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Original Article |
Bone Metabolism Group, University of Sheffield (A.R., R.A.H., D.G., R.E.), Sheffield S5 7AU; and Division of Biomedical Sciences, Sheffield Hallam University (G.S.), Sheffield S1 1WB, United Kingdom
Address all correspondence and requests for reprints to: Dr. Angela Rogers, Clinical Sciences Centre (North), Northern General Hospital, Herries Road, Sheffield S5 7AU, United Kingdom. E-mail: angela.rogers{at}sheffield.ac.uk.
Abstract
Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of NF
B ligand. OPG has been shown to be an important inhibitor of osteoclast differentiation and activation in rodent models. Estrogen is known to suppress bone resorption, and the action of estrogen on bone may be mediated by OPG. The relationship between endogenous estrogen and circulating OPG levels and bone status in human populations is unclear. Thus, the aim of this study was to investigate the relationship between biochemical markers of bone turnover and bone density and circulating OPG and endogenous estradiol levels in a population-based cohort of postmenopausal women.
Subjects were 180 women ages 55–91 yr (mean age, 67 yr). Serum estradiol was measured using an auto-analyzer. Serum concentrations of OPG were determined by ELISA. Markers of bone formation and resorption were measured by standard methods. Bone mineral density at total body, total hip, femoral neck, and lumbar spine was measured by dual energy x-ray absorptiometry.
There was a significant inverse relationship between estradiol and all bone turnover markers (r-values from -0.46 to -0.23; P < 0.05). Serum estradiol was positively related to absolute bone density at all sites and to change in bone density at the hip and femoral neck by univariate analysis (r-values from 0.15–0.29; P < 0.05). We observed a weak inverse association between OPG and serum-based bone turnover markers (r-values -0.18 and -0.16; P < 0.05). There was a significant positive relationship between OPG and bone mineral density at total body, total hip, and femoral neck (r-values from 0.17–0.2; P < 0.05) by univariate analysis, which was lost after adjustment for age and body mass index. There was a significant weak positive relationship between circulating OPG and serum estradiol (r = 0.18; P < 0.02). We observed no significant relationships between OPG and bone turnover markers measured in urine.
We conclude that the variation in circulating endogenous estradiol levels is an important factor contributing to levels of bone turnover and bone density at the menopause. Our observations also suggest that circulating levels of OPG may reflect OPG activity in bone and are related to circulating endogenous levels of estradiol. We have previously reported high levels of variability in urine markers of bone resorption, and we suggest that this could account for the absence of a significant association between these markers and circulating OPG.
IT IS WELL RECOGNIZED that estrogen deficiency is a major contributory factor to postmenopausal osteoporosis. There is a dramatic decrease in the level of circulating estradiol at the menopause in all women, although there remains some considerable variation in residual endogenous estradiol levels. Since the introduction of more sensitive and reproducible assays, it is possible to measure the low levels of circulating 17ß-estradiol found in postmenopausal women. This has resulted in new studies that have examined the association between endogenous estradiol concentrations and bone turnover after menopause. These studies showed that decreased levels of serum estradiol are associated with increased levels of bone turnover markers (1, 2, 3). The amount of variance explained by estradiol in these studies, however, was small (<2%), and the importance of residual endogenous estradiol levels is uncertain.
The mechanism of action of estrogen on bone is still not fully understood. There is evidence to suggest that estrogen may suppress the production of locally produced proinflammatory cytokines (4, 5, 6). Recent studies in vitro have shown that estradiol increases the production of the novel peptide osteoprotegerin (OPG) in human osteoblast-like cells (7). This may be an important signaling pathway for the action of estrogen on bone.
The discovery of OPG in 1997 has enhanced our understanding of the way in which the processes of bone remodeling are regulated (8, 9). In vitro and animal studies have unambiguously revealed the role of OPG as a decoy receptor for receptor activator of NFB ligand (RANKL), neutralizing the effect of RANKL on the differentiation and proliferation of osteoclasts.
The critical importance of this cytokine is observed in OPG knockout mice that develop severe osteoporosis (10) and also in transgenic mice designed to overexpress OPG, which develop osteopetrosis (8, 11).
Since this early work was performed, there have been several studies designed to assess the importance of OPG to the skeleton in human populations. The results of these epidemiology studies have been conflicting. In one study, women with osteoporosis were shown to have higher circulating levels of OPG than controls (13). Another study has shown no difference between serum OPG levels in osteoporotic vs. healthy postmenopausal women (14). OPG has also been administered as a therapeutic agent, resulting in a dramatic reduction in bone turnover state, but little is known of its long-term effect on bone density (15). The relationship between serum concentrations of endogenous OPG and bone turnover is uncertain, with different studies yielding different results. It is unclear whether circulating concentrations of OPG reflect the activity of OPG in the bone microenvironment. A relationship between circulating OPG and estradiol in vivo in humans has yet to be established.
In this study, we examine the association between circulating levels of OPG and estradiol, bone turnover, bone density, and rate of bone loss in a population-based cohort of 180 postmenopausal women.
Subjects and Methods
Study cohort
A population-based sample of 375 postmenopausal women was recruited by age-stratified randomization from three general practitioners in Sheffield, United Kingdom, as part of the European Vertebral Osteoporosis Study group’s work (16). Strata were half decades of age. Subjects were recruited to take part in a longitudinal study of the epidemiology of osteoporosis. Women were excluded if they were too ill to take part (e.g. terminal illness) or if they were unable to give informed consent. A total of 242 women returned after 5 yr. Bone density measurements were made at baseline and at 5 yr. Serum and urine samples for measurements of biochemical markers of bone turnover were performed at the 5-yr visit. Serum levels of OPG and estradiol were measured in a subset of 180 women in which samples were available. The mean age of this group at the 5-yr visit was 67 yr (8). None of the women in this subset were taking or had ever taken estrogen replacement therapy at the time of sampling. All participants gave their written informed consent, and the studies were approved by the North Sheffield Local Research Ethics Committee (Sheffield, UK).
Sample collection
All samples were collected between 0900 and 0945 h after an overnight fast. Blood samples for serum were collected in serum separator tubes (Vacutainer, Cowley, Oxford, UK). Blood was allowed to clot for 30 min at room temperature and was then centrifuged at 2500 x g for 10 min at 4 C and stored at -70 C until assay. Twenty-four-hour urine samples were collected and stored at -20 C until assay.
Laboratory analyses
Markers of bone turnover. Bone-specific alkaline phosphatase (Bone ALP), a marker of bone formation, was measured by ELISA in serum [Alkphase-B, Metra Biosystems Inc., Mountain View, CA; intra-assay coefficient of variation (CV), 2.5%; interassay CV, 4.5%].
Immunoreactive free deoxypyridinoline (IFDPD), a marker of bone resorption, was measured by ELISA in urine (Pyrilinks-D, Metra Biosystems Inc., Mountain View, CA; intra-assay CV, 5.3%; interassay CV, 8.8%). Urinary cross-linked N-telopeptides of type I collagen (U-NTX), a marker of bone resorption was measured by ELISA (Osteomark, Ostex International, Inc., Seattle, WA; intra-assay CV, 6.9%; interassay CV, 10.8%). Serum cross-linked C-telopeptides of type I collagen (S-CTX), a marker of bone resorption, was measured using an Elecsys Autoanalyzer (Roche Diagnostics, Basel, Switzerland; intra-assay CV, 3%; interassay CV, 6%).
Measurements made in urine were corrected for creatinine using dry chemistry methods in the Clinical Chemistry Department, Northern General Hospital (Sheffield, UK).
Serum OPG measurement. OPG concentration was measured in serum by a sandwich ELISA (Immundiagnostik, Bensheim, Germany) according to the manufacturer’s protocol (intra-assay CV, 7.8%; interassay CV, 9.3%). The assay includes two highly specific antibodies against OPG. Monoclonal IgG antibody was used as a capture antibody. This was produced by a mouse hybridoma cell line derived from a mouse immunized with recombinant human OPG. The antibody is able to neutralize the biological activity of recombinant human OPG. The detection antibody was a biotin-labeled polyclonal antihuman OPG antibody derived from a goat, immunized with human recombinant OPG. Standard curves were generated by using recombinant human OPG purchased from Research Diagnostics, Inc. (Flanders, NJ). The concentrations of serum OPG were calculated on the basis of the protein concentration given by the manufacturer. This assay was designed to detect monomeric, dimeric, and ligand-bound forms of circulating OPG.
Serum estradiol measurement. Estradiol was measured using Elecsys 2010 autoanalyzer (intra-assay CV, 10.2%; interassay CV, 4.4–6.0%). This assay measures total estradiol. Cross reactivity with other estrogen metabolites was less than 1%. The lower limit of detection was 5 pg/ml.
Bone density measurements. Bone mineral density (BMD) was measured at the lumbar spine (L2-L4; LSBMD), femoral neck (FNBMD), total hip (THBMD), and total body (TBBMD) by dual energy x-ray absorptiometry (Lunar DPX, Lunar Corp. Inc., Madison, WI). Measurements were made at baseline and after 5 yr, from which the percentage change in BMD was calculated.
Statistical analysis
Correlations between biochemical markers and serum levels of OPG and estradiol were made using Pearson’s correlation with and without an adjustment for age and body mass index (BMI). Values of biochemical markers and OPG and estradiol were not normally distributed and so were log-transformed before analysis. Correlations between bone density and serum OPG and estradiol were made with and without adjustment for age and BMI. ANOVA was used to determine differences in OPG and estradiol between tertiles of hip and spine bone density.
A P value of 0.05 or less was considered significant for all analyses. Analyses were performed using Statgraphics for Windows software (Manugistics, Inc., Rockville, MD).
Results
Table 1
shows descriptive statistics for the subjects taking part in the study at the 5-yr visit. Associations between variables were made using Pearson’s correlations.
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). This association remained significant after adjustment for age. Serum estradiol was also weakly positively correlated to bone density at all sites measured (r-values from 0.14–0.20; Table 2) and to percentage change in bone density at the total hip and femoral neck (r-values of 0.28 and 0.15, respectively; Table 2) using a univariate analysis. This association was lost after adjustment for age and BMI, with the exception of change in THBMD (r = 0.21; P = 0.03).
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). Serum estradiol was also higher in the upper tertile of THBMD although this was not significant (P = 0.09; Table 3).
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). Analysis of bone resorption markers measured in urine (U-NTX, U-IFDPD) showed no significant relationships with serum OPG (Fig. 2). Multiple regression analysis revealed age as a possible confounder of bone marker measurements. When adjustment for age was made, all significant associations remained significant, with a P value of less than 0.05.
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When assessed as a continuous variable, there was no association between change in BMD and OPG. However, those women with a reduction in BMD of more than 3% over 5 yr at the lumbar spine (n = 50) had lower levels of serum OPG compared with the rest (P < 0.05). There was no association between bone loss at the femoral neck or total hip in relation to serum OPG concentration. Serum OPG was not significantly related to age in this cohort (r = 0.12; P = 0.10)
Serum OPG was higher in the upper tertile of LSBMD and also in the upper tertile of THBMD compared with the middle and lower tertiles; however, this did not reach significance (P = 0.06 and 0.1, respectively; Table 3
).
Discussion
In this study we examined the relationships between the concentration of circulating OPG and estradiol, and bone turnover and bone density in a cohort of postmenopausal women. We were able to detect estradiol in all women using an automated electrochemiluminescent assay. Estradiol levels were highly significantly correlated to markers of bone turnover and explained up to 16% of the variation in bone turnover. The relationship with bone density and change in bone density was weaker. Estradiol explained less than 3% of the variation in bone density in this cohort and was also related to change in bone density, especially in the region of the hip. Interestingly, the relationship between estradiol and bone turnover in this study was stronger than that observed in previous studies (1, 2, 3). The reasons for this are not clear, but the use of a sensitive reproducible assay and the relative ages of the populations may be influencing factors.
We have shown an inverse relationship between circulating levels of OPG and serum-based markers of bone turnover; the r-values, although significant, were small (r -0.16 and -0.18), indicating a rather weak relationship between these variables. Although urine-based bone turnover markers showed a similar relationship, they did not reach significance. We believe that this may be due to the high level of biological variability encountered when making measurements of bone resorption in urine (17). Previous studies have observed inverse associations between serum OPG and bone turnover markers. In a study by Browner et al. (18), a significant negative correlation between serum osteocalcin and OPG was observed in a cohort of 490 elderly women. Szulc et al. (19) observed a significant negative association between U-IFPD and OPG in men over the age of 40, but not in younger men. Bone formation markers, however, were not associated with OPG in this male study.
Serum levels of OPG have been shown to increase with age in both male and female cohorts (13, 18, 19). However, in our study there was no correlation between age or menopausal age and OPG concentration. The age band occupied by the women in our study is narrower than that of other studies that may account for this observation.
It has been suggested that circulating OPG levels are higher in osteoporotic women, compared with controls, and that this occurs as a protective mechanism to slow down the increased bone resorption and subsequent bone loss seen in osteoporosis (13, 20). Our results do not appear to endorse this observation. In our study, higher levels of OPG were related to higher bone density, whereas in the study by Browner et al. (18), there was no relationship between OPG and bone density. These differences may be due to the different study populations and design and also the assays used. In the study by Yano et al. (13), those women with the severest osteoporosis had substantially higher levels of OPG. In our study, none of the women had osteoporosis as defined by the World Health Organization criteria, i.e. a T score of less than -2.5, which may be an explanation for our discrepant findings. When we used a cut point analysis to compare levels of OPG by tertiles of LSBMD and THBMD, we observed higher levels of OPG in the upper tertiles of bone density.
There is evidence from studies in vitro that 17ß-estradiol stimulates the expression of OPG (7). In this study, there was a positive relationship between OPG and serum estradiol levels. This finding is in accordance with that of Szulc et al. (19), who also showed a positive correlation between serum estradiol and OPG in a cohort of 252 men. In this study, multiple regression analysis revealed that estradiol levels were more closely related to bone turnover and bone density than to OPG. This suggests that 17ß-estradiol may inhibit osteoclastogenesis by other pathways independent of OPG/RANKL.
Circulating OPG levels may not fully reflect the activity of OPG within the bone microenvironment. OPG is synthesized by both skeletal and nonskeletal cell types and is regulated by a variety of hormones and cytokines (21). It is also likely that the biological activity of OPG is dependent on the relative levels of both OPG and its ligand (RANKL; Ref. 22). Measurement of the ratio of OPG to RANKL may thus provide more insight into the significance of circulating OPG concentrations in humans.
The limitations of these serum measurements may also partially explain some of the apparent discrepancies in the findings of different studies. The methodology for the detection of OPG in serum in still under development, and different assay designs are currently being tested. OPG circulates in monomeric, dimeric, and ligand-bound forms. Assays are being developed that use OPG ligand as a capture protein as an alternative to an antihuman OPG monoclonal antibody (23). These assays may preferentially bind the dimeric form of OPG. The assay used in this study was designed to detect all circulating forms of OPG. However, as yet it remains unclear as to the appropriate fraction of circulating OPG to measure.
In conclusion, on the basis of the results of this study, we propose a model whereby relatively higher endogenous estradiol levels lead to higher circulating concentrations of OPG, which inhibits osteoclastogenesis, this by coupling leads to suppression of osteoblast activity resulting in overall suppression of bone turnover. Lower rates of bone turnover are in turn related to a reduced rate of bone loss and higher bone mass. The relationship between estradiol and bone turnover seen in this study is stronger than that previously observed and may reflect the importance of residual endogenous estradiol in protecting the skeleton after menopause. In relation to OPG, the limitations of using peripheral measurements to define processes that occur in a very localized environment and also the limitations of currently available methods for assay must be acknowledged before making strong assertions about the importance of circulating OPG in relation to bone turnover.
Acknowledgments
We acknowledge the help of the nurses and radiographers at the Osteoporosis Centre, Northern General Hospital (Sheffield, UK) for their help in the recruitment and scanning of the subjects for this study.
Footnotes
This work was funded in part by Programme Grant E0510 from the Arthritis Research Council UK.
Abbreviations: BMD, Bone mineral density; BMI, body mass index; Bone ALP, bone-specific alkaline phosphatase; CV, coefficient(s) of variation; FNBMD, femoral neck BMD; IFDPD, immunoreactive free deoxypyridinoline; LSBMD, lumbar spine BMD; OPG, osteoprotegerin; RANKL, receptor activator of NFB ligand; S-CTX, serum cross-linked C-telopeptides of type I collagen; TBBMD, total body BMD; THBMD, total hip BMD; U-NTX, urinary cross-linked N-telopeptides of type I collagen.
Received March 13, 2002.
Accepted June 27, 2002.
References
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