Abstract

Background Osteoprotegerin (OPG) is a cytokine essential for the regulation of bone resorption, but large longitudinal studies on its relationship to fracture risk in humans are lacking. In this population-based study of 2740 men and 2857 post-menopausal women, it was examined whether serum OPG was associated with hip fracture incidence. The participants were followed for 15 years.

Methods Baseline measurements included height, weight and serum OPG, and information about lifestyle, prevalent diseases and use of medication.

Results Men with OPG in the highest quartile were 2.79-fold [95% confidence interval (CI) 1.34–5.82] more likely to have a hip fracture during follow-up, compared with those with OPG in the lowest quartile (P-trend over OPG quartiles ≤0.001, after adjustments for age and other confounders). In women not using post-menopausal hormone therapy (HT), the risk of hip fracture was 1.64-fold higher (95% CI 0.94–2.86) in the highest quartile compared with the lowest OPG quartile (P-trend over OPG quartiles = 0.05). No relationship was found in post-menopausal women using HT (P-trend over OPG quartiles = 0.23).

Conclusions In men, OPG was positively associated with the incidence of hip fracture. In post-menopausal women not using HT a similar, but weaker, relationship was found.

Introduction

Osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) are cytokines essential for the regulation of bone resorption and bone mass.1,2 The osteoblasts and the bone marrow stromal cells produce RANKL, which activates the osteoclasts by binding to its RANK receptor. This binding may, however, be prevented by OPG, which competitively binds RANKL, whereby osteoclast differentiation, activation and survival are inhibited.3

Low bone mass is strongly related to the risk of fracture, but only a few studies have examined the relationship between OPG levels and fracture risk in humans and the results differ. Cross-sectional studies have found both decreased4 and increased5 serum OPG levels in post-menopausal women with prevalent fractures, and a prospective study of post-menopausal women found no association between OPG and all types of self-reported fractures.6 Hip fracture is regarded to be the most severe osteoporotic fracture. However, no longitudinal studies have examined the relationship between serum OPG levels and hip fracture incidence.

In a previous population-based study, we showed that serum OPG is associated with bone loss in post-menopausal women not using post-menopausal HT.7 Moreover, reduction in height (as a proxy for vertebral fractures) was related to OPG levels in men and post-menopausal women not using HT.8

The aim of the present study was to examine whether serum OPG predicts hip fractures in men and post-menopausal women during 15 years of follow-up.

Materials and Methods

Study population

The Tromsø Study is a population-based, longitudinal study with repeated health surveys of inhabitants in the municipality of Tromsø, Norway. It focuses on chronic and lifestyle-related conditions, such as atherosclerosis and osteoporosis. The regional ethical committee approved the survey, and the participants gave written informed consent.9

In the fourth survey of the Tromsø Study, conducted in 1994–95, all inhabitants aged 55–74 years, and 5–10% random samples of the other 5-year birth cohorts aged 25–84 years, were invited to participate in a two-part survey. The first part included standardized measurements of height, body weight and non-fasting serum lipids; the second part (4–12 weeks later) included more advanced measurements such as carotid ultrasonography. A total of 6727 subjects (74% of the eligible population) completed this examination. Serum OPG was assessed in this group of subjects.

The subjects eligible for our analyses consisted of a group of 5866 persons aged 50 years and above who had reliable measurements of OPG. Pre-menopausal women and women with missing or inconsistent information about menstruation (n = 42), subjects with a previous hip fracture (n = 125) and persons with pathological fractures during follow-up (n = 5) were excluded. Also, we did not include 97 persons whose information about body mass index (BMI), prevalent cardiovascular disease or physical activity was missing. Thus, 5597 persons—2740 men and 2857 women (among whom 451 used HT)—were included in the analyses.

Measurements

In 1994–95, information about physical activity, prevalent cardiovascular diseases and for women use of HT and menopausal status was collected from self-administered questionnaires.10 In addition, participants were asked which medication they had used during the last week. Persons were defined as being physically inactive if they reported that they never were so active in their leisure time that they were sweating or out of breath, and that they had been lightly active only (not sweating or out of breath) <3 h a week during the past year. Height was measured to the nearest centimetre, and weight was measured to the nearest half-kilogram. For both measurements, the subjects were wearing light clothing and no shoes. BMI was calculated as weight in kilograms divided by the square of height in metres (kg/m2).

The concentrations of total OPG were analysed in frozen serum aliquots stored at −70°C. OPG was analysed by an ELISA assay (R&D Systems, Abingdon, UK) with mouse anti-human OPG as capture antibody. Biotinylated goat anti-human OPG and streptavidin horseradish peroxidase were used for detection. The OPG assay was performed according to the instructions by the manufacturer. The detection limit was 62.5 pg/ml. The intra- and interassay coefficients of variation in our laboratory were 3.2 and 6.8%, respectively. Between-assay variation in OPG was adjusted for by use of an internal standard.

All analyses were performed on coded samples without knowledge of status regarding hip fracture incidence by the person performing the assays. All samples were analysed in duplicate and the mean value is used in this report.

Areal bone mineral density (aBMD) of the distal forearm was measured in 2686 of the men and 2797 of the women. It was assessed as previously described11 using single X-ray absorptiometry (DTX-100, OsteometerMediTech, Inc., Hawthorne, CA, USA). All scans were reviewed in order to detect and correct possible artefacts, and systematic bone mineral density differences between the two densitometers were adjusted before analysis.11 A total of 111 subjects had repeated measurements performed within a week. The median coefficients of variations for two scans performed 1 week apart by two different operators were 0.79% at the distal site of the forearm.12

Fracture registration and follow-up time

Hip fractures that occurred in the study population were registered from the radiographic archives of the University Hospital of North Norway. All fractures are registered here as there is no other radiology service in the city or within 250 km. The only exception to this would be fractures occurring while travelling with no control radiograph after returning home, or fractures that were never radiographically examined.

The computerized records in the radiographic archives of the University Hospital contain codes for information about fractures in addition to the national personal identification number and time of investigation. All radiographic examinations coded abnormal (any pathology) were reviewed to ascertain the fracture code and to capture fractures that had not been coded correctly as fractures. In those with fractures, the exact anatomical location of the fracture was identified, the trauma mechanism was categorized into high energetic (fall from a height or traffic accident), low energetic or pathological (tumour or metastasis), and consecutive fracture cases were distinguished from one another. In addition, the discharge records were checked with respect to hip fractures. A similar registration of fractures in participants in the second and third Tromsø Study surveys has been performed, validated and described by Joakimsen et al.13

Follow-up time was assigned from the date of the screening to the date of the first hip fracture or migration from Tromsø, death or end of follow-up (31 December 2009), whichever came first.

Statistical analysis

Characteristics of the study population are presented as means (SD) for continuous variables or percentages for dichotomous variables.

The participants were divided into sex-specific groups according to quartiles of OPG.

We estimated the relative risks (RRs) for hip fracture among subjects at different OPG levels by use of Cox regression analysis. The assumption of the proportional hazard was tested by use of time-dependent covariate, and found to be met. Adjustments were made for age (and sex, when relevant), BMI, physical inactivity and prevalent cardiovascular disease. Age (and sex) was introduced in the model first. Thereafter, the variables inactivity, BMI and prevalent cardiovascular disease were introduced together (in one block). In a subanalysis further adjustments for aBMD were done. The interaction term ‘OPG × sex’, was included in the model in a separate analysis.

The Cox analysis also generated curves, adjusted for age and sex and taking censoring into account, described the proportion of the cohort without hip fracture as a function of follow-up time.

The data were analysed using Windows 17.0 version of SPSS.

Results

The study consisted of 2740 men and 2857 post-menopausal women aged ≥50 years. Selected baseline characteristics by sex and HT use are presented in Table 1.

Table 1

Characteristics of the study population

  Post-menopausal women
 
Characteristics Men (n = 2740) All (n = 2857) Users of HT (n = 451) Non-users of HT (n = 2406) 
Age (years)a 62.7 (6.5) 63.7 (6.2) 61.1 (5.9) 64.1 (6.1) 
Height (cm)a 174.8 (6.8) 160.9 (6.0) 161.6 (5.7) 160.8 (6.1) 
BMI (kg/m2)a 26.1 (3.4) 26.3 (4.5) 25.6 (3.8) 26.4 (4.6) 
Physically inactive (%) 35 49 39 51 
Self-reported cardiovascular diseases (%) 20 12 13 
Age at menopause (years)a – 48.5 (4.8) 49.0 (5.0) 48.4 (4.7) 
OPG (ng/ml)a 3.35 (1.04) 3.56 (1.04) 3.10 (1.00) 3.65 (1.02) 
OPG, median (ng/ml) 3.18 3.39 2.92 3.47 
Number of hip fractures during follow-up 113 219 24 195 
  Post-menopausal women
 
Characteristics Men (n = 2740) All (n = 2857) Users of HT (n = 451) Non-users of HT (n = 2406) 
Age (years)a 62.7 (6.5) 63.7 (6.2) 61.1 (5.9) 64.1 (6.1) 
Height (cm)a 174.8 (6.8) 160.9 (6.0) 161.6 (5.7) 160.8 (6.1) 
BMI (kg/m2)a 26.1 (3.4) 26.3 (4.5) 25.6 (3.8) 26.4 (4.6) 
Physically inactive (%) 35 49 39 51 
Self-reported cardiovascular diseases (%) 20 12 13 
Age at menopause (years)a – 48.5 (4.8) 49.0 (5.0) 48.4 (4.7) 
OPG (ng/ml)a 3.35 (1.04) 3.56 (1.04) 3.10 (1.00) 3.65 (1.02) 
OPG, median (ng/ml) 3.18 3.39 2.92 3.47 
Number of hip fractures during follow-up 113 219 24 195 

aValues are means (SD).

A total of 113 (4%) of the men and 219 (8%) of the women sustained a hip fracture during 32 782 and 35 995 person-years of follow-up, respectively. The mean follow-up time was 12.3 years and the median 14.7 years. The median follow-up time (interquartile range) to the first hip fracture was 9.5 years (5.3–12.4). The unadjusted incidences of hip fracture in men and women were 3.4 and 6.1/1000 person-years, respectively.

As displayed in Table 2 and Figure 1, serum OPG was positively associated with risk of hip fracture when men and women were merged in one analysis. Sex-specific analyses showed that serum OPG was associated with an increased risk of fracture in men [RR in the highest quartile vs the lowest=2.79 (95% CI 1.34–5.82), P-value for trend over the OPG quartiles = 0.001]; whereas in women the association was weaker (Table 2). However, the relationship did not differ between men and women (P = 0.85). When the women were stratified into users and non-users of HT, there was a linear relationship in the latter group (P-trend = 0.03, after adjustments for age) with a slightly weaker relationship after multiple adjustments (P = 0.05). In women who used HT, there was no relationship between OPG levels and fracture risk (P = 0.23) (Table 2).

Figure 1

Estimated age- and gender-adjusted proportion of the cohort without a hip fracture as a function of follow-up time

Figure 1

Estimated age- and gender-adjusted proportion of the cohort without a hip fracture as a function of follow-up time

Table 2

RR of hip fractures in relation to OPG levels

 OPG quartilesa
 
 
 P-trend 
All      
    Number of subjects 1399 1399 1400 1399  
    Number with hip fracture 34 67 102 129  
    RRb (95% CI) 1.37 (0.90–2.09) 1.64 (1.09–2.46) 1.81 (1.19–2.76) 0.004 
    RRc (95% CI) 1.34 (0.88–2.04) 1.56 (1.04–2.35) 1.70 (1.12–2.59) 0.01 
Men, all      
    Number of subjects 685 685 685 685  
    Number with hip fracture 11 19 41 42  
    RRb (95% CI) 1.44 (0.68–3.05) 2.81 (1.40–5.65) 2.85 (1.37–5.93) 0.001 
    RRc (95% CI) 1.43 (0.67–3.03) 2.81 (1.40–5.65) 2.79 (1.34–5.82) 0.001 
Women, all      
    Number of subjects 714 714 715 714  
    Number with hip fracture 25 45 64 85  
    RRb (95% CI) 1.22 (0.74–2.00) 1.42 (0.88–2.29) 1.52 (0.93–2.48) 0.08 
    RRc (95% CI) 1.19 (0.73–1.96) 1.34 (0.83–2.17) 1.44 (0.88–2.35) 0.13 
Users of HT      
    Number of subjects 212 107 73 59  
    Number with hip fracture  
    RRb (95% CI) 0.95 (0.33–2.79) 0.61 (0.18–2.09) 0.49 (0.13–1.84) 0.23 
    RRc (95% CI) 1.05 (0.36–3.07) 0.55 (0.16–1.91) 0.52 (0.14–1.92) 0.23 
Non-users of HT      
    Number of subjects 502 607 642 655  
    Number with hip fracture 18 38 59 80  
    RRb (95% CI) 1.29 (0.73–2.27) 1.59 (0.92–2.73) 1.78 (1.02–3.08) 0.03 
    RRc (95% CI) 1.23 (0.70–2.17) 1.48 (0.86–2.55) 1.64 (0.94–2.86) 0.05 
 OPG quartilesa
 
 
 P-trend 
All      
    Number of subjects 1399 1399 1400 1399  
    Number with hip fracture 34 67 102 129  
    RRb (95% CI) 1.37 (0.90–2.09) 1.64 (1.09–2.46) 1.81 (1.19–2.76) 0.004 
    RRc (95% CI) 1.34 (0.88–2.04) 1.56 (1.04–2.35) 1.70 (1.12–2.59) 0.01 
Men, all      
    Number of subjects 685 685 685 685  
    Number with hip fracture 11 19 41 42  
    RRb (95% CI) 1.44 (0.68–3.05) 2.81 (1.40–5.65) 2.85 (1.37–5.93) 0.001 
    RRc (95% CI) 1.43 (0.67–3.03) 2.81 (1.40–5.65) 2.79 (1.34–5.82) 0.001 
Women, all      
    Number of subjects 714 714 715 714  
    Number with hip fracture 25 45 64 85  
    RRb (95% CI) 1.22 (0.74–2.00) 1.42 (0.88–2.29) 1.52 (0.93–2.48) 0.08 
    RRc (95% CI) 1.19 (0.73–1.96) 1.34 (0.83–2.17) 1.44 (0.88–2.35) 0.13 
Users of HT      
    Number of subjects 212 107 73 59  
    Number with hip fracture  
    RRb (95% CI) 0.95 (0.33–2.79) 0.61 (0.18–2.09) 0.49 (0.13–1.84) 0.23 
    RRc (95% CI) 1.05 (0.36–3.07) 0.55 (0.16–1.91) 0.52 (0.14–1.92) 0.23 
Non-users of HT      
    Number of subjects 502 607 642 655  
    Number with hip fracture 18 38 59 80  
    RRb (95% CI) 1.29 (0.73–2.27) 1.59 (0.92–2.73) 1.78 (1.02–3.08) 0.03 
    RRc (95% CI) 1.23 (0.70–2.17) 1.48 (0.86–2.55) 1.64 (0.94–2.86) 0.05 

aOPG quartiles, range (ng/ml). All: ≤2.76, 2.76–3.28; 3.28–3.95, ≥3.95 Men: ≤2.65, 2.65–3.18, 3.18–3.83, ≥3.84 Women (all, users and non-users of HT): ≤2.86, 2.86–3.39, 3.39–4.04, ≥4.04.

bAdjusted for age and sex in all subjects, and for age in men and women.

cAdditional adjustments for BMI, physical inactivity, prevalent cardiovascular disease.

In the subgroup of persons with measurements of aBMD, the estimates were very similar after additional adjustments for this variable. In men (n = 2686) and post-menopausal women not using HT (n = 2352), the risk of hip fracture was 2.94-fold (95% CI 1.38–6.30) and 1.60-fold (95% CI 0.92–2.78) higher, respectively, in the highest compared with the lowest OPG quartile (P-trend over OPG quartiles = 0.001 for men and 0.06 for the women). No relationship was found in post-menopausal women using HT (n = 445) (P-trend over OPG quartiles = 0.14).

In a separate set of analyses, OPG was included in analyses as a continuous variable (OPG logarithmically transformed). For a 1 SD higher value for the log-transformed OPG, the RR for hip fracture in all subjects was 1.18 (95% CI 1.04–1.35) (P = 0.01). In men, the RR was 1.27 (95% CI 1.02–1.59) (P = 0.03), and in women not using or using HT, it was 1.16 (95% CI 0.99–1.37) (P = 0.07) and 1.01 (95% CI 0.62–1.64) (P = 0.9), respectively, after multiple adjustments.

Discussion

In this population-based study, higher serum OPG levels were associated with increased risk of hip fracture in men. In post-menopausal women not using HT, we found a similar, but weaker, relationship, whereas there was no relationship in post-menopausal women using HT.

The present study is the first population-based study that examines the relationship between serum OPG and incident hip fractures. Previously, a post hoc analysis of 490 post-menopausal women showed that those who sustained a hip fracture had higher OPG levels.6 Several cross-sectional studies have examined the relationship between bone mineral density (a strong predictor of fracture) and OPG, finding a positive,14 no,6,1525 or an inverse5,2628 relationship between the two. Only few longitudinal studies are available and whereas we found that high OPG levels were associated with bone loss in post-menopausal women not using HT,7 two much smaller studies found no relationship.23,29 In the present study, high OPG levels were clearly related to an increased risk of hip fracture in men. We have previously reported that OPG is positively associated with height loss (a proxy for vertebral fractures) in men and in post-menopausal women not using HT.8

Experimental data from animals have shown that the deletion of the OPG gene promoted severe osteoporosis in mice, and that in transgenic mice over-expression of OPG from mid-gestation completely prevented the lesions.30 Moreover, mice with targeted deletion of the RANKL gene developed osteopetrosis,31 whereas parenteral administration of RANKL led to massive osteoporosis.32 OPG inhibited osteoclastogenesis in a dose-dependent manner by binding to RANKL in cell cultures.3 In humans, the balance between OPG and RANKL within the bone microenvironment influences bone turnover33 and modulation of the OPG–RANKL system has a significant impact on BMD and fracture risk. Recently, treatment with denosumab, a fully human monoclonal antibody to RANKL that blocks its binding to RANK, was shown to reduce bone resorption and increase BMD, but most importantly to reduce the risk of vertebral, non-vertebral and hip fractures in post-menopausal women with osteoporosis.34

Based on the findings of these studies, our observation that high serum OPG is associated with an increased risk of hip fracture is unexpected and difficult to interpret. It is, however, consistent with our previous findings of an inverse relationship between serum OPG and BMD, and serum OPG and reduction of height.7,8 One hypothesis is that the high serum OPG levels may reflect an attempt to counterbalance the development of osteoporosis. This view may be supported by the increase in OPG with age,35 signalling an attempt to counter the age-related decrease in bone strength. Interestingly, the relationship between OPG and hip fracture was not explained by aBMD in the present study. Other factors of bone strength, such as the microarchitecture, may therefore have been affected. A recent study by Szulc et al.36 seems to support this hypothesis. In a population-based study of 1149 men, they examined the association between serum OPG and bone microarchitecture. The study showed that men with higher serum OPG levels had lower cortical thickness and volumetric BMD and higher levels of bone resorption markers. The authors suggested that ‘high OPG concentrations may reflect the compensatory activation of OPG secretion by osteoblasts to protect cortical bone, the main determinant of bone strength in older men.’ 39 In contrast, a study by Samelson et al.14 showed that OPG was positively associated with greater femoral neck aBMD in women (but not in men) and greater estimated bending strength in men (but not in women). Both studies found greater bone width in men with high serum OPG levels. The study by Samelson et al. indicates that OPG may associate differently to indices of bone strength in men and women. In the present study, measures of the bone microarchitecture may have added valuable information about the relationship between OPG and fracture risk and the sex differences. In addition, it would have been of interest to examine the influence of bone turnover markers. Unfortunately, we had no measures of these possible explanatory factors. On the other hand, as shown in a recent review,35 the relationship between OPG and bone turnover markers is unclear, with findings of a positive, a negative or no correlation. One may also speculate whether one potential mechanism of the association between OPG and fracture risk is related to sex hormones. The development of osteoporosis is clearly related to estrogen deficit in women, but recent studies have shown that estradiol also plays an important role regarding the risk of fracture in elderly men.3739 Older men have higher circulating estradiol levels than older women. However, as for bone turnover markers, the direction of the relationship between OPG and estrogen levels differs considerably between studies.35

The study has some limitations. The first is related to the fact that OPG is produced by many tissues and that the serum concentrations do not fully reflect the local milieu within the bone microenvironment. Secondly, measurements of RANKL could have been of interest, since the ratio of RANKL to OPG may be an important factor in determining associations with fracture. On the other hand, most RANKL is cell bound and not detected in the circulation. Furthermore, the interpretation of the relationship between fracture risk and the OPG/RANKL ratio estimated from serum measurements is limited due to the relatively high percentage of participants with undetectable RANKL. The third is related to the possibility of bias. Although our study group was large and had a high attendance rate, severely ill or disabled individuals were under-represented. However, if such bias should influence the findings of a positive relationship between OPG and hip fracture, fracture risk should be strongly associated to low OPG levels in the non-participants. We believe that this is unlikely and that it is more plausible that non-participation may have weakened the actual relationship. Finally, although single X-ray absorptiometry is one of the most precise methods of measuring aBMD,12 and the ability to predict any fracture in women is considered to be as good as for other measurement sites,40 the study would have been strengthened if we had measured aBMD at other sites of the body (e.g. the hip) as well. Measures of other indices of bone strength than aBMD, such as bone geometry and bone microarchitecture, could also have added valuable information. Moreover, we had no information about falls, a major risk factor for fractures.

We conclude that in this study, serum OPG was positively associated with hip fracture incidence in men. In post-menopausal women not using HT, we found a similar, but weaker, relationship, whereas there was no relationship in post-menopausal women using HT.

Funding

Northern Norway Regional Health Authority.

Conflicts of interests: None declared.

KEY MESSAGES

  • In this population-based study, higher serum osteoprotegerin levels were associated with an increased risk of hip fracture in men.

  • In women not using post-menopausal HT, the relationship was in the same direction but weaker.

  • In women using HT no relationship was found.

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