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Serum Estradiol, Testosterone, and Sex Hormone-Binding Globulin as Regulators of Peak Bone Mass and Bone Turnover Rate in Young Finnish Men

Division of Endocrinology (V.-V.V., M.J.V.), Department of Medicine, and Department of Clinical Chemistry (H.A., U.-H.S.), Helsinki University Central Hospital, FIN-00290 Helsinki; and Department of Biotechnology (K.K.I., K.P.), and Department of Statistics (E.L.), University of Turku, FIN-20014 Turku, Finland

Address all correspondence and requests for reprints to: Matti Välimäki, M.D., Ph.D., Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland. E-mail: matti.valimaki{at}hus.fi.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To study the role of serum testosterone (T), estradiol (E₂), and SHBG as regulators of peak bone mass and bone turnover rate in males, a cross-sectional study with data on lifestyle factors collected retrospectively was performed in 204 young Finnish men, 18.3–20.6 yr old. One hundred fifty-four men were recruits of the Finnish Army, and 50 were men of similar age who had postponed their military service for reasons not related to health. Bone mineral content, density, and scan area were measured in lumbar spine and upper femur by dual-energy x-ray absorptiometry. Blood was sampled for determination of serum total and free T, total and free E₂, SHBG, type I procollagen aminoterminal propeptide (PINP), total osteocalcin (TOC) and carboxylated osteocalcin (COC), and tartrate-resistant acid phosphatase 5b (TRACP5b); and urine was collected for determination of type I collagen aminoterminal telopeptide (NTX). Serum sex steroid concentrations did not associate with bone mineral content, scan area, or bone mineral density, adjusted for anthropometric and lifestyle factors at any measurement site. Instead, serum total (r = 0.23; P = 0.008) and free (r = 0.15; P = 0.023) T were positive predictors of serum TOC, whereas serum free E₂ correlated inversely with serum PINP (r = –0.20; P = 0.0039), TOC (r = –0.12; P = 0.086), COC (r = –0.14; P = 0.036), and urinary NTX (r = –0.15; P = 0.041). Interestingly, serum SHBG correlated positively with all the bone markers studied, the correlation coefficients being 0.18 for serum PINP (P = 0.012), 0.24 for TOC (P = 0.0006), 0.24 for COC (P = 0.0005), 0.27 for serum TRACP5b (P < 0.0001), and 0.21 for urine NTX (P = 0.0031). Serum SHBG was also a positive predictor of serum 25-hydroxyvitamin-D level (r = 0.20; P = 0.0036). The correlations of SHBG persisted after adjusting for weight, free E₂, and free T. We conclude that single measurements of serum E₂ and T were not determinants of peak bone mass in this population of young men. However, E₂ and T contributed to bone turnover rate, with serum T increasing bone formation, and serum E₂ suppressing both bone formation and resorption. Moreover, serum SHBG appeared to be an independent positive predictor of bone turnover rate, which also positively associated with serum 25-hydroxyvitamin-D levels.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
RECENT EVIDENCE FROM a male with a disruptive mutation of the estrogen (E) receptor gene and E resistance (1) and from men with aromatase deficiency and low serum estradiol (E₂) levels (2, 3, 4) has established that Es are essential for the normal growth and maturation of the male skeleton. These individuals had unfused epiphyses, osteopenia, and elevated markers of bone turnover (1, 2, 3, 4), which abnormalities were reversed by E treatment in aromatase-deficient men (3, 4, 5). In elderly men, several cross-sectional studies have, in general, demonstrated significant relationships between serum E levels and bone mineral density (BMD) (6, 7, 8, 9, 10, 11, 12). A recent interventional study showed that E played a dominant role in regulating bone resorption in elderly men, but testosterone (T) contributed to the maintenance of bone formation together with E (13). All these studies left open the question of whether the main effect of sex steroids is in the achievement of peak bone mass in childhood and adolescence or in age-related bone loss after completion of the growth. To our knowledge, hitherto only two studies have addressed the relationship between sex steroids and peak bone mass in young men (14, 15). In men of age 22–39 yr, the rate of increase in BMD at the forearm sites, but not at the hip and the spine, was significantly correlated to serum total and bioavailable E₂ but not with total or bioavailable T levels (14). Among 90 Caucasian boys with a mean age of 16.9 yr, the levels of serum E₂ did not predict BMD at the spine and the hip or in the total body (15). Moreover, in boys of age 9–10 yr, BMD was not correlated with E₂ levels (16). In previous studies, SHBG has been considered as a determinant of serum bioavailable E₂ and T only; its independent role in the regulation of bone turnover rate has not been evaluated.

The present study was undertaken to investigate the role of serum sex steroids as a determinant of peak bone mass and bone turnover rate among young Finnish men. In terms of age, our study population was very uniform and consisted of 204 men, 18.3–20.6 yr old, at which age peak bone mass mostly has been achieved (17). We carefully recorded the lifestyle of the participants and adjusted possible relationships between the hormones and peak bone mass with respect to these confounding factors. Special attention was paid to studying the independent role of SHBG as a regulator of bone turnover rate.

	Subjects and Methods

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Subjects

The study population comprised 204 young men, 18.3–20.6 yr old. They were participants in a study aimed at elucidating the role of genes, hormones, and lifestyle factors as determinants of peak bone mass, and studying exercise-induced changes in bone mass during military service. One hundred fifty-four men were recruits of the Finnish Army; and 50 men of similar age, who had postponed their military service for reasons unrelated to health, formed a control group in the exercise part of the study. For the purpose of the present study, the two groups were combined. The study was approved by the Ethical Committee of Department of Medicine, Helsinki University Central Hospital, and a written consent was obtained from the participants.

Study design

Both groups were examined at the same time, in the very beginning of the military service of the recruits. Bone mineral content (BMC) and BMD were measured, and blood was sampled in the morning, before 1000 h, for determination of serum T, E₂, SBHG, type I procollagen aminoterminal propeptide (PINP), total osteocalcin (TOC), carboxylated osteocalcin (COC), tartrate-resistant acid phosphatase 5b (TRACP5b), and 25-hydroxyvitamin-D (25-OHD). Second-void urine samples were collected for the determination of type I collagen aminoterminal telopeptide (NTX). The samples were stored at –70 C until assayed.

Lifestyle

Current exercise, smoking, calcium intake, and alcohol consumption were recorded using a questionnaire. The participation in various physical activities over the last year was recorded by questions addressing 30 different types of physical activity and training, which were weighted according to their bone loading effect. Light or non-weight-bearing activities such as walking, cycling, and swimming scored 1, activities producing repetitive weight-bearing impact (e.g. endurance running and skiing) 1.5, and high-impact or high-magnitude loading (e.g. jumping, sprint running, and weight-lifting) 2. For each type of exercise practiced, the subjects were asked to indicate the average number of occasions per month and the duration (minutes) and intensity (1 = light, 2 = moderate, 3 = heavy) of each occasion. An exercise index was then calculated by summing up the products of frequency, duration, intensity, and bone loading effect of different activities in summer and wintertime. Calcium intake was calculated on the basis of the supply from dairy products only, by using the following estimations: 1 dl milk, sour milk, and yogurt contains 120 mg calcium, and one slice of cheese contains 100 mg. In the calculations, 1.8 dl was used as the volume of one glass.

Bone mass measurements

BMC and BMD of the lumbar spine and three femoral sites (femoral neck, trochanter, and total hip) were measured by dual-energy x-ray absorptiometry using a Lunar Corp. (Madison, WI) Prodigy densitometer. The scan areas of the lumbar spine and the femoral neck were taken also into account in the calculations.

Biochemical measurements

T and E₂ were quantitated with time-resolved fluoroimmunoassays (AutoDELFIA, Wallac, Turku, Finland). The detection limit of the T assay was 0.3 nmol/liter. The intraassay coefficient of variation (CV) was 4% at the level of 3.5 nmol/liter and 4% at the level of 21 nmol/liter. The corresponding interassay CVs were 7% and 5%. The cross-reaction with 5-dihydrotestosterone was 27%. In the E₂ assay, 150 µl serum was extracted with a 16-fold volume of diethyl ether before assay. The dried residue was solubilized in 75 µl assay buffer. The detection limit of the E₂ assay was 20 pmol/liter. The intraassay CVs were 20, 5.8, and 4.0% at the levels of 30, 71, and 118 pmol/liter, respectively. The interassay CVs were 13.0% at the level of 106 pmol/liter and 10.8% at the level of 289 pmol/liter. SHBG was quantitated with a time-resolved immunofluorometric assay (AutoDELFIA). Samples were prediluted 1:100. The detection limit of the assay was 0.5 nmol/liter. The intraassay CV was 1.4% at the level of 20 nmol/liter and 1.8% at the level of 130 nmol/liter. The corresponding interassay CVs were 8.2 and 10.1%. Free T was calculated from the concentrations of T and SHBG by the method of Anderson et al. (18), and free E₂ by the method of Södergård et al. (19).

Urinary NTX was measured with NTX Reagent kits (Vitros Immunodiagnostics Products, Ortho-Clinical Diagnostics, Amersham, UK). The intraassay CV was 8.0% at the level of 55 nM bone collagen equivalents (BCE)/liter and 3.0% at the level of 187 nM BCE/liter. The interassay CV was 9.8% at the level of 60 nM BCE/liter and 5.3% at the level of 403 nM BCE/liter. The NTX values were corrected for creatinine excretion measured by a standard laboratory method. Serum intact PINP was determined by a competitive RIA with a commercial kit (Intact PINP RIA) from Orion Diagnostica (Oulunsalo, Finland). Analytical sensitivity of this assay was 2 µg/liter, and the intra- and interassay CVs ranged from 2–6%. Serum TOC and COC were measured with in-house immunoassays as previously reported (20). The intraassay CV was less than 5% and the interassay CV less than 8% for both assays. Serum TRACP5b activity was measured by an immunoextraction method with BoneTRAP reagents from Suomen Bioanalytiikka Oy (Turku, Finland) (21). The analytical sensitivity of this assay was 0.1 U/liter, and the intra- and interassay CVs were 6% or less at relevant concentrations. Serum 25-OHD was measured by RIA kits from DiaSorin, Inc. (Stillwater, MN). At vitamin D levels of 30 nmol/liter and 100 nmol/liter, the intraassay CVs were 8.9% and 5.9%, and the interassay CVs were 12.8% and 8.8%, respectively.

Statistics

The relationships between BMC, scan area, BMD, and serum sex hormone concentrations were examined by multiple regression models, which included age, height, weight, smoking, exercise, alcohol consumption, and calcium intake as covariants. Pearson correlation coefficients were calculated between serum sex hormone concentrations and bone turnover markers as well as 25-OHD. The relationships between bone turnover markers and SHBG were first studied with Pearson correlation coefficients. Thereafter, multiple regression models were created by adding first weight; then weight and free E₂; and finally weight, free E₂, and free T as adjusting factors. For all bone turnover markers, natural logarithm transformation was used to achieve normality assumption in the models. All tests were performed as two-sided, with a 0.05 significance level. All analyses were done with an SAS System (version 8.02 for Windows; SAS Institute, Cary, NC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The baseline characteristics of the study population are given in Table 1.

In Table 2 lumbar spine and femoral neck BMC, BMD, and the respective scan areas, as well as trochanter and total hip BMC and BMD, adjusted for anthropometric and lifestyle factors, are related to serum sex steroid and SHBG concentrations in a multiple regression model. As can be seen, no significant correlations existed between bone and hormone parameters. To study possible relationships between bone parameters and serum total and free E₂ levels more thoroughly, we divided the men into quartiles of total and free E₂. After the above mentioned adjustments, bone parameters were similar for the quartiles (data not shown). Finally, we studied the Pearson correlation coefficients for the same relationships in the lowest quartiles of total and free E₂, but all of them were insignificant (data not shown).

As presented in Table 3 serum total (r = 0.23; P = 0.008) and free (r = 0.15; P = 0.023) T were positive predictors of serum TOC but not of another marker of bone formation, serum PINP. Serum free E₂ correlated inversely with serum PINP (r = –0.20; P = 0.0039), TOC (r = –0.12; P = 0.086), COC (r = –0.14; P = 0.036), and urinary NTX (r = –0.15; P = 0.041). Similar associations were observed for serum total E₂, but they were statistically less significant. Interestingly, serum SHBG correlated positively and most strongly with all the markers studied, the correlation coefficients being 0.18 for serum PINP (P = 0.012), 0.24 for serum TOC (P = 0.0006), 0.24 for serum COC (P = 0.0005), 0.27 for serum TRACP5b (P < 0.0001), and 0.21 for urine NTX (P = 0.0031). Serum SHBG was also a positive predictor of serum 25-OHD level (r = 0.20; P = 0.0036).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Serum total or bioavailable T and total or bioavailable E₂ did not correlate with peak bone mass of young men in the present study population, which, in terms of age, was very uniform. Limitations in our cross-sectional study were that we measured serum concentrations of sex steroids only once and that we were not able to correlate them to bone gain or loss in a longitudinal study model. It is very evident that a single value for sex steroids is a rough measure of the T or E status of individuals studied, e.g. due to day-to-day variation; however, diurnal variation was carefully taken into account by collecting blood samples by 1000 h. It is also clear that one individual measurement did not describe the total dose of T or E action to which the single man had been exposed during his previous life. Despite these limitations, or just due to them, our results corroborated those on the nonassociation of a single serum E₂ measurement with bone mass in young boys of age 9–10 yr in the study of Klein et al. (16) or in older boys with a mean age of 16.9 yr in the study by Lorentzon et al. (15). In contrast, Khosla et al. (14) found that in 22- to 39-yr-old men, the rate of increase in BMD over 4 yr, at the forearm sites, was significantly and positively correlated to serum total and bioavailable E₂ and estrone levels but not with total or bioavailable T. However, there existed no correlation to changes in spine and hip BMD, and the baseline correlations at the beginning of follow-up were not reported (14). As in elderly men, who seem to have a threshold concentration for serum E₂ above or below which they are either E sufficient or deficient with respect to suppression of bone resorption by E (14, 22), there may exist a similar level regarding bone mass acquisition by means of E in young men. Indeed, Khosla et al. (14) were able to demonstrate that kind of threshold for serum bioavailable E₂, above and below which men of age 22–39 yr had significantly different rates of change in radius and midlateral spine BMD. Unfortunately, despite detailed analyses, we were not capable of finding that kind of threshold for acquisition of peak bone mass. Possibly most of the present men were above the threshold and thus E sufficient. On the other hand, it is quite possible that sex steroids affect bone gain or loss even after the initial consolidation of the skeleton.

Consistent with previous observations (13, 14), serum total OC levels were positively associated with T levels in the present study, but a similar relationship was not found for another marker of bone formation, serum PINP. PINP is produced by early osteoblasts, and OC is considered as a marker of more differentiated and mature osteoblasts, and this may, in part, explain some differences observed in these two formation markers (23). However, OC and PINP, as well as urinary NTX levels, were negatively associated with E₂ levels, indicating a reduction in bone turnover in men with higher E levels. This finding corroborates that by Khosla et al. (14) in men 22–39, 40–59, or 60–80 yr old. Thus, from early adulthood to late senescence, serum E is an inhibitor of bone turnover in the male skeleton.

Most interestingly, serum SHBG levels correlated positively and statistically most significantly with all the bone markers studied, i.e. serum PINP, TOC, COC, TRACP5b, and urinary NTX levels. Importantly, although lessened, the relationships persisted after adjusting for weight, free E₂, and free T levels. To our knowledge, this is the first report to suggest an independent role for serum SHBG in the regulation of bone turnover in healthy men. Indeed, a recent abstract supported this view by demonstrating higher SHBG levels to be a predictor of lower total hip BMD among 348 men, 23–90 yr old, even after adjusting for bioavailable sex steroids, growth factors, and body mass (24). Moreover, in comparison with controls, serum SHBG levels were significantly higher in men with primary or secondary osteoporosis and correlated positively with bone marker levels but inversely with hip BMD. However, no similar correlations were found for T or E₂ levels (25). Serum SHBG is a determinant of serum free E and T, but more important may be its role at the tissue level in regulating the supply of E and T from the circulation to bone cells (26). On the other hand, because serum SHBG is raised by E and lowered by T, its actual level describes the net effect of these two important regulators of bone metabolism. The seasonal and diurnal variation of serum T is not evident for serum SHBG (27). Accordingly, due to this stability, serum SHBG may be a more reliable measure of the long-term exposure of the male skeleton to sex steroids than their single measurements in serum. Finally, SHBG has been shown to effect cellular function directly by acting through specific membrane receptor (SHBG-R), and this effect may independent of E or T (28). Through this receptor, SHBG specifically inhibits E₂-induced cell proliferation in E-dependent breast cancer (29).

Interestingly, serum SHBG levels were positively associated also with serum 25-OHD levels, even after adjusting for weight, which is known to suppress both SHBG and 25-OHD concentrations in serum (30, 31). The explanation for this association remains open; but in obesity, low 25-OHD-levels have been attributed to the decreased bioavailability of vitamin D from cutaneous and dietary sources because of its deposition in body fat compartments (30). Furthermore, in obesity, hepatic synthesis of 25-OHD may be suppressed by the increased serum 1,25-dihydroxyvitamin-D levels associated with secondary hyperparathyroidism (31). On the other hand, the positive association between SHBG levels and bone turnover rate cannot go through serum 25-OHD levels, because high 25-OHD levels result in low PTH concentrations and, consequently, in low bone turnover rate; in the present study, high SHBG levels were associated with both high 25-OHD and high turnover rates.

In summary, single measurements of serum E₂ and T were not determinants of peak bone mass among young Finnish men but contributed to bone turnover rate, with serum T increasing bone formation, and serum E₂ suppressing both bone formation and resorption. Serum SHBG appeared to be an independent positive predictor of bone turnover rate, which positively correlated also with serum 25-OHD levels.

	Footnotes

 
This work was supported by a grant from the Ministry of Education, Helsinki, Finland; research funding from Helsinki University Central Hospital (Erityisvaltionosuus); and Paulo Foundation, Helsinki, Finland.

Abbreviations: BCE, Bone collagen equivalents; BMC, bone mineral content; BMD, bone mineral density; COC, carboxylated osteocalcin; CV, coefficient of variation; E, estrogen; E₂, estradiol; NTX, type I collagen aminoterminal telopeptide; 25-OHD, 25-hydroxyvitamin-D; PINP, type I procollagen aminoterminal propeptide; T, testosterone; TOC, total osteocalcin; TRACP5b, tartrate-resistant acid phosphatase 5b.

Received December 22, 2003.

Accepted May 7, 2004.

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