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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-1832
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3230-3238
Copyright © 2007 by The Endocrine Society

The Circadian Rhythm of Osteoprotegerin and Its Association with Parathyroid Hormone Secretion

F. Joseph1, B. Y. Chan1, B. H. Durham, A. M. Ahmad, S. Vinjamuri, J. A. Gallagher, J. P. Vora and W. D. Fraser

Departments of Diabetes and Endocrinology (F.J., A.M.A., J.P.V.) and Nuclear Medicine (S.V.), Royal Liverpool University Hospital, Liverpool L7 8XP, United Kingdom; Department of Clinical Biochemistry and Metabolic Medicine (B.Y.C., B.H.D., W.D.F.), Royal Liverpool University Hospital, Liverpool L69 3GA, United Kingdom; and Human Bone Cell Research Group (J.A.G.), Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3GE, United Kingdom

Address all correspondence and requests for reprints to: Dr. Franklin Joseph, Department of Diabetes and Endocrinology, Link 7C, Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP, United Kingdom. E-mail: drfrankjoseph{at}yahoo.co.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Osteoclast resorptive activity, which is known to demonstrate circadian rhythmicity, is regulated by various endocrine hormones and cytokines. PTH suppresses osteoprotegerin (OPG), a regulator of osteoclast activity that has recently been shown to have a circadian rhythm in healthy controls. We studied the differences in the relationship between PTH, OPG, and type I collagen C-telopeptide (ßCTX) over a 24-h period in premenopausal women, elderly postmenopausal women, and elderly men.

Methods: Hourly peripheral venous blood samples were obtained from 18 healthy non-osteoporotic volunteers: premenopausal women (n = 6; mean age, 30.2 ± 2.2 yr), postmenopausal women (n = 6; mean age, 68.2 ± 2.6 yr), and elderly men (n = 6; mean age, 68.2 ± 2.3 yr). Plasma PTH (1–84), OPG, ßCTX, and calcium were measured on all samples. Cosinor analysis was performed to analyze the circadian rhythm parameters. Cross-correlation analysis was used to determine the relationship between the time series of the variables.

Results: The 24-h mean PTH, OPG, and ßCTX concentrations were significantly higher in postmenopausal women as compared with premenopausal women and elderly men (P < 0.001). Significant circadian rhythms were observed for PTH (P < 0.05), OPG (P < 0.05), and ßCTX (P < 0.001) in all subjects. PTH secretion was characterized by two peaks in premenopausal women and elderly men and by a sustained increase in PTH concentration in postmenopausal women. OPG secretion was circadian with a daytime increase and nocturnal decrease, and a greater percent decrease in OPG secretion was observed in the postmenopausal women between 1600 and 2400 h. OPG secretion was inversely related to PTH (r = –0.4) and ßCTX (r = –0.6) secretion over a 24-h period.

Conclusion: This report confirms a circadian rhythm for circulating OPG. The nocturnal decline in circulating OPG is greater in postmenopausal women as compared with premenopausal women and elderly men. Altered PTH secretion may contribute to the OPG secretory pattern in postmenopausal women resulting in increased nocturnal bone resorption.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OSTEOCLAST RESORPTIVE ACTIVITY demonstrates circadian rhythmicity and is controlled by various endocrine hormones and cytokine factors. PTH is one of the most important of these systemic regulators, controlling bone mineral homeostasis. PTH circadian rhythmicity is well established in healthy individuals (1, 2, 3, 4, 5), and there is increasing evidence that fluctuations in PTH secretion may have an important effect in governing normal bone health, bone turnover, and bone remodeling. The nocturnal rise in PTH secretion is blunted in osteoporotic women (6), and PTH circadian rhythm is lost in primary hyperparathyroidism and returns after parathyroid surgery (7). PTH rhythm abnormalities have also been demonstrated in adult GH-deficient patients who have a high incidence of osteoporosis (8, 9). PTH has a concentration- and duration-dependent biphasic effect and can induce opposing responses resulting in both net bone loss and net bone formation (10, 11, 12, 13, 14), and the manipulation of the PTH rhythm presents an important therapeutic possibility for the treatment of osteoporosis by affecting bone turnover (10). At a cellular level, the effect of PTH on bone turnover is mediated by the production of factors from osteoblasts that target osteoclast activity (15, 16, 17, 18, 19). The recent discovery of osteoprotegerin (OPG) and receptor activator for nuclear factor {kappa}B ligand (RANKL) as the fundamental factors controlling bone turnover has significantly advanced the understanding of the processes involved in osteoclastogenesis and bone remodeling (15, 16, 17, 18, 19). Bone remodeling requires the synthesis of bone matrix by osteoblasts along with coordinated resorption by osteoclasts. This process is controlled by osteoblasts through the expression of RANKL and OPG (15, 16, 17, 18, 19, 20, 21). OPG acts as a decoy receptor for RANKL and prevents osteoclastogenesis and bone resorption by inhibiting the signals induced by RANKL-RANK interaction (16, 17) and has recently been shown to demonstrate a possible circasemidian rhythm in healthy subjects (22).

Subcutaneous administration of human PTH (1–38) peptide has been shown to induce a rapid and transient decrease in OPG mRNA in both metaphyseal and diaphyseal bone of rats (23). In animal osteoblast cell culture, PTH stimulates RANKL and suppresses OPG expression (23, 24, 25, 26). A negative association between OPG and PTH concentrations has been demonstrated in men who are over 40 (27), and human PTH (1–34) administered to postmenopausal women with glucocorticoid-induced osteoporosis decreases the circulating concentration of OPG and increases RANKL (28). Animal and human studies have also shown the importance of intermittent PTH injections in increasing trabecular bone mass, whereas continuous PTH infusions favor bone resorption (29, 30, 31). The catabolic effect of continuous administration of human PTH (1–38) is associated with inhibited OPG production (11).

The circadian rhythm of PTH correlates significantly with the circadian rhythms of bone resorption marker type I collagen C-telopeptide (ßCTX) (8), but the factors mediating the rhythm-related effect are still unexplained. The recent demonstration of the circasemidian rhythm of OPG raises the possibility of this as a putative pathway mediating this effect. We have investigated the dynamic relationship between circulating PTH, OPG, and ßCTX over a 24-h period in premenopausal women, postmenopausal women, and elderly men.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Volunteers

Subjects were recruited from hospital personnel and from a database of volunteers willing to participate in medical research. Eighteen subjects were recruited for the study: six healthy premenopausal women (mean age 30.2 ± 2.2 yr), six healthy postmenopausal non-osteoporotic elderly women (mean age 68.2 ± 2.6 yr), and six healthy elderly men (mean age 68.2 ± 2.3 yr). Postmenopausal status was confirmed by serum FSH/LH concentrations above 40 U/liter. Patients with diabetes, ischemic heart disease, heart failure, renal disease, cancer, chronic illness, vertebral fracture, or any disease or medication such as corticosteroids affecting the skeleton were excluded. Subjects were excluded if they were receiving hormone replacement therapy or had received hormone replacement therapy in the year before start of the study, were on calcium and vitamin D supplements, or had ever been exposed to bisphosphonate therapy.

All volunteers underwent bone densitometric evaluation using a Prodigy Oracle Fan-Beam bone densitometer (GE Medical Systems, Milwaukee, WI). T-scores were calculated against a reference population of United Kingdom subjects 20–39 yr of age. Mean age, lumbar spine (L2–L4), and femoral neck T-score are summarized in Table 1Go.


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  		TABLE 1. Characteristics of study population

 
The local ethics research committee approved the study, and all patients provided written informed consent before recruitment.

Volunteers were hospitalized at 1300 h for a 25-h period. Blood samples were obtained hourly from 1400–1400 h via an indwelling venous cannula inserted in the antecubital fossa at the time of admission. Each time, 7 ml blood was collected, the samples were immediately centrifuged, and the plasma was aliquoted and stored at –80 C before analysis. Subjects remained recumbent during 2300–0800 h and slept during this period. Each patient was served preset standardized plated hospital meals at 0800, 1200, and 1800 h. The serving sizes and combinations of foods contained recommended daily allowances of all nutrients including calcium and phosphate. Patients were provided with 1.5 liters water and encouraged to drink at fairly frequent intervals to maintain hydration. Urine samples were collected at 3-hourly intervals between 1400–2300 h and 0800–1400 h with a 24-h urine volume estimation to assess fluid balance, and no significant variability was observed in the hydration of individuals.

Biochemistry assays

Plasma PTH (1–84), OPG, ßCTX, and calcium were measured on all samples. PTH (1–84) was measured using a commercial assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) with inter- and intraassay coefficients of variation (CV) of less than 7% across the working range (0.5–90 pmol/liter). OPG was measured by a direct sandwich-type ELISA (Biomedica, Oxford Biosystems, Oxford, UK) with inter- and intraassay CV of less than 10% across the working range (0.14–30 pmol/liter). ßCTX was measured using an electrochemiluminescence assay (ELECSYS; Roche Diagnostics, Lewes, UK). The inter- and intraassay CV for ßCTX was less than 5 and less than 4%, respectively, across the working range (0.01–6 µg/liter). Calcium was measured by standard procedures on an automated platform (Hitachi 747) (Roche Diagnostics) and was adjusted for albumin (32). Serum adjusted calcium (ACa) has been shown to strongly correlate with ionized calcium and has been found to be precise in subjects with calcium and albumin within the reference range (32, 33).

Statistical analysis

Individual and population mean cosinor analysis was used first to confirm circadian rhythmicity and determine the circadian rhythm parameters of PTH, OPG, and ßCTX using CHRONOLAB 3.0 (Universdade de Vigo, Vigo, Spain), a software package for analyzing biological time series by least-squares estimation (8, 9, 34). The package has previously been well validated and used to analyze PTH and bone marker circadian rhythms in various groups of patients (6, 8, 9). The software thus provides the following circadian parameters: 1) midline estimate statistic of rhythm (MESOR), defined as the rhythm-adjusted mean or the average value of the rhythmic function fitted to the data; 2) amplitude, defined as half the extent of rhythmic change in a cycle approximated by the fitted cosine curve (difference between the maximum and MESOR of the fitted curve); and 3) acrophase, defined as the lag between a defined reference time (1400 h of the first day in our study when the fitted period is 24 h) and time of peak value of the crest time in the cosine curve fitted to the data. A P value for the rejection of the zero-amplitude (no rhythm) assumption is also determined for each individual series and for the group. The method used by the program allows analysis of hybrid data (time series sampled from a group of subjects, each represented by an individual series). Given k individual series, the program fits the same linear model with m different frequencies (harmonics or not from one fundamental period) to each series. This fit provides estimations for 2m + 1 parameters, namely, the amplitude and acrophase of each component, as well as the rhythm-adjusted mean. The population parameter estimates are based on the means of estimates obtained from individuals in the sample. The confidence intervals depend on the variability among individual parameter estimates. The variance-covariance matrix is then estimated on the basis of the sample covariances. Confidence intervals for the rhythm-adjusted mean, as well as for the amplitude-acrophase pair, of each component is then computed using the estimated covariance matrix. The P values for testing the zero-amplitude assumption for each component as well as for the global model is finally derived using those confidence intervals and the t and F distributions (35). Bingham’s test, developed for testing cosinor parameters and part of CHRONOLAB 3.0 software, was used to determine the significance of the differences of cosinor-derived circadian rhythm parameters between subjects.

After the confirmation of concerted circadian rhythms, further analysis of the more extensively studied PTH rhythm was performed as the next step. Over and above its diurnal variation, PTH secretion demonstrates two discrete peaks in healthy individuals (early evening and nocturnal). It has been shown that in pathological conditions, the PTH rhythm is altered during the time periods that these peaks occur (6, 8, 9, 36). Based on these previous data, time points for further analysis were selected for the individual peaks, and using additional statistical techniques, we further analyzed our data. Based on recent work by Luboshitzky et al. (37), the time of onset was defined as the time of first occurrence of at least three consecutive samples exceeding the mean levels of PTH obtained between 0800 and 1400 h by more than 1 SD. Changes in OPG and ßCTX concentration were then analyzed during corresponding time periods in relation to the changes in PTH circadian rhythm.

Cross-correlation analysis was performed to determine the relationships between the 24-h profiles of PTH, OPG, and ßCTX. Cross-correlation analysis determines the correlation between two time series of equal length that have been paired, data point by data point, and then one of the time series is shifted by one or more time points (lag time) and the correlation process is repeated. This process can be repeated with the time series shifted backward and forward, as many times as there are time points minus one. PTH, OPG, and ßCTX time series for the group were derived by calculating the mean value at each time point for all subjects (8, 9, 38, 39). Thus, 25 means were determined for PTH, OPG, and ßCTX. To determine whether one time series led another, for instance whether changes in serum PTH preceded changes in OPG, we computed the cross-correlation functions at 12 lag time points (up to 12 h) (40). Previous studies using half-hourly sampling have revealed significant interactions between PTH and other bone-related metabolites using a 6-h lag (8, 9, 39). To allow for the hourly sampling frequency and the lack of prior documentation of the circadian pattern of OPG, we used a 12-h lag time for cross-correlation analysis.

Cross-correlation with log-transformed values and Monte Carlo simulation were performed to establish statistical replicability of the cross-correlation analysis between variables. In the Monte Carlo simulation, statistical procedure was based on simulated samples of varying sizes (12, 24 and 48, multiples of number of patients and controls) repeated 100 times. The type I error was estimated by samples consisting of time series representing healthy controls and for a type I error of 0.05, a cutoff point of 5% was selected. To determine whether the time or lag difference between the original and simulated data were similar, z-score-transformed r values were obtained from each simulated sample. Significant cross-correlation values at any particular lag were then tested against the null hypothesis of purely random associations applied to the z-score-transformed r values, assuming that uncorrelated data show a unit normal z-score distribution with 0 mean unit-variances.

The differences between groups were determined using ANOVA for repeated measures taking into account the 25 measurements for each of the six individuals in each group. This method has been previously validated for similar comparisons (8, 41). Repeated-measures ANOVA assumes normally distributed errors, equal variances, and sphericity. The Kolmogorov-Smirnov test was used to confirm normal distribution and Levenes’s test for equality of variances. Mauchly’s test indicated that the sphericity assumption was violated, and degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity ({epsilon} = 0.62). The between-group comparisons of circadian parameters, however, was performed with n = 6 values, one for each of the individuals in each group, using Student’s t test for unpaired data. Significant differences are highlighted in Table 2Go. This comparison is subject to type II error given the limited number of individuals in the study, and the values of circadian parameters presented in Table 2Go are MESOR, amplitude, and acrophase from the population mean cosinor analysis (CHRONOLAB 3) for the analytes in all three groups. Values are expressed as the mean ± SEM. P < 0.05 was considered significant.


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  		TABLE 2. Cosinor analysis of circadian rhythm parameters of OPG, PTH, and ßCTX

 

   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Premenopausal women vs. elderly men

The difference in 24-h mean PTH concentrations between premenopausal women (4.7 ± 0.1 pmol/liter) and elderly men (5.4 ± 0.1 pmol/liter) approached significance with P = 0.08. The 24-h mean OPG concentration (4.1 ± 0.1 vs. 3.7 ± 0.1 pmol/liter; P < 0.001) and 24-h mean ßCTX concentration (0.21 ± 0.02 vs. 0.14 ± 0.03 µg/liter; P < 0.001) was significantly higher in elderly men compared with premenopausal women. The 24-h mean ACa concentration was also significantly higher in elderly men (2.36 ± 0.004 mmol/liter; P < 0.01) compared with premenopausal women (2.34 ± 0.004 mmol/liter).

Postmenopausal women vs. premenopausal women

PTH concentration was highest in postmenopausal women (5.8 ± 0.1 pmol/liter) and was significantly higher compared with premenopausal women and elderly men (P < 0.001) as was circulating OPG concentration (4.8 ± 0.1 pmol/liter; P < 0.001). ßCTX concentration was again highest in postmenopausal women (0.33 ± 0.03 µg/liter) and was significantly higher than in premenopausal women and elderly men (P < 0.001). The 24-h mean ACa concentration, however, was significantly higher in postmenopausal women (2.36 ± 0.004 mmol/liter; P < 0.01) compared with premenopausal women (2.34 ± 0.004 mmol/liter) with no difference observed when compared with elderly men.

Circadian rhythm analysis

Individual and population mean cosinor analyses demonstrated significant circadian rhythms for PTH (P < 0.05), OPG (P < 0.05), and ßCTX (P < 0.001) in all patients. Cosinor-derived population mean circadian rhythms of OPG, PTH, and ßCTX are presented in Fig. 1Go, and population mean parameters for OPG are shown in Table 2Go. The individual cosinor parameters are shown in Table 3Go. No significant circadian rhythm was observed for ACa.


Figure 1
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  		FIG. 1. Cosinor-derived circadian rhythmometry (CHRONOLAB) for PTH, OPG, and ßCTX in non-osteoporotic healthy elderly men, premenopausal women, and postmenopausal women. Arrows indicate meal times.

 

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  		TABLE 3. Circadian rhythm parameters of OPG for individuals in each group

 
Although the circadian rhythm was maintained in all groups, we observed an initial increase in PTH concentration beginning at 1600 h (individual range, 1400–1700 h) followed by a later increase beginning at 2400 h (individual range, 2200–2400 h) in premenopausal women, consistent with previous reports in healthy subjects (6). Elderly men also demonstrated a PTH increase beginning at 1700 h (individual range, 1400–1700 h) followed by a second increase beginning at 0100 h (individual range, 2400–0200 h). To highlight further the differences in secretory pattern between groups, we compared the mean percent nocturnal increase from 1600 h [value at each time point – 1600-h concentration)/1600-h concentration x 100] between 1600 and 2400 h (times of onset of the PTH peaks in young, premenopausal, healthy women) and 2400 and 0800 h (end of overnight fast) (8, 9), and no significant difference between men and premenopausal women was observed (1600–2400 h, 7 ± 2 vs. 6 ± 2%; P = 0.4) (2400–0800 h, 16 ± 2 vs. 11 ± 2%; P = 0.7).

A single sustained increase in PTH secretion between 1600 and 0800 h was observed in the postmenopausal women. The percent increase in PTH secretion between 1600 and 2400 h was 14 ± 2% in postmenopausal women and was significantly higher compared with premenopausal women (7 ± 2%; P < 0.05). No significant difference in mean PTH percent increase was observed between 2400 and 0800 h when postmenopausal women (15 ± 2%) were compared with premenopausal women (16 ± 2%; P = 0.7).

In all subjects, the circadian rhythm of OPG secretion was characterized by higher daytime concentrations and a nocturnal decrease. Time of onset of OPG decrease was defined as the time of first occurrence of at least three consecutive samples lower than the mean levels obtained between 0800 and 1400 h by more than 1 SD. The nocturnal decrease began at 1800 h in premenopausal women (individual range, 1500–1900 h), 1600 h in elderly men (individual range, 1500–1900 h), and 1600 h in postmenopausal women (individual range, 1600–1800 h). We calculated the percent nocturnal decrease in OPG between 1600 and 2400 h (time period during which significant changes in PTH secretion were observed). A greater percent decrease [(value at each time point – 1600-h concentration)/1600-h concentration x 100] in nocturnal OPG secretion was observed in the postmenopausal women (15 ± 2%) compared with the premenopausal women (2 ± 2%; P < 0.01) and the men (7 ± 2%; P < 0.05) between 1600 and 2400 h. The percent decrease was also greater between 2400 and 0800 h in postmenopausal women (23 ± 2%) as compared with the premenopausal women (3 ± 2%; P < 0.01) and the elderly men (6 ± 2%; P < 0.01). No significant difference was observed between the men and premenopausal women.

ßCTX concentrations demonstrated a nocturnal increase beginning at 2100 h in premenopausal women and men (individual range, 2000–2200 h) and at 2000 h in postmenopausal women (individual range, 2100–2200 h). The percent increase in ßCTX concentration between 1600 and 2400 h in postmenopausal women (58 ± 8%) was higher than in premenopausal women (16 ± 8%; P < 0.05) and elderly men (19 ± 8%; P < 0.05), with no significant difference seen between premenopausal women and elderly men. The percent increase in ßCTX concentration between 1600 and 0800 h was higher in postmenopausal women (103 ± 12%) compared with premenopausal women (57 ± 12%; P < 0.05) and elderly men (60 ± 12%; P < 0.05).

Cross-correlation analysis

Secretory patterns of PTH and OPG were inversely related and mirrored each other during a 24-h period (Fig. 2Go). Maximal negative correlation between PTH and OPG was observed when PTH changes preceded OPG changes in the opposite direction by 2 h in premenopausal women and elderly men (r = –0.5). A shift in maximal negative correlation between PTH and OPG rhythms was observed in postmenopausal women to zero lags (r = –0.6), suggesting that changes in PTH and OPG occurred at the same time in opposite directions. Maximal negative cross-correlation between OPG and ßCTX was observed when OPG changes preceded ßCTX changes by 3 h in premenopausal women (r = –0.6) and men (r = –0.6) and 2 h in postmenopausal women (r = –0.7). PTH and ßCTX demonstrated a positive correlation (r = 0.7) with a constant lag time of 2 h in all three groups. Cross-correlation with log-transformed values confirmed these findings.


Figure 2
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  		FIG. 2. Cross-correlation analysis of PTH, OPG, and ßCTX in non-osteoporotic healthy elderly men, premenopausal women, and postmenopausal women.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Changes in hormonal concentrations are important for physiological and pathophysiological effects (42, 43). Circadian rhythms are known to exist for bone turnover and bone mineral metabolism and can be altered by changes in PTH circadian rhythm (2, 4, 5, 6, 44, 45). Intermittent PTH injections increase trabecular bone mass, whereas continuous PTH infusion favors bone resorption with inhibited OPG production (11, 29, 30, 31). The significant differences in the circadian rhythms, temporal interactions, and concentrations of PTH, OPG, and ßCTX secretion we observed when postmenopausal women were compared with elderly men and premenopausal women suggest that the rhythmicity of osteoclast activity and the increased osteoclast activity in postmenopausal women may be mediated by this pathway.

A circasemidian rather than circadian rhythm for OPG has been reported with a bimodal variation in a mixed group of nine subjects (mean age of 32.5 ± 7.8 yr). The findings were based on 4-hourly samples, and a circadian rhythm could not be demonstrated using cosinor analysis due to the limited number of samples, and results were based on data expressed as percentages of the arithmetic mean of each time series (22). Our data confirm a circadian rhythm for OPG with a pattern different from that reported previously. The difference in age and gender of the groups studied, as well as the difference in OPG assay used may have been contributory factors, but more importantly, cosinor analysis is dependent on the sampling frequency and biokinetic properties of the analyte. Despite its short half-life (46), the circadian characteristics of PTH have been demonstrated with hourly sampling in this study, but the characteristics cannot, however, be demonstrated with samples greater than 2 h apart (47). Thus, increased sampling frequency provides a more accurate representation of circadian rhythmicity. The half-life of OPG has been shown to be approximately 20 min in rats (17), but no human data are available. All studies measuring circulating OPG are also limited by the multiplicity of its extraskeletal sources and the possible discrepancy between circulating concentrations and concentrations in the bone microenvironment. Despite these factors, the increased sampling frequency in our study results in a more accurate representation of circulating OPG concentrations over a 24-h period.

The profiles of OPG and PTH in our patients were inversely related and mirrored each other, suggesting that the expression of PTH and OPG may be linked to common rhythm-generating or common regulatory factors and that fluctuations in PTH concentration over a 24-h period may directly or indirectly regulate OPG production, consistent with the known suppressive effect of PTH on OPG production in vitro and in vivo. PTH (1–34) suppresses OPG and stimulates RANKL both in vitro and in vivo, and an inverse correlation between PTH and OPG has been established (11) (23, 24, 25, 26, 28, 48). Pharmacological administration of human PTH (1–34) decreased circulating OPG levels in postmenopausal women with glucocorticoid-induced osteoporosis (28), and PTH has also been shown to negatively correlate with serum OPG levels in men beyond 40 yr after adjustment for age and body weight (27).

Osteoclast activity is regulated via cross-talk between osteoblasts and osteoclasts mediated by OPG and RANKL, and blocking RANKL signaling with OPG is able to block the pro-resorptive effects of PTH. Although controversial, PTH may also directly affect osteoclast activity via PTH receptors (49, 50). The maximum correlation between PTH and ßCTX remained constant in all three groups, but in postmenopausal women, the temporal relationship between PTH and OPG and OPG and ßCTX was altered. It is possible that the constant time lag between PTH and ßCTX is a reflection of the direct effect of PTH on osteoclasts, which remains unaltered. The altered temporal relationship between PTH, OPG, and ßCTX may reflect changes in the indirect effect of PTH on osteoclasts via osteoblast cross-talk. In vitro, PTH (1–34) administration suppresses OPG and stimulates RANKL gene expression within 1 h (11, 23, 24, 25, 26, 28, 48), and in vivo sc administration of a single injection of PTH (1–38) at 80 µg/kg has been shown to induce a rapid and transient decrease in OPG mRNA in both metaphyseal and diaphyseal bone of rat that is evident by 1 h (23). Our data indicate that changes in OPG concentration follow changes in PTH by 2 h in premenopausal women and in healthy elderly men. Observational data must be interpreted with caution because they do not entirely reflect data after pharmacological administration. In postmenopausal women, the temporal relationship between PTH and OPG was altered, with changes in PTH and OPG occurring concurrently. The altered effect of PTH on OPG may be a consequence of the altered hormonal milieu, including the lack of estrogen after the menopause and/or the declining GH and IGF-I concentrations with increasing age (51, 52, 53, 54, 55, 56, 57) either interfering with the effect of PTH on OPG production by osteoblasts or affecting osteoblast production of OPG directly and thus altering the temporal relationship. The 3-h delay between changes in OPG and ßCTX probably reflects a separate osteoblast-mediated component of osteoclast activity that is altered in postmenopausal women via similar mechanisms. These temporal alterations may contribute to the significantly greater nocturnal decrease in OPG concentrations and increased osteoclast activity in postmenopausal women. Interpretation of the temporal relationships reported in our study is limited by the cross-sectional design of the study and dependency of the statistical technique on sampling frequency of the analytes. Additional interventional studies designed to induce shifts in the analyte concentrations and more frequent sampling would be required to confirm the exact time taken for PTH to alter circulating OPG and ßCTX in these different patient groups.

OPG is a major biological factor that inhibits osteoclast differentiation, formation, and function (16, 17, 58, 59), and circulating OPG concentrations are altered in conditions of abnormal bone remodeling (15, 17, 21, 60, 61, 62, 63, 64, 65). Results from previous studies using single-time-point sampling methodology have been inconsistent, and although some studies have demonstrated a negative correlation between circulating OPG and bone resorption markers (60, 62, 64), others have not (66, 67, 68, 69, 70). The negative correlation of serum OPG concentrations with bone mineral density has also been reported by some (63, 71, 72) but not others (66, 68, 69, 70). High serum OPG concentrations have been observed in patients with Paget’s disease (60) but not from patients with severe osteolysis (73). These discordant results may reflect the single-time-point (fasting sample) methodology in sample collection. Our observation that plasma OPG is subject to diurnal variation may help explain some of these inconsistencies. Future studies on circulating OPG must take into consideration this variability.

The majority of data currently supports higher circulating OPG in conditions where bone resorption predominates over bone formation. Higher OPG concentrations have been reported in elderly and postmenopausal women with and without osteoporosis (61, 62, 63, 64, 74, 75), and our findings are in keeping with this. Because serum OPG was positively correlated with biochemical markers of bone resorption in these studies, this was thought to be a somewhat ineffective counterregulatory mechanism to prevent additional bone loss (21). Alternative explanations are that OPG clearance is decreased in the elderly or there is enhanced release from bone with aging due to microfractures. Because of the contradiction between local OPG levels, which decrease with aging, and the unambiguous findings of increased circulating OPG serum levels with aging, it is unclear whether circulating OPG adequately reflects local OPG production within the bone microenvironment, especially during aging (27). Several reports have also established an inverse relationship between OPG and bone resorption markers (64, 65, 76). A significantly higher mean nocturnal percent decline in OPG may result in increased nocturnal bone-resorptive activity by osteoclasts as observed in our postmenopausal subjects and explain the apparent contradiction and ineffectiveness of higher fasting or 24-h mean OPG concentration in postmenopausal women. The dynamic change in OPG concentration we have observed may be the important regulatory factor for osteoclast function rather than absolute circulating values.

OPG activity goes hand in hand with RANKL activity, with the ratio between the two being more relevant to bone cellular activity than absolute values of either taken individually. Most RANKL activity is provided by its expression on the osteoblast cell surface. Current RANKL assays are problematic and not sufficiently sensitive because they measure soluble RANKL that is unstable, is degraded rapidly, or binds to OPG to form large stable conglomerates (77). RANKL profiles were investigated in several individuals from each group using a current available assay (ELISA measurement provided by Biomedica, Oxford Biosystems, UK), but the concentrations of RANKL obtained were all close or below the assay detection limit, and no statistically significant variability was observed, and these data were not included in the study. Because OPG is a decoy receptor for RANKL and they have an inverse relationship, it is possible that total RANKL may demonstrate diurnal variation opposite to that of OPG but similar to PTH. Future studies with more sensitive RANKL assays would be required to confirm or refute this hypothesis.

In conclusion, we have confirmed a significant circadian rhythm for OPG in different subject groups and propose that altered OPG circadian rhythm may be an additional factor contributing to postmenopausal bone loss. The circadian rhythm of PTH is recognized as a significant regulator of bone turnover, and the rhythm-mediated effect may be, at least in part, mediated by the rhythm of OPG. Significantly greater nocturnal decline in circulating OPG in postmenopausal women may in part alter the circadian pattern of osteoclast activity on a daily basis resulting in higher nocturnal resorption and net bone loss. The rate of change of OPG concentration could contribute to the rate of bone resorption over and above the absolute circulating OPG concentration. Manipulation of the endogenous rhythm of OPG presents another possible therapeutic target for osteoporosis, and our findings warrant future studies aimed at identifying factors that may be used to modify the circadian secretion of OPG to prevent bone loss.


   Acknowledgments
 
We are grateful to the endocrinology specialist nurses and all the nursing and clerical staff on the Metabolic Bone Unit, Royal Liverpool University Hospital, for all their help and support. We also thank Dr. Nick Hoyle (Roche) for the provision of bone marker kits.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online June 5, 2007

1 F.J. and B.Y.C. have contributed equally to this work. Back

Abbreviations: ACa, Adjusted calcium; ßCTX, type I collagen C-telopeptide; CV, coefficients of variation; MESOR, midline estimate statistic of rhythm; OPG, osteoprotegerin; RANKL, receptor activator for nuclear factor {kappa}B ligand.

Received August 22, 2006.

Accepted May 24, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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