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Endocrine Care |
Department of Growth and Development (A.J.K.), and the Department of Medicine and Physiology and Division of Endocrinology at the VA Medical Center (B.H.), University of California at San Francisco, California 94143-0438
Address all correspondence and requests for reprints to: Dr. Arnold Kahn, Department of Growth and Development, University of California-San Francisco School of Dentistry, 707 Parnassus Avenue, D-1018, San Francisco, California 94143-0438. E-mail: . olbones{at}itsa.ucsf.edu
Abstract
In the present placebo-controlled, double-blind study, we assessed the effect of dehydroepiandrosterone (DHEA) supplementation (90 mg orally/d) on bone turnover in 43 healthy men, 56–80 yr old. Placebo or steroid was given for 6 months, followed by a 1-month washout period and then a further 6 months of the opposite agent. Serum samples were collected at baseline 3, 6, 7, and 13 months and assayed for procollagen peptide, bone-specific alkaline phosphatase, and osteocalcin, all markers of bone formation. Measurements were also made of serum cortisol, DHEA/DHEA-S, E2 and free and total T. First void, fasting urine was collected at baseline, 6, 7, and 13 months and assessed for deoxypyridinoline, a marker of bone resorption. Mean serum DHEA and DHEA-S levels in treated men were increased approximately 3-fold (
2.2 ng/ml to 6 ng/ml) and 4.5-fold (1000 ng/ml to 4500 ng/ml), respectively, after 6 months and returned to baseline after washout. Similarly, circulating E2 concentrations were also increased 1.4-fold (from 16–23 pg/ml; P < 0.001), a finding not observed with any other measured hormone. Bone marker levels remained remarkably constant at each sampling interval; procollagen peptide at approximately 8.0 ng/ml; bone-specific alkaline phosphatase at approximately 21.0 U/liter; deoxypyridinoline at 4.5 nmol/mmol Cr. Osteocalcin showed a transient reduction from approximately 10.2- 6.2 ng/ml, P < 0.005 to P < 0.001, at 3 months, but this decline was observed in both treated and controls. Stratifying the marker levels by age or baseline DHEA/DHEA-S levels did not affect the findings. We conclude that oral DHEA does not affect bone turnover in middle-aged to elderly men when used for a 6-month period at doses targeted to restore circulating levels of the steroid to that seen in young adults.
OSTEOPOROSIS IS AN increasingly common, worldwide medical problem characterized by the loss of bone mass and load-bearing architecture resulting in an increased susceptibility to fracture (1, 2). While immobility and pain are among the more common consequences of fracture, death within 6 months also occurs in about 12–20% of hip fracture cases (1, 3, 4). Osteoporosis affects millions of late middle-aged to elderly individuals of both sexes but is more common in women than men (1, 2). In women, a major predisposing factor is the loss of estrogen, typically from natural menopause (1, 5). Circulating estrogen levels may also play a critical role in men (6, 7). For both sexes, however, the single, most important, shared risk factor is advanced age (1, 2, 8). While a number of drugs have been widely available in the clinical management of postmenopausal, osteoporotic women (1), only relatively recently have treatment protocols been assessed for osteoporotic men (9, 10). Indeed, the U.S. Food and Drug Administration has now approved the antiresportive agent Alendronate (Fosamax, Merck \|[amp ]\| Co. Inc., Whitehorse, NJ) (9) for use in men.
Dehydroepiandrosterone (DHEA) and its sulfated derivative, DHEA-S, are adrenal steroids that achieve peak circulating levels in young adults and decline thereafter reaching an average of approximately 10% of maximal by ages 85 and older (11, 12). Because of this decline with age and the implicated spectrum of action of this hormone, it has been suggested that it is the relative absence of DHEA and DHEA-S that is responsible for many of the senescent changes seen in the elderly. For example, lower DHEA/DHEA-S levels have been associated with reduced immune function and an increased incidence of cardiovascular disease, diabetes mellitus, and dementia (13, 14, 15). In bone, a correlation between DHEA(S) levels and bone mass or bone mineral content in older women has been reported by some (lower hormone levels equating with less bone) (16, 17, 18, 19, 20, 21) but not all investigators (22, 23, 24). It has also been reported that topical application of a 10% DHEA cream increases bone mineral density in the hips of postmenopausal women and elicits changes in several of the important biochemical markers of bone remodeling (25). Some of these changes occurred as early as 3–6 months after DHEA treatment was started.
In the present study, we tested the hypothesis that DHEA given orally at a dose sufficient to restore its circulating levels to those seen in young adults would stimulate bone formation and reduce bone resorption in middle-aged to elderly men. If the results support the hypothesis, they would help establish this readily available steroid as a candidate drug for treating osteoporosis in men.
Materials and Methods
Study population
A total of 508 healthy elderly male subjects were screened from the local community. Sixty-one subjects were enrolled, and 43 subjects completed the study protocol (average age 66 yr, range 56–80 yr). Apparent side effects (insomnia, depression) were among the reasons that a few of the subjects withdrew. Inclusion criteria included baseline DHEA serum levels that were less than the 50th percentile DHEA level for their gender and age group, English as a primary language, Mini Mental Status Exam score of 28 (i.e. normal, nondemented range), and capability to reliably take medication on their own. Exclusion criteria included: abnormal age-adjusted prostate-specific antigen levels during the year before entering the study; prostatic cancer or malignant melanoma; significant history of seizure disorder, stroke, focal brain lesions; Parkinson’s Disease; significant head trauma with sustained loss of consciousness; uncorrected thyroid disorder; vitamin B12 or folate deficiency; uncontrolled diabetes mellitus; or significant drug or alcohol (>14 drinks/week) abuse. Subjects were also excluded if receiving medications such as steroids (e.g. prednisone, pregnenolone, beclamethasone, dexamethasone, estrogen, progesterone, T), antiresorptive medications, anticonvulsants, antidepressants, mood stabilizers, or antipsychotics. In addition, men were excluded if they were taking Gingko, vitamin C (if >600 mg/d), Saw Palmetto, Lipoic Acid, St. John’s Wort, vitamin E (if >500 IU/day), or antihistamine within 24 h of a study visit. The study was performed at the General Clinical Research Center (GCRC), under a protocol approved for use by the Committee on Human Research, University of California, San Francisco. Informed consent was obtained from each subject.
Study design
To determine the effects of DHEA on bone turnover, we conducted a randomized, double-blind, controlled cross-over trial of DHEA vs. placebo administration. Randomization was done in groups of ten, with half of each group randomly assigned to placebo or DHEA using a random numbers chart. Half of the subjects were given DHEA for 6 months, followed by a 1-month-washout period where no medication was given. These subjects were then switched to placebo for 6 months. The other half of the study subjects were first given placebo followed by a 1-month-washout and were then treated for 6 months with DHEA.
The dose of DHEA (90 mg/d given as two equally divided doses of 45 mg each) was chosen to yield circulating DHEA(-S) levels in elderly men in the mid-to-high physiological range for healthy young men (26, 27). Subjects reported to the GCRC on d 1, 90, and 180 during each of the two 6-month periods that they were taking DHEA or placebo. On d 1, the men received their first dose of DHEA or placebo and were given the remainder of their medication to take at home on a twice-daily basis. After an overnight fast, at 0830 h on each study day (baseline, 3 months, 6 months, 7 [washout], 10 and 13 months [cross-over]), blood was drawn from an arm vein for determination of: serum bone-specific alkaline phosphatase activity (BSAP), osteocalcin (OC), procollagen peptide (PCP), DHEA, DHEA-S, prostate-specific antigen, T, free T, E2, and cortisol. Overnight urine was collected for determination of urinary creatine and deoxypyridinoline (DPD). All samples were stored at -80 C until processed.
Laboratory methods
The Drug Product Services Laboratory of the Department of Clinical Pharmacy, University of California, San Francisco, prepared DHEA and placebo for subject use. The DHEA was purchased from Spectrum Quality Products (Gardena, CA) and was certified to be 99.58% pure.
Serum concentrations of BSAP, OC, PCP, and DPD were measured in duplicate using commercial assay kits (Alkphase-B, Novocalcin, Prolagen-C and Pryilinks-D, respectively) from Metra Biosystems, Inc. (Mountain View, CA). Urinary creatinine was measured by autoanalyzer. To minimize the risk of sample degradation during storage and the effects of interassay variation, serum and urine samples were stored for no more than 7 months before assay. In addition, samples from a given subject were included in one of two groups for analysis (group 1: baseline, 3 and 6 months; group 2: 7, 10, and 13 months) and assayed together.
The Endocrine Research Laboratory at the Harbor-UCLA Medical Center performed all other hormone assays. All hormones were measured in duplicate. Serum DHEA was measured by a direct assay without extraction with reagents from Diagnostic Systems Laboratories, Inc. (Webster, TX). Cross-reactivities of the DHEA antibody are 0.02% for DHEA-S, 0.7% for isoandrosterone, 0.5% for androstendione, and <0.01% for all other steroids tested. The lower limit of quantitation of serum DHEA is 0.02 ng/ml. All values below this value are reported as 0.02 ng/ml. Intra and interassay coefficients of variation for DHEA are 3.9 and 9.9%, respectively, for the normal adult male range (1.4–12.5 ng/ml).
Serum DHEA-sulfate (DHEA-S) was measured by a direct assay without extraction with reagents from DSL. Cross reactivities of the DHEA-S antibody are 41% for DHEA, 7.3% for androsterone, 2.9% for androstendione, 0.8% dihydrotestosterone, 0.3% for T, 0.2% for progesterone, 0.2% for estrone, and <0.01% for all other steroids tested. The lower limit of quantitation of the serum concentration of DHEA-S was 34 ng/ml. All values below this value are reported as 34 ng/ml. Intra and interassay coefficients of variation for DHEA-S are 7.8 and 9.9%, respectively, for the normal adult male range (1280–6050 ng/ml).
Serum T was measured after extraction with ethylacetate and hexane by a specific RIA using reagents from ICN Biomedicals (Costa Mesa, CA). Cross reactivities of the antiserum used in the T RIA were 2.0% for DHT, 2.3% for androstenedione, 0.8% for 3-androstanediol, 0.6% for etiocholanolone and <0.01% for all other steroids tested. The lower limit of quantitation of serum T measured by this assay is 25 ng/dl. The mean recovery of the T assay, determined by spiking steroid free serum with varying amounts of T (25–1500 ng/ml), is 104% (range 92–117%). Intra and interassay coefficients of variation for T are 7.3 and 11.1% for the normal adult male range which in our laboratory is 298 to 1043 ng/dl.
Serum free T was measured using overnight equilibrium dialysis. Percent free T was calculated as the labeled T in the dialysate/labeled T in the serum inside the dialysis cell. From the serum total T concentration and the % free T, the free T concentration was calculated and expressed in ng/dl. The lower limit of quantitation of serum free T, using this method, is estimated to be 0.1%. Intra and interassay coefficients of variation for free T are 6.2 and 16.4% for the normal adult male range (percent free T:0.86% to 1.91%, free T concentration 5.2–17.8 ng/dl).
Serum E2 was measured by a direct assay without extraction with reagents from DSL. Cross-reactivities of the E2 antibody are 6.9% for estrone, 0.4% for equilenin, and <0.01% for all other steroids tested. The lower limit of quantitation for E2 was 4.9 pg/ml. All values below this value are reported as 4.9 pg/ml. The recovery of E2 was assessed by spiking steroid free serum with increasing amounts of E2 (4.9–74.6 pg/ml). The mean recovery of E2 compared with the amount added was 99.1% (range 95–101%). Intraassay and interassay coefficients of variation for E2 are 3.9 and 9.4%, respectively, for the normal adult male range (17.1–46.1 pg/ml).
Serum cortisol was measured by RIA using anti cortisol antibody and I125 labeled trace from ICN Biomedicals. Cross reactivities of the cortisol antibody are 31.7% for corticosterone, 16.3% for 11-deoxycortisol, 20% for 21-deoxycortisol, 42% for prednisolone, 1.3% for progesterone, and <1.0% for all other steroids tested. The lower limit of quantitation of the cortisol assay is 1.0 µg/dl. All values below this value are reported as 1.0 µg/dl. The recovery of cortisol assay was assessed by spiking cortisol-free plasma with increasing amounts of cortisol from 1–50 µg/dl. The mean recovery of cortisol compared with the amount added was 100.5% (range 95.9–110%). Intra and interassay coefficients of variation for cortisol are 4.7 and 10.4%, respectively, for the adult male range (7–22 µg/dl for AM samples and 1.5–15 µg/dl for PM samples).
Data analysis
Data from subjects who completed the study (43 of 61) were used in the present analysis and are presented as the mean ± SD. Statistical significance between treated and control measurements was assessed using one-way repeated measures ANOVA. The relationships between age and serum variables were determined using linear regression analysis and are reported with the 5 and 95% confidence limits. All these analyses were performed using SigmStat (Jandel Scientific, San Rafael, CA).
Results
The demographic characteristics of the study cohort are shown in Table 1
. This profile indicates that the members of the study cohort are of average size but above average educational level (5 yr beyond high school). The majority of men are in stable relationships, consume moderate amounts of alcohol (if they drink at all), and are currently nonsmokers.
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[DHEA] and 2 [DHEA-S]). Daily supplementation with DHEA significantly (P < 0.001) increased the circulating levels of both DHEA and DHEA-S, reaching levels approximately 2- to 3-fold and 4- to 5-fold higher, respectively, after 6 months of treatment (Table 2). These elevated values returned to normal after a 1-month washout period. No change was seen in the placebo group at any sampling interval (Table 2). Supplementation with DHEA also elevated serum E2 levels by approximately 20% (P < 0.001) (Table 2). This stimulatory effect of DHEA was not observed, however, with free or total T or cortisol. Table 2, which shows the values for total T at various sampling intervals, is representative of our findings (other data not shown).
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). Throughout the trial, the values remained at approximately 8 ng/ml for PCP, at approximately 21 U/liter for BSAP, and approximately 4.5 nmol/mmol Cr for DPD. The only puzzling finding involved serum osteocalcin (OC), which in both DHEA and placebo-treated subjects showed a statistically significant decline of approximately 40% at the 6-month sampling interval. By 6 months, however, OC levels returned to levels indistinguishable from baseline (Table 3).
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If DHEA is a positive, regulatory factor in bone accretion and/or retention, it should be possible to demonstrate such action in studies involving DHEA supplementation or replacement. Indeed, in earlier work, it was found that DHEA introduced through the diet, by sc pellet or by injection, reduced the osteopenia induced in both ovariectomized and cancer-bearing rats (21, 28, 29). Moreover, Labrie et al. (25) reported that the topical application of a 10% DHEA cream to osteoporotic women elicited changes in some of the biochemical markers of bone remodeling within 3–6 months and increased bone mass in the hip within 6 months. Although the number of subjects involved in this latter study was small (n = 14), these data provided early, direct evidence that DHEA might be a useful drug in the treatment of osteoporosis.
Given these previous findings, we were surprised that DHEA supplementation apparently did not alter bone turnover in healthy middle-aged to elderly men. This absence of response occurred despite clear evidence that treatment with DHEA did significantly and substantially increase serum DHEA and DHEA-S levels, as well as elicit a moderate but statistically significant increase in serum E2. Indeed, the augmented levels of DHEA-S seen in response to supplementation are in good general agreement with those previously reported for younger men, approximately 3,300 ng/ml vs. 4,600 ng/ml (11). Thus, with the exception of an as yet unexplained and transient diminution in level of osteocalcin at three months in both DHEA and placebo groups, the levels of all of the other markers of turnover remained remarkably constant for each individual (and for the cohort) throughout the 13-month trial. Stratifying the data by age and baseline levels of DHEA did not affect the findings or provide any additional insight. Our results, therefore, are in agreement with the recent reports of Baulieu et al. (30) and Arlt et al. (31). Baulieu et al. (30) found that older men receiving daily supplementation of DHEA (50 mg/d for 1 yr ) showed no change in two biochemical markers of bone turnover (osteocalcin, C-terminal peptide of type I collagen) or in bone density. Similarly, Arlt et al. (31) failed to find changes in serum osteocalcin and urinary pypridinoline/DPD in older men given 50 mg/d of DHEA for 4 months. Importantly, markers of bone turnover are lower in normal older than younger men (32, 33), so that successful treatment with DHEA should (and could) have been reflected in these measurements.
In contrast to these negative findings, there are other circumstances where supplementation with the steroid elicits a clear response (reviewed by Svec and Porter; Ref. 34). Thus, in women, Baulieu et al. (30) not only found that oral DHEA increased bone mass but also improved libido and skin health in older women; results that are in keeping with the earlier report of Labrie et al. (25) on postmenopausal women and of Gordon et al. (33) on young women with anorexia nervosa. In addition, Morales et al. (26) observed increases in serum IGF-I in both middle-aged men and women given 100 mg/d DHEA for 6 months and evidence for positive change in fat body mass and muscle strength in men. However, these investigators (26) saw no change in bone mineral density or urinary collagen cross-links in either treated men or women. Finally, in what is, perhaps, the clearest circumstance justifying the possible clinical use of DHEA, van Vollenhoven and his colleagues (36, 37) showed that treatment with this steroid significantly improved the condition of patients with mild to moderate systemic lupus erythematosus. The DHEA was well tolerated, with only one common side effect, acne.
Even though the present study provides no evidence that DHEA given alone and for a relatively short period of time is likely to be of benefit in treating osteoporosis in healthy older men, the hormone may yet prove to be valuable under some circumstances. For example, a number of recent studies in men indicate that E2, rather than T, is positively associated with bone mineral density (6), a reduced risk of vertebral fracture (38) and lower levels of bone resorption (39). Because both we and Baulieu et al. (30) have shown that DHEA raises the level of E2 in male subjects, albeit modestly, it remains possible that by increasing the dose of DHEA and/or duration of supplementation with the steroid, beneficial indirect effects on the skeleton might occur. In addition, as suggested by the in vitro findings of Scheven and Milne, (40) DHEA may have to be administered together with another therapeutic agent, like vitamin D3, to be effective. Finally, given the ability of DHEA to modulate the action of cortisol (41) particularly in neural tissue (42), the hormone may prove useful as a countermeasure to control the loss of bone associated with glucocorticoid-induced osteopenia (43).
Acknowledgments
Footnotes
This research was supported in part by the University of California-San Francisco Academic Senate Grant (to A.J.K.), the Merit Review Program of the Veterans Administration (to B.H.) and the Ellison Medical Research Foundation (to Dr. Louann Brizendine, PI). These studies were conducted, in part, at the General Clinical Research Center, Moffit Hospital, UCSF with funds provided by the National Center for Research Resources, 5 Mo1 RR-00079, USPHS.
Abbreviations: BSAP, Bone-specific alkaline phosphatase activity; DHEA, dehydroepiandrosterone; DHEA-S, sulfated derivative of DHEA; DPD, deoxypyridinoline; OC, osteocalcin; PCP, procollagen peptide.
Received June 12, 2001.
Accepted January 4, 2002.
References
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