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Department of Health Sciences (Q.W., M.A., P.H.F.N., H.S., S.C.), University of Jyväskylä, FIN-40014, Jyväskylä, Finland; Institute of Biomedicine (J.M.H., S.L.A.), Department of Anatomy, University of Turku, FIN-20014, Turku, Finland; Pharmatest Services Ltd. (J.M.H.), FIN-20520, Turku, Finland; Finnish Red Cross (S.L.A.), Blood Service, FIN-00140, Helsinki, Finland; and Division of Endocrinology (C.O.), Department of Internal Medicine, Sahlgrenska University Hospital, 41345 Göteborg, Sweden
Address all correspondence and requests for reprints to: Sulin Cheng, Department of Health Sciences, University of Jyväskylä, P.O. Box 35 (LL), FIN-40014, Jyväskylä, Finland. E-mail: cheng{at}sport.jyu.fi.
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Abstract |
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Objective: This study examines the association of sex hormones and bone markers with bone geometry and density in pubertal girls.
Design: The study was designed as a 2-yr longitudinal study in pubertal girls. Measurements were performed at baseline and at 1- and 2-yr follow-ups.
Setting: The study was conducted in a university laboratory.
Participants: A total of 258 10- to 13-yr-old healthy girls at the baseline participated.
Methods: Peripheral quantitative computed tomography was used to scan the left tibial shaft. Serum 17ß-estradiol (E2), testosterone (T), SHBG, osteocalcin (OC), bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase isoform 5b were assessed. Data were analyzed using hierarchical linear models with random effect.
Results: E2 was a positive predictor for total bone mineral density (BMD), cortical thickness, and a negative predictor for endocortical circumference but had no predictive value for total bone cross-sectional area or periosteal circumference. T was a positive predictor for total cross-sectional area and periosteal circumference as well as endocortical circumference, and a negative predictor for total BMD. OC was negatively correlated with cortical BMD (R2 = 0.325; P < 0.001).
Conclusions: In pubertal girls, E2 and T have different influences on bone properties at the long bone shaft. The results suggest that, at the endocortical surface, E2 inhibits bone resorption during rapid growth, and later, after menarche, acts at higher concentrations to promote bone formation. At the periosteal surface, T promotes bone formation, whereas E2 does not affect it. In addition, OC might be used as a predictor of cortical BMD.
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Introduction |
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In the long bone shaft, it has been generally postulated that androgens promote periosteal bone formation, whereas estrogens inhibit periosteal bone formation as well as endosteal resorption (1). However, such hypotheses have mostly been extrapolated from animal studies and on the basis of sexual dimorphism. In a previous study we reported evidence indicating that 17ß-estradiol (E2) has an inhibitory effect on endocortical resorption, but not on periosteal formation at the tibial shaft in early pubertal girls (2). Testosterone (T) is another important determinant of bone growth, but its effect on bone mass and size is more elusive in pubertal girls than that of E2.
One methodological problem in investigating the association between fluctuating variables, such as serum hormones, and gradually but not linearly growing variables, such as bone mass in children, is the difficulty in adjusting for the confounding influence of growth. In early pubertal girls, older children will have higher serum E2 concentration and bigger bone size than their younger counterparts. Without controlling for growth, a positive correlation between the two variables will undoubtedly be observed. Age, body height and weight, as well as Tanner Stage are the variables that have generally been used in previous studies as covariates. However, because bone mass or size does not increase during growth as a linear function of chronological age, and the bone growth curve differs from that of body height and weight, linearly adjusting for these covariates will distort the real phenomenon.
One solution for this problem is to use longitudinal data and to control for an indicator of growth using a curvilinear model. In pubertal girls, time relative-to-menarche has been used widely as a feasible chronologic indicator of growth. We have examined the growth patterns of bone density and geometry with respect to time relative-to-menarche using hierarchical linear models in our previous report (3). In the current study we introduced sex hormones into these models to advance our understanding of the relationship between sex hormones and bone density/geometry.
Biochemical markers of bone turnover, such as osteocalcin (OC), bone-specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b), are collective indicators of the metabolic activity of the skeleton in the whole body. Their concentrations during puberty are much higher than thereafter (4). However, detailed information about the change in bone biomarkers with respect to time relative-to-menarche during puberty is scarce. Such information may be clinically relevant in terms of interpretation of laboratory tests. Furthermore, the bone markers might be not only indicative of bone turnover, but also related to the bone mass and size, although this has not yet been studied in healthy pubertal girls.
In this study we first examined the changing patterns of E2, T, their binding globulin, and bone biomarkers (OC, BALP, and TRACP 5b) with respect to time relative-to-menarche to give a descriptive picture of these factors’ behavior in pubertal girls. Then the association of sex hormones and bone biomarkers with bone density/geometry was investigated using hierarchical linear models controlling for time relative-to-menarche with curvilinear functions.
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Subjects and Methods |
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The study population has been described previously (3). Briefly, 258 healthy Finnish girls aged 10–13 yr at the time of baseline measurement were enrolled in the study. Sixty and 37 girls were absent for the 1- and 2-yr follow-up measurements, respectively. The main reason for dropout was loss of interest. Menarche date was collected by a questionnaire or retrospective phone call. Eight girls already had menarche at the baseline after initial recruitment. There were 25 girls who had not reached menarche at the time of data analysis, and there were another 26 girls who did not provide information on menarche. As a result, of the 258 girls, 207 were available for inclusion in the data analysis.
Written informed consents for the girls were provided by the girls and their parents in accordance with the ethical committees of University of Jyväskylä, the Central Hospital of Central Finland, and the Finnish National Agency of Medicines.
Laboratory assays
Blood samples were collected in the morning between 0730 and 0900 h after an overnight fasting at the baseline and 1- and 2-yr follow-ups. If the girls began menstruation, the blood samples were collected between 2 and 5 d after menstrual bleeding started. Serum was extracted from blood by centrifugation and stored immediately at –70 C until analyzed. The samples from different time points were analyzed by one technician at the same time.
E2, T, and SHBG were assessed using time-resolved fluoroimmunoassays (Delfia; Wallac Oy, Turku, Finland). The detection limits of the E2 and T assays were 0.05 nmol/liter (13.6 pg/ml) and 0.3 nmol/liter (0.09 ng/ml), respectively. Inter- and intraassay coefficients of variation (CVs) were 5.2 and 5.1% for E2, 9.2 and 9.4% for T, and 1.1 and 1.1% for SHBG, respectively. Free E2 was calculated using the equation: free E2 (pmol/liter) = E2 (pmol/liter)/(K x SHBG (nmol/liter) + 1), and free T using the equation: free T (pmol/liter) = T (nmol/liter) x 1000/(K x SHBG (nmol/liter) + 1), according to Ekins (5). where K represents the equilibrium constant for E2 and T binding to SHBG (0.68 x 109 liters/mol and 1.6 x 109 liters/mol, respectively)
The serum level of intact OC, a bone formation marker, was measured using an in-house immunofluorometric assay as previously described (6). The inter- and intraassay CVs of the assay were less than 8% and less than 5%, respectively. Serum activity of BALP, a bone formation marker, was assessed using competitive enzyme immunoassay (Metra Biosystems, Inc., Mountain View, CA). The inter- and intraassay CVs were less than 5.2% and less than 5.8%, respectively. Serum TRACP 5b (7), a bone resorption marker, was determined using a commercial immunoassay (BoneTRAP; SBA-Sciences, Oulu, Finland). The inter- and intraassay CVs were 5.7 and 2.7%, respectively.
Bone mineral density/geometry
A detailed description for the bone scan has been presented earlier (3). Briefly, a peripheral quantitative computed tomography device (XCT-2000; Stratec Medizintechnik, GmbH, Pforzheim, Germany) was used to scan the tibial shaft at 60% of lower leg length up from the lateral malleolus using the research scan mode. Image processing and calculation of bone parameters were performed using the manufacturer’s software package (version 5.40) and Geanie 2.1 (Bonalyse Oy, Jyväskylä, Finland).
A threshold of 280 mg/cm3 was used to determine the outer bone border. Total cross-sectional area (CSA) was then defined as the area enclosed within the outer bone border. Bone mineral content (BMC) was calculated as the mineral contained within a volume defined by the product of the CSA and 1 mm thickness. Volumetric total bone mineral density (BMD) was then defined as the quotient of BMC on the volume (Fig. 1A
). Hence, total BMD was a measure of bone density at organ rather than tissue level, because the medullary cavity was included in the volume of interest along with the bone tissue itself.
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). Periosteal circumference (PC), endocortical circumference (EC), and cortical thickness were obtained from the Stratec software version 5.4 using the circular ring model in which the bone was assumed to be an ideal cylinder with the same CSA as the real bone. Therefore, the cortical thickness was calculated as (total bone CSA/
)0.5 – [(total bone CSA – cortical bone CSA)/]0.5, PC was calculated as 2(total bone CSA/)0.5, and EC was calculated as 2[(total bone CSA – cortical bone CSA)/]0.5. The cortical proportion was calculated as the ratio of cortical CSA to total CSA. The reproducibility (CV) of these measurements was 1% for CSA and BMC and less than 1% for BMD and cortical thickness. Statistical analysis
All data were checked for normality using Shapiro-Wilk test in SPSS 11.0 for Windows. Because E2 and T were not normally distributed, their natural logarithms were used in further analysis. We first explored the change of E2, T, SHBG, OC, BALP, and TRACP 5b using hierarchical linear model to provide basis for understanding of their association with bone variables in growing subjects. Data were analyzed using MLwiN 1.1 (Multiple Project, Institute of Education, University of London, London, UK).
Second, E2, T and SHBG were introduced into the growth models of bone variables constructed earlier (3) to investigate their predictive value for bone parameters. A representative regression model used to calculate the predictive value of free E2 and free T for a certain bone variable (for instance, CSA) of the ith measurement from the jth individual was expressed as: CSAij = f(time ij) + ß1 x (free E2)ij + ß2 x (free T)ij + ß3 x SHBGij, where "time" denotes time relative-to-menarche, and f(time) denotes the curvilinear function constructed previously (3). The correlations between bone biomarkers and bone variables were calculated using multivariate hierarchical linear models controlling for time relative-to-menarche.
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Results |
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The serum levels of E2 and T as well as free E2 and T increased from early puberty up to menarche. At the same time, the SHBG level declined linearly and then maintained a low level thereafter (Fig. 2). Time relative-to-menarche explained 34.6, 42.1, 38.5, 30.6, and 30.8% of the between-measurement variation of E2, free E2, T, free T, and SHBG, respectively.
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, A and B). TRACP 5b maintained a relatively high level until 1 yr premenarche, and declined in the same manner as OC and ALP thereafter (Fig. 3C). Time relative-to-menarche explained 28.5, 33.0, and 35.2% of the between-measurement variation of OC, BAP, and TRAP 5b, respectively.
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At tibial shaft, free E2 was a positive predictor for cortical CSA, cortical thickness, and cortical proportion as well as total BMD, a negative predictor for EC, but had no predictive value for total bone CSA and PC. In contrast, free T was a positive predictor for total CSA, PC, and EC, a negative predictor for the total BMD and cortical proportion, but no predictive value for cortical thickness, cortical CSA, and cortical BMD. Total T and E2 showed similar correlations with bone variables as their bioactive forms. SHBG was negatively associated with total and cortical CSA, cortical thickness, and cortical proportion as well as PC. The regression coefficients of sex hormones on bone parameters are shown in Table 1.
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BALP and TRACP 5b were not correlated with any of the measured bone variables. However, OC was negatively correlated with total BMD, cortical thickness, and cortical BMD of the tibial shaft (r = –0.29, –0.19, and –0.57, respectively; P < 0.01) (Fig. 4).
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Discussion |
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By adopting the statistical approach used in this study, it was possible to develop flexible models that could account for the underlying effects of growth. Chronological age is not a good indicator of growth status and controlling for it in linear fashion will create artificially a distorted set of residuals for the chosen explanatory variable(s) to account for. Body height and weight are considered to be other necessary covariates, but similar problems exist as for age because the growth pattern of body height is different to that of bone mass (8, 9). Using time relative-to-menarche in a spline polynomial function is an elegant and robust solution to this problem. Another advantage of our study is that longitudinal data make it possible to follow each participant over 2 yr, and by controlling for the growth effect on the bone variables and hormone concentration, the association of sex hormones and bone density and geometry can be investigated reliably.
BALP is thought to have a specific function involved with, and may play an essential role in the initiation of bone mineralization (10). Serum OC is a sensitive and specific marker for osteoblast activity and mineralization, and its serum level reflects the rate of bone formation (11). TRACP 5b is formed in transcytotic vesicles of osteoclasts by cleavage with cathepsin K (12), and secreted into the blood circulation as an index of osteoclast number and bone resorption (13, 14). We found that the serum level of TRACP 5b was sustained up to 1 yr before menarche, unlike the changing patterns of OC and BALP, which increased first then declined. The changing patterns of the two bone formation markers were in agreement with the earlier report by Cadogan et al. (15) using similar study design.
We found that OC, but not BALP or TRACP 5b was negatively associated with cortical BMD. This may be due to differences in the production, distribution, or clearance of these biomarkers. OC and BALP are produced exclusively by osteoblasts in their different stages; BALP is secreted by immature and OC by mature osteoblasts. Their distributions are also different; OC is rich in cortical bone, whereas BALP is present in the growth plate (16). Osteoclasts, the source of TRACP 5b, are predominately present at sites of active bone resorption, such as metaphysis of long bone. Therefore, it is not surprising that OC is more indicative to the properties of the cortex of the long bone shaft than is either BALP or TRACP 5b. Although the physiological function of OC is not yet clear, recent evidence has suggested that some circulating fragments of OC may be released from bone matrix during resorption (17). Ducy et al. (18) have reported that OC-deficient mice demonstrated higher bone mass and improved functional bone quality. Our result is in line with this animal study and provides the first evidence that OC is correlated negatively with cortical BMD in human.
Previously, we found that the medullary cavity of tibial shaft remained unchanged before menarche and decreased after that (3). Putting the results of the current study with the previous observation together, we now have evidence that, at the endocortical surface, E2 inhibits bone resorption during rapid pubertal growth and later (after menarche) the higher concentration of E2 promotes bone formation. Hence E2 is an important determinant of cortical thickness at long bone shaft. This is in agreement with the findings that E2 deficiency is correlated with thin cortex in girls, women, and even men (19, 20). E2 is a vital determinant for the development of reproductive system. With the maturation of the female, it is necessary to develop the ability to store calcium in preparation for the large calcium demand during pregnancy (21). The most efficient way of achieving this might be through inhibiting bone resorption in the prematurational stage.
Our findings do not support the view that E2 inhibits bone formation at the periosteal surface. The sexual dimorphism in bone morphology might be due to the fact that E2 is a major determinant in the fusion of the growth plate (19). As long as the bone elongates, its radial size increases proportionately. Thus, the sexual dimorphism in bone size is more likely due to the combination of earlier fusion of bone growth plate and the weaker proformative effect of androgens on the periosteal surface in girls compared with boys.
The effect of androgens on bone mineralization in pubertal girls is not clear. Androgens may act directly on bone cells after binding to its receptor or by E2 after its aromatization (1). The result that T is a positive predictor for the total CSA as well as PC of tibial shaft is in agreement with the notion that T promotes bone formation at the periosteal surface (1). The oddest finding, that T was a negative predictor for total BMD of the tibial shaft, can be understood within the context of bone mechanical demands. Bone "design" needs to fulfill a tradeoff between two contradictory requirements: withstanding the load applied on it (more bone mass) and facilitating locomotion efficiency (less bone mass). As long as the diaphyseal bone size is enlarged, the bone tissue at the endocortical surface is resorbed and thus, the medullary cavity occupies an increased part of bone. Total BMD is the combination of the density of the three compartments: cortex, subcortex, and medullary cavity in the transverse plane. With increased medullary proportion, it is not surprising that T is negatively associated with total BMD. This finding is in agreement with the observation that the total BMD at long bone shaft measured by peripheral quantitative computed tomography is higher in women than men (22).
The association between SHBG and bone variables might have three alternative explanations. First, SHBG influences bone properties indirectly by modifying the bioavailability and metabolism of sex steroids. Second, SHBG is a more stable indicator for sex hormone exposure than the measurement of sex steroids themselves, because the time-to-time variation of sex steroids is much higher than that of SHBG. With a smaller variation, the association will be easier to detect. Third, SHBG receptor has been found in the cell membrane of some estrogen target cells. It binds to its receptor, after which estrogen is attached to the SHBG-receptor compound to induce antiestrogenic effect through cAMP and PKA pathway (23). However, this phenomenon has not yet been revealed in bone cells.
The serum levels of sex steroids fluctuate diurnally, and one snapshot measurement can not possibly reflect the real hormonal exposure. This limitation makes the calculated predictive value of sex hormones on bone parameters and bone markers much weaker than the expected value. It would be valuable if objective and stable indicators for sex hormone exposure, as the glycosylated hemoglobin used in monitoring long-term blood glucose, could be defined in the future.
We conclude that, in pubertal girls, E2 and T have different influences on bone properties at long bone shaft. At the endocortical surface, it appears that E2 inhibits bone resorption during rapid growth and then acts later, after menarche, at higher concentrations to promote bone formation. At the periosteal surface, T promotes bone formation, whereas E2 does not affect it. In addition, OC might be used as a predictor of cortical BMD.
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Acknowledgments |
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Footnotes |
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First Published Online October 25, 2005
Abbreviations: BALP, Bone-specific alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; CSA, cross-sectional area; CV, coefficient of variation; E2, 17ß-estradiol; EC, endocortical circumference; OC, osteocalcin; PC, periosteal circumference; T, testosterone; TRACP 5b, tartrate-resistant acid phosphatase isoform 5b.
Received July 19, 2005.
Accepted October 13, 2005.
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