture with LPS at 100 ng/mL. PBMC were obtained from middle-aged subjects consuming either placebo or supplement at the time points indicated. Results are expressed as mean ± SEM.

 
In Vitro Effect of DHEA, Androstenedione or Supplement on Lymphocyte-Induced Proliferation
PBMC were cultured with DHEA, androstenedione or the complete dietary supplement ± PHA. No significant proliferation was observed in wells containing hormones or supplement without PHA (results not shown). As expected, PHA did induce proliferation, but the addition of DHEA, androstenedione or the complete supplement did not alter proliferation at any of the concentrations tested (10-5 M, 10-7 M, 10-9 M) (Fig. 5).



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  		Fig. 5. PBMC obtained from middle-aged men and incubated with PHA (5 µg/mL) plus media, the dietary supplement (containing androstenedione and DHEA at 10-7 M), androstenedione (10-7 M) or DHEA (10-7 M). Results are expressed as mean ± SEM.

 
In Vitro Effect of DHEA, Androstenedione or Supplement on Cytokine Production
PHA was added to cultures to induce cytokine production. Wells that contained hormones or supplement but not PHA did not produce detectable levels of any of the cytokines measured (results not shown). The in vitro addition of DHEA (10-7 M), androstenedione (10-7 M) or supplement (10-7 M) did not alter the production of IL-2, IFN-{gamma}, or IL-10 (Figs. 6, 7). However, IL-4 production was enhanced by the addition of the supplement to PBMC cultures (p = 0.004) (Fig. 7). There was a trend towards enhanced IL-4 production in wells containing DHEA (p = 0.065) and in wells containing androstenedione (p = 0.094).



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  		Fig. 6. IL-2 and IFN-{gamma} in supernatant from PBMC obtained from middle-aged men and incubated with PHA (5 µg/mL) plus media, the dietary supplement (containing androstenedione and DHEA at 10-7 M), androstenedione (10-7 M) or DHEA (10-7 M). Results are expressed as mean ± SEM.

 


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  		Fig. 7. IL-10 and IL-4 in supernatant from PBMC obtained from middle-aged men and incubated with PHA (5 µg/mL) plus media, the dietary supplement (containing androstenedione and DHEA at 10-7 M), androstenedione (10-7 M) or DHEA (10-7 M). Results are expressed as mean ± SEM. Asterisk indicates significant difference (p < 0.05) between cultures containing media alone and cultures containing dietary supplement. A trend (p < 0.1) towards a difference between cultures containing media alone and cultures containing DHEA or androstenedione is indicated by +.

 
Serum Hormone Levels
Although serum levels of androstenedione, free testosterone, DHT and estradiol did not differ between groups at baseline, dietary supplementation resulted in increased serum levels of androstenedione, free testosterone, DHT and estradiol at weeks 1 to 4 (Table 1). Total testosterone was not altered by dietary supplementation. Total cholesterol and LDL-cholesterol did not change (results not shown); however, a significant decrease in HDL-cholesterol concentration was observed in subjects consuming the supplement (Table 1).


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  		Table 1. Serum Hormone and HDL-Cholesterol Concentrations in Placebo- and Supplement-Treated Groups

 

   DISCUSSION
 
Although some rodent studies have noted an enhancement of immune function following administration of weak androgens, our results in humans using a supplement containing androgens and herbal extracts designed for maximal "androgenic effect" do not strongly support the use of oral androgen plus herbal supplementation with respect to immune benefits. It has been suggested that weak androgens such as DHEA may reverse the age-associated dysregulation of the Th1/Th2 cytokine balance by increasing the production of Th1 cytokines, IL-2 and IFN-{gamma}, with little or no effect on the Th2 cytokines, IL-4 and IL-10 [4,5]. However, it is important to note that a DHEA-induced enhancement of Th1 cytokine production is not consistently found among rodent studies and decreases of Th1 cytokines have also been reported [25]. In our study we examined the effect of an "androgenic dietary supplement" containing DHEA on both Th1 and Th2 cytokine production in healthy older adults. Four weeks of oral intake of a supplement containing DHEA (150 mg/day), androstenedione (300 mg/day), plus herbal extracts, had no effect on PHA stimulated production of IL-2, IL-4, IL-10 or IFN-{gamma}. We did observe an effect of age on Th1 and Th2 cytokine production that is consistent with other research (decreased Th1, increased Th2). The Th1 cytokines, IL-2 and IFN-{gamma}, were lower among the 50 to 59 year old subjects as compared to younger subjects whereas production of the Th2 cytokine, IL-4, was greater among the older subjects. We have also consistently observed that the variability in cytokine production is greater in older subjects than young subjects. Differing rates of immunosenescence among older subjects may explain this larger degree of variability. Based on our findings, it appears that four weeks of oral intake of a complex dietary supplement containing DHEA, androstenedione and herbal extracts was not sufficient to reverse the age-related changes in Th1/Th2 cytokine balance. However, given that we did not test the effect of DHEA or androstenedione supplementation alone, it remains possible that oral intake of these steroids alone or in combination may have altered Th1/Th2 cytokines. We did document an increase in serum levels of androstenedione in those subjects consuming the supplement compared to those subjects consuming placebo, yet this change in serum level of androstenedione did not appear to be associated with altered cytokines.

In addition, we added DHEA, androstenedioene or supplement in vitro to cultures containing PBMC from middle-aged men and found no effect on the production of IL-2, IL-10 and IFN-{gamma}. However, we did observe an increase in IL-4 production in cultures containing the supplement and a trend towards increased IL-4 in cultures containing DHEA or androstenedione. An increase in IL-4 (Th2 cytokine) does not support the theory that these steroids can reverse the age-associated change in cytokine balance. Instead, IL-4 has been reported to increase with age. Therefore, our results suggest that oral intake of a supplement containing DHEA and androstenedione, or addition of these steroids to mononuclear cell cultures in vitro, do not reverse the age-related dysregulation of Th1/Th2 cytokine balance.

The findings from our study differ with the results from a study by Khorram et al. [14] in that we did not observe increased IL-2 production; however, subjects in the Khorram study only consumed DHEA, not an "androgenic dietary supplement." In their study, men 53 to 69 years of age consumed 50 mg of DHEA per day for 20 weeks. However, the increase in IL-2 was observed after 20 weeks of treatment, but not seen after four weeks of supplementation. Although these authors reported an increase in several immune parameters during the 20-week trial [14], it has been suggested that some of these effects may reflect a multiple comparisons artifact [10]. A separate study involving postmenopausal women reported no change in IL-2 production after three weeks of DHEA supplementation (50 mg/day) [15]. Based on these two studies and our results, it is difficult to make definite conclusions regarding the effects of DHEA and "androgenic dietary supplements" of IL-2.

The ability of T cells to proliferate in response to mitogen or antigen also declines with age [2]. We have also observed that mitogen-induced proliferation is greater in young subjects than in older subjects. Again, the variability in the amount of proliferation tends to be greater among older subjects. With respect to mitogen-induced T cell proliferation, we observed an increase in PHA-stimulated proliferation only at week one of supplementation. Although this increase was statistically significant, it was a small effect and did not restore the amount of proliferation to the level seen in younger subjects. Also, given the responses over time, with higher levels of proliferation at week 4 in both groups and a decrease only in placebo at week 1, it is doubtful that the statistical difference at week 1 is physiologically meaningful. In the study by Khorram et al. [14], an increase in PHA-induced proliferation was observed in subjects consuming 50 mg/day of DHEA at week 12 of supplementation, but not at week 4 or week 20. The other human study involving postmenopausal women reported a decrease in mitogen-induced T cell proliferation after three weeks of DHEA supplementation [15]. Taken together these studies and our results do not provide strong support for enhanced T cell proliferation following either DHEA supplementation or intake of "androgenic dietary supplements."

This is the first study to our knowledge to examine oral intake of any supplement containing androstenedione on immune responsiveness in humans. The results from several studies using rodents suggest that weak androgens such as androstenediol and/or androstenetriol have more potent immunomodulatory effects than DHEA [17,18]. It has been suggested that the metabolic conversion of DHEA to other androgens is necessary for regulation of immune function. Based on our results, it appears that the addition of androstenedione to a supplement containing DHEA and other herbal extracts thought to prevent the conversion of androgens to DHT or estrogen does not enhance immune function in older men. It is possible that the combination of steroids and/or extracts contained in the supplement inhibited any immunoenhancing properties of androstenedione. However, our in vitro results are not consistent with this possibility since androstenedione alone did not have significant immunomodulatory effects. Also, we did observe elevated plasma levels of androstenedione in the supplement group, but this alteration in serum level did not appear to be associated with changes in lymphocyte proliferation.

Although we did not independently test the potential immunomodulatory properties of the herbal extracts by separate oral administration of these compounds, the herbal extracts contained in the supplement are not known to significantly impact lymphocyte function. Indole-3-carbinol has been shown in several studies to inhibit tumor cell growth, but this appears to be via a direct effect on the tumor cell itself rather than via activation of immune response [26,27]. In one study, large doses of indole-3-carbinol were fed to rats, and no significant changes were seen in lymphocyte proliferation, Natural Killer cell activity or delayed type hypersensitivity (DTH) response [28]. In a separate study, chrysin was reported to enhance killing of L-929 tumor cells by increasing tumor necrosis factor (TNF-{alpha}) cytotoxicity, but no effects on lymphocyte function were reported [29]. The extract Serenoa repens derived from saw palmetto has been shown to reduce symptoms of benign prostatic hyperplasia, but the mechanism of action involves inhibition of 5-alpha-reductase and inhibition of DHT binding to androgen receptors in prostate cells, rather than involvement of immune cells [30]. Therefore, there is not strong evidence that any of the herbal extracts contained in the supplement have direct effects on lymphocyte function.

Although enhancement of the immune response has been reported in many, but not all, studies involving rodents and DHEA, the majority of human studies do not observe large immune benefits [11–13,15,31]. Perhaps one explanation for the different findings is that the adrenal glands of mice do not synthesize androgens, and the circulating level of DHEA in mice has been reported to be very low or undetectable [32]. The dose of androgens administered either via diet or injection in many rodent studies likely results in pharmacological levels of androgen. In contrast, DHEA levels in humans altered by DHEA administration may increase twofold to tenfold and are generally considered to be within the physiological range [33,12]. At higher doses of androgen there may be negative health consequences, such as the decline in serum HDL-cholesterol noted in this study, found as part of a larger investigation on androgenic steroid intakes in men 30 to 59 years of age [34]. Therefore, in rodents, supraphysiological doses of androgen may confer some immune benefit, and it is possible that very large doses of androgen may enhance immune function in humans. However, there may be negative health effects in humans associated with supraphysiological doses such as reduced HDL-cholesterol and increased risk of breast or ovarian cancer in women [34,35]. As part of a larger study on androgenic dietary supplement intake in men 30 to 59 years of age, the intake of supplements containing the same ingredients, total daily dose of 150 mg DHEA, 300 mg androstenedione, 750 mg Tribulus terrestris, 625 mg chrysin, 300 mg indole-3-carbinol and 540 mg saw palmetto [34] or a total daily dose of 300 mg androstenedione [36], demonstrated the negative health consequences of steroid intake, in particular, the decrease in HDL-cholesterol found in all age groups with all androgenic supplement combinations. In addition, four weeks of oral intake of this supplement (DHEA (150 mg/day), androstenedione (300 mg/day) plus herbal extracts) did not reverse the age-related decrease in serum total testosterone. The detailed serum hormone concentrations and serum lipid concentrations resulting from oral intake of this supplement are reported elsewhere [34].

Taken together, the findings from this study demonstrate that oral intake of a supplement containing DHEA (150 mg/day), androstenedione (300 mg/day) plus herbal extracts for four weeks does not enhance immune response among middle aged men. Serum steroid hormones and HDL-cholesterol are altered in subjects consuming the supplement, but these changes may lead to negative health consequences [34]. Therefore, it is possible that higher doses or a longer time period of consumption of the supplement given in this study or some other combination of the ingredients found in the supplement may enhance immune function, but the health risks may outweigh any potential health benefits.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
This study was funded in part by a grant from Experimental and Applied Sciences, Golden, CO.

Received May 24, 2002. Accepted December 12, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Gray A, Feldman HA, McKinlay JB, Longcope C: Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study.J Clin Endocrinol Metab73 :1016 –1025,1991 .[Abstract]
  2. Miller RA: The aging immune system: primer and prospectus.Science273 :70 –73,1996 .[Abstract]
  3. Shearer GM: Th1/Th2 changes in aging.Mech Ageing Dev94 :1 –5,1997 .
  4. Inserra P, Zhang Z, Aredestani SK, Araghi-Nikman M, Liang B, Jiang S, Shaw D, Moliter M, Elliott K, Watson RR: Modulation of cytokine production by dehydroepiandrosterone (DHEA) plus melatonin (MLT) supplementation of old mice.Proc Soc Exp Biol Med218 :76 –82,1998 .[Abstract]
  5. Daynes RA, Dudley DJ, Araneo BA: Regulation of murine lymphokine production in vivo II. Dehydroepiandrostenedione is a natural enhancer of interleukin-2 synthesis by helper T cells.Eur J Immunol20 :793 –802,1990 .[Medline]
  6. Daynes RA, Araneo BA: Prevention and reversal of some age associated changes in immunologic responses by supplemental DHEAS therapy.Aging Immunol Infect Dis3 :135 –154,1992 .
  7. Hernandez-Pando R, De La Luz Streber M, Orozco H, Arriaga K, Pavon L, Al-Nakhli SA, Rook GAW: The effects of androstenediol and dehydroepiandrosterone on the course and cytokine profile of tuberculosis in BALB/c mice.Immunol95 :234 –241,1998 .[Medline]
  8. Ben-Nathan D, Lustig S, Kobiler D, Danenberg HD, Lupu E, Feurstein G: Dehydroepiandrosterone protects mice inoculated with West Nile Virus and exposed to cold stress.J Med Virol38 :159 –166,1992 .[Medline]
  9. Loria RM, Inge TH, Cook SS, Szaki AK, Regelson W: Protection against lethal viral infection with the native steroid dehydroepiandrostenedione.J Med Virol26 :301 –314,1988 .[Medline]
  10. Miller RA, Chrisp C: Lifelong treatment with oral DHEA sulfate does not preserve immune function, prevent disease, or improve survival in genetically heterogenous mice.J Am Geriatr Soc47 :960 –966,1999 .[Medline]
  11. Danenberg HD, Ben-Yehuda A, Zakay-Rones Z, Gross DJ, Friedman G: Dehydroepiandrosterone treatment is not beneficial to the immune response to influenza in elderly subjects.J Clin Endocrinol Metab82 :2911 –2914,1997 .[Abstract/Free Full Text]
  12. Degelau J, Guay D, Hallgren MS: The effects of DHEAS on influenza vaccination in aging adults.J Am Geriatr Soc45 :747 –751,1997 .[Medline]
  13. Evans TG, Judd ME, Dowell T, Poe S, Daynes RA, Araneo BA: The use of oral dehydroepiandrosterone sulfate as an adjuvant in tetanus and influenza vaccination of the elderly.Vaccine14 :1531 –1537,1996 .[Medline]
  14. Khorram O, Vu L, Yen SS: Activation of immune function by dehydroepiandrosterone in age-advanced men.J Gerontol A Biol Sci Med Sci52 :M1 –7,1997 .[Abstract]
  15. Casson PR, Andersen RN, Herod HG: Oral dehydroepiandrosterone treatment in physiologic doses modulates immune function in postmenopausal women.Am J Obstet Gynecol169 :1536 –1539,1993 .[Medline]
  16. Araneo BA, Dowell T, Diegel M, Daynes RA: Dihydrotestosterone exerts a depressive influence on the production of interleukin-4 (IL-4), IL-5, and {gamma}-interferon, but not IL-2 by activated murine T cells.Blood78 :688 –699,1991 .[Abstract/Free Full Text]
  17. Padgett DA, Loria RM: In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol.J Immunol153 :1544 –1452,1994 .[Abstract]
  18. Loria RM, Padgett DA, Huynh PN: Regulation of the immune response by dehydroepiandrosterone and its metabolites.J Endocrinol150 :S209 –S220,1996 .[Medline]
  19. Ben-Nathan D, Padgett DA, Loria RM: Androstenediol and dehydroepiandrosterone protects mice against lethal bacterial infections and lipopolysaccharide toxicity.J Med Microbiol48 :425 –431,1999 .[Medline]
  20. Mooradian AD, Morley JE, Korenman SG: Biological actions of androgens.Endocr Rev8 :1 –28,1987 .[Medline]
  21. Mean F, Pellaton M, Magrini G: Study on the binding of dihydrotestosterone, testosterone and oestradiol with sex hormone binding globulin.Clin Chim Acta80 :171 –180,1977 .[Medline]
  22. Sultan C, Terraza A, DeVillier C, Carilla E, Briley M, Lorre C, DeScomps B: Inhibition of androgen metabolism and binding by a liposterolic extract of "Serona repens B" in human foreskin fibroblasts.J Steroid Biochem20 :515 –519,1984 .[Medline]
  23. Kellis JT, Vivkery LE: Inhibition of human estrogen synthetase (aromatase) by flavones.Science225 :1032 –1034,1984 .[Abstract/Free Full Text]
  24. Bradlow HL, Sepkovic DW, Telang NT, Osborne MP: Indole-3-carbinol. A novel approach to breast cancer prevention.Ann NY Acad Sci768 :180 –200,1995 .[Medline]
  25. Moynihan JA, Callahan TA, Kelley SP, Campbell LM: Adrenal hormone modulation of type 1 and type 2 cytokine production by spleen cells: dexamethasone and dehydroepiandrosterone suppresses interleukin-2, interleukin-4, and interferon-gamma production in vitro.Cell Immunol184 :58 –64,1998 .[Medline]
  26. Meng Q, Yuan F, Goldberg ID, Rosen EM, Auborn K, Fan S: Indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells.J Nutr130 :2927 –2931,2000 .[Abstract/Free Full Text]
  27. Chinni SR, Li Y, Upadhyay S, Koppolu PK, Sarkar FH: Indole-3-carbinol induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells.Oncogene24 :2927 –2936,2001 .
  28. Exon JH, South EH, Magnuson BA, Hendrix K: Effects of indole-3-carbinol on immune responses, aberrant crypt foci, and colonic crypt cell proliferation in rats.J Toxicol Environ Health62 :561 –573,2001 .
  29. Habtemariam S: Flavonoids as inhibitors or enhancers of the cytotoxicity of tumor necrosis factor-alpha in L929 tumor cells.J Nat Prod60 :775 –778,1997 .[Medline]
  30. Plosker GL, Brogden RN: Serenoa repens (Permixon). A review of its pharmacology and therapeutic efficacy in benign prostatic hyperplasia.Drugs Aging9 :379 –395,1996 .[Medline]
  31. Dyner TS, Lang W, Geaga J: An open-label dose-escalation trial of oral DHEA tolerance and pharmacokinetics in patients with HIV disease.J AIDS6 :459 –465,1993 .
  32. van-Weerden WM, Bierings HG, van-Steenbrugge GJ: Adrenal glands of mouse and rat do not synthesize androgensLife Sci50 :857 –861,1992 .[Medline]
  33. Ben-Yehuda A, Danenberg HD, Zakay-Rones Z, Gross DJ, Friedman G: The influence of sequential annual vaccination and of DHEA administration on the efficacy of the immune response to influenza vaccine in the elderly.Mech Ageing Dev102 :299 –306,1998 .[Medline]
  34. Brown GA, Vukovich MD, Martini ER, Kohut ML, Franke WD, Jackson DA, King DS: Effects of androstenedione-herbal supplement on serum sex hormone concentrations in 30–59 year old men.Int J Vitam Nutr Res71 :27 –31,2001 .
  35. Sirrs SM, Bebb RA: DHEA:panacea or snake oil?Can Fam Physician45 :1723 –1728,1999 .[Medline]
  36. Brown GA, Vukovich MD, Martini ER, Kohut ML, Franke WD, Jackson DA, King DS: Endocrine responses to chronic androstendione intake in 30–56 year old men.J Clin Endocrinol Metab85 :4074 –4080,2000 .[Abstract/Free Full Text]




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