Journal of Bone and Mineral Metabolism

, Volume 31, Issue 5, pp 579–584

Serum sclerostin levels in healthy men over 50 years of age

Authors

    • Department of Laboratory Medicine, Medical and Health Science CenterUniversity of Debrecen
    • Regional Osteoporosis Center, Department of Obstetrics and Gynecology, Medical and Health Science CenterUniversity of Debrecen
  • John Wamwaki
    • Department of Laboratory Medicine, Medical and Health Science CenterUniversity of Debrecen
  • Edit Kalina
    • Department of Laboratory Medicine, Medical and Health Science CenterUniversity of Debrecen
  • Roza Foldesi
    • Department of Laboratory Medicine, Medical and Health Science CenterUniversity of Debrecen
  • Adam Balogh
    • Regional Osteoporosis Center, Department of Obstetrics and Gynecology, Medical and Health Science CenterUniversity of Debrecen
  • Peter Antal-Szalmas
    • Department of Laboratory Medicine, Medical and Health Science CenterUniversity of Debrecen
Original Article

DOI: 10.1007/s00774-013-0451-z

Cite this article as:
Bhattoa, H.P., Wamwaki, J., Kalina, E. et al. J Bone Miner Metab (2013) 31: 579. doi:10.1007/s00774-013-0451-z

Abstract

The aim of this study is to evaluate the relationship of serum sclerostin levels with age, cystatin C, bone mineral density (BMD) and biochemical markers of bone turnover in healthy Hungarian men >50 years of age. We determined serum levels of sclerostin and examined its relationship to age, cystatin C, osteocalcin, C-terminal telopeptides of type-I collagen, procollagen type 1 amino-terminal propeptide, 25-hydroxyvitamin D, parathyroid hormone, and L1–L4 (LS) and femur neck (FN) BMD data available from 194 randomly selected ambulatory men belonging to the HunMen cohort. In the study population as a whole [n = 194; age (median, range) 59 (51–81) years], statistically significant correlation was found between sclerostin and age (r = 0.211; p = 0.003), cystatin C (r = 0.246; p = 0.001), FN BMD (r = 0.147; p = 0.041) and LS BMD (r = 0.169; p = 0.019). Compared to middle-aged men (age ≤59 years, n = 98), elderly men (age >59 years, n = 96) had significantly higher serum sclerostin levels (67.8 ± 15.9 vs 63.5 ± 14 pmol/L; p = 0.047). Among men with normal (T score >−1.0) FN BMD, the elderly had significantly higher serum sclerostin levels compared to the middle-aged men (70.4 ± 17 vs 63.9 ± 11.5 pmol/L; p = 0.019). Furthermore, among the elderly men cystatin C was the only significant predictor of serum sclerostin levels (standardized regression coefficient (β) = 0.487; p < 0.001). In the studied healthy elderly cohort, this study reports a significant increase in sclerostin levels with increasing age and deteriorating kidney function as determined by plasma cystatin C levels.

Keywords

SclerostinCystatin cMarkers of bone turnoverBone mineral densityMen

Introduction

Sclerostin is a non-classical bone morphogenetic protein antagonist, produced by the osteocytes and has an inhibitory effect on the lifetime of the osteoblast. Sclerostin has been identified as binding to low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 receptors and inhibiting the Wnt signaling pathway and does this by moving through the canaliculi into the bone marrow and reduces osteoblast differentiation and bone formation through its inhibition of frizzled/LRP5/6 transmembrane signaling [1, 2]. Mutations in the gene encoding sclerostin (Sost) result in a high bone mass disease (van Buchem disease or sclerosteosis syndrome), as such sclerostin inhibition might have a therapeutic potential for conditions associated with low bone mass [3]. Indeed results of a recently published phase I study have reported statistically significant increases in lumbar spine and total hip bone mineral density (BMD) in healthy individuals receiving sclerostin monoclonal antibody. Furthermore, a phase II trial is still ongoing [4, 5].

Data on sclerostin in healthy humans is accumulating. Although the majority of studies examined pre- and postmenopausal women, there are two published reports on men and one on children [615]. To date there is only one study that included a population-based sample of elderly men and, as such, we have sought to evaluate the determinants of serum sclerostin levels in men >50 years of age [11]. The aim of the present study is to evaluate serum sclerostin levels in a randomly selected male cohort >50 years of age and its association with age, kidney function as determined by cystatin C measurements, BMD and biochemical markers of bone turnover.

Materials and methods

The fasting serum samples (n = 194) for sclerostin measurement belonged to participants in the HunMen study [16]. Briefly, the HunMen study was a local initiative to evaluate the bone health of randomly selected healthy men who confirmed to the inclusion and/or did not confirm to the exclusion criteria. Inclusion criteria were >50 years of age, male, ambulatory, community dwelling and generally regarded as healthy. Exclusion criteria were known prevalent metabolic bone disease, liver or renal disease and use of medication influencing bone metabolism (excluding calcium and vitamin D supplementation). The study was approved by the ethics review board of the institution in compliance with the Declaration of Helsinki, and all subjects gave written informed consent.

Dual energy X-ray absorptiometry examination was performed using the LUNAR Prodigy (GE-Lunar Corp., Madison, WI, USA) densitometer. BMD was measured at L1–L4 lumbar spine (LS) and femur neck (FN). The coefficient of variation (CV) of the technique at our institute was 0.8 % using the anatomical spine phantom measured daily. BMD was expressed as T score—the number of standard deviations from the mean of young men attaining peak bone mass using the normative reference values (German) provided by the manufacturer. Normality, osteopenia and osteoporosis were defined according to the World Health Organization (WHO) classification [17].

Serum sclerostin was measured using an enzyme-linked immunosorbent assay (Biomedica Medizinprodukte GmbH & Co KG, Wein, Austria). This assay uses a polyclonal goat anti-human sclerostin antibody as capture antibody and a biotin-labeled mouse monoclonal antibody for detection. Plasma 25-hydroxyvitamin D (25-OH-D) was analyzed by high pressure liquid chromatography (HPLC) using a Jasco HPLC system (Jasco, Tokyo, Japan) and Bio-Rad reagent kit (Bio-Rad Laboratories, Hercules, CA, USA). Serum parathyroid hormone (PTH), osteocalcin (OC), C-terminal telopeptides of type-I collagen (CTX-I), procollagen type 1 amino-terminal propeptide (PINP) were measured using electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany). The inter-assay CV was <6 % for sclerostin <3.5 % for 25-OH-D, <7 % for PTH, <4 % for OC, <7 % for CTX-I and <6 % for PINP. Plasma cystatin C was measured using particle-enhanced nephelometric immunoassay (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), and its inter-assay CV was <4 %. The 4-variable modification of diet in renal disease (MDRD) study equation was used for calculating estimated glomerular filtration rate (eGFR) [18].

The Kolmogorov–Smirnov test was used to check for normality of distribution. Parameters not showing normal distribution were log transformed. Correlation was tested using Pearson’s correlation analysis. Associations were tested using linear regression analysis. Multivariate analysis of variance was used to control for confounders. A value of p < 0.05 was considered statistically significant. All analyses were performed with the SPSS Statistics software, version 19.0 (IBM Corps., Armonk, NY, USA).

Results

Serum sclerostin levels showed statistically significant correlation between age (r = 0.211; p = 0.003), cystatin C (r = 0.246; p = 0.001), LS BMD (r = 0.169; p = 0.019) and FN BMD (r = 0.147; p = 0.041). No significant correlation was found between serum sclerostin and PTH, body mass index (BMI), markers of bone turnover and 25-OH-D (Table 1). Multivariate linear regression analysis was carried out to determine the statistically significant predictors of serum sclerostin levels. In order to avoid adjustment twice for a similar confounder, LS and FN BMD were used individually as covariates in two separate models. As such, by including age, cystatin C and FN BMD as independent variables, age (standardized regression coefficient (β) = 0.167; p = 0.029) and cystatin C (standardized regression coefficient (β) = 0.187; p = 0.013) were statistically significant predictors of serum sclerostin levels. In the second model, where age, cystatin C and LS BMD were included as independent variables, only cystatin C (standardized regression coefficient (β) = 0.200; p = 0.008) was the significant predictors of serum sclerostin levels.
Table 1

Correlation coefficients (with p values) between serum sclerostin levels and the studied parameters

 

All men (n = 194)

Middle-aged men (≤59 years of age, n = 98)

Elderly men (>59 years of age, n = 96)

Age

0.211 (0.003)

0.047 (0.648)

0.259 (0.011)

BMI

0.043 (0.485)

0.066 (0.286)

0.096 (0.121)

PTH

−0.008 (0.908)

0.094 (0.359)

−0.112 (0.288)

25-OH-D

−0.088 (0.225)

−0.048 (0.641)

−0.128 (0.215)

OC

−0.066 (0.369)

−0.080 (0.437)

−0.029 (0.780)

CTX-I

−0.005 (0.940)

0.054 (0.833)

−0.071 (0.501)

PINP

−0.115 (0.110)

−0.113 (0.267)

−0.101 (0.327)

Cystatin C

0.246 (0.001)

−0.103 (0.327)

0.522 (<0.001)

Creatinine

0.172 (0.022)

−0.067 (0.522)

0.387 (<0.001)

eGFR

−0.162 (0.033)

0.067 (0.522)

−0.396 (<0.001)

LS BMD

0.169 (0.019)

0.160 (0.540)

0.150 (0.145)

FN BMD

0.147 (0.041)

0.145 (0.153)

0.170 (0.098)

BMI body mass index, PTH parathyroid hormone, 25-OH-D 25-hydroxy-vitamin D, OC osteocalcin, CTX-I C-terminal telopeptides of type-I collagen, PINP total procollagen type 1 amino-terminal propeptide, eGFR estimated glomerular filtraton rate, LS BMD L1–L4 bone mineral density, FN BMD femur neck bone mineral density

In the over-all group, plasma cystatin C (r = 0.246; p = 0.001), serum creatinine (r = 0.172; p = 0.022) and eGFR (r = −0.162; p = 0.033) all showed statistically significant correlation with serum sclerostin levels. Although this was not the case in the middle-aged men, where no significant correlation was found, in the elderly men, plasma cystatin C (r = 0.522; p < 0.001), serum creatinine (r = 0.387; p < 0.001) and eGFR (r = −0.396; p < 0.001) all showed even better statistically significant correlation with serum sclerostin levels. The correlation coefficient (r) and the degree of significance (p) were the strongest for plasma cystatin C. It needs to be mentioned here that the MDRD study equation which is used to calculate eGFR, comprises the age of the patient, therefore the GFR and the age are not independent variables, as such eGFR dependence of the outcome may still represent the effect of age and not true kidney function. Hence, cystatin-C level provides a more sensitive indicator of GFR than the serum creatinine concentration, and is not affected by age, diet or nutritional state.

In order to reduce the confounding effect of age, using the median age of 59 years, the study population was divided into a middle-aged (those ≤59 years of age, n = 98) and an elderly (those >59 years of age, n = 96) sub-group. Compared to middle-aged men, elderly men had significantly higher serum sclerostin (67.8 ± 15.9 vs 63.5 ± 14 pmol/L; p = 0.047) and plasma cystatin C (0.736 ± 0.127 vs 0.657 ± 0.126 mg/L; p < 0.001) levels (Table 2). The middle-aged men showed no significant correlation between serum sclerostin levels and the other studied parameters (age, cystatin C, BMI, LS BMD, FN BMD, PTH, 25-OH-D, OC, CTx, P1NP). The elderly men showed a significant correlation between serum sclerostin levels and age (r = 0.259; p = 0.011) and cystatin C (r = 0.522; p < 0.001) (Fig. 1). Including cystatin C and age as independent variables in a regression model, showed that cystatin C was the only significant predictor of serum sclerostin levels (standardized regression coefficient (β) = 0.487; p < 0.001).
Table 2

Subject characteristics

 

All men (n = 194)

Middle-aged men (≤59 years of age, n = 98)

Elderly men (>59 years of age, n = 96)

Age, years (mean, range)

60.4 (51–81)

55.6 (51–59)a

65.4 (60–81)a

BMI, kg/m2 (mean, range)

29.2 (17.3–41.7)

28.9 (18–38.5)

29.5 (17.3–41.7)

Sclerostin, pmol/L (mean, range)

65.6 (27.2–114.6)

63.5 (39.2–114.6)b

67.8 (27.2–110.2)b

PTH, pmol/L (mean, range) (reference range 1.6–6.9 pmol/L)

4.2 (1–10.2)

4.2 (1–10.2)

4.3 (1.6–8.3)

25-OH-D, nmol/L (mean, range) (reference range >75 nmol/L)

73.1 (11–185)

72.1 (11–137)

74.0 (14–185)

OC, μg/L (mean, range) (reference range 14–46 μg/L)

14.6 (5–35)

15.2 (5–33)

14.0 (6–35)

CTX-I, μg/L (mean, range) (reference range <0.704 μg/L)

0.22 (0.01–0.77)

0.24 (0.01–0.77)

0.20 (0.04–0.70)

PINP, μg/L (mean, range)

38.3 (10.5–98.6)

39.2 (10.5–98.6)

37.3 (13.1–81.3)

Cystatin C, mg/L (mean, range) (reference range 0.5–0.96 mg/L)

0.697 (0.381–1.150)

0.657 (0.381–1.060)c

0.736 (0.485–1.150)c

Creatinine, μmol/L (mean, range) (reference range 62–106 μmol/L)

78 (46–109)

77 (46–109)

79 (52–105)

eGFR, mL/min/1.73 m2 (mean, range) (reference range >90 mL/min/1.73 m2)

91 (60–165)

94 (60–165)d

87 (62–140)d

LS BMD, gm/cm2 (mean ± SD)

1.172 ± 0.185

1.148 ± 0.167

1.197 ± 0.199

FN BMD, gm/cm2 (mean ± SD)

0.970 ± 0.137

0.980 ± 0.131

0.960 ± 0.142

BMI body mass index, PTH parathyroid hormone, 25-OH-D 25-hydroxy-vitamin D, OC osteocalcin, CTX-I C-terminal telopeptides of type-I collagen, PINP total procollagen type 1 amino-terminal propeptide, eGFR estimated glomerular filtraton rate, LS BMD L1–L4 bone mineral density, FN BMD femur neck bone mineral density

a,cp < 0.001

bp = 0.047

dp = 0.017 between middle-aged and elderly men

https://static-content.springer.com/image/art%3A10.1007%2Fs00774-013-0451-z/MediaObjects/774_2013_451_Fig1_HTML.gif
Fig. 1

Dot-plot for serum sclerostin levels with plasma cystatin C in middle-aged and elderly healthy men

Apart from the correlations already mentioned, in the over-all group, plasma cystatin C correlated with creatinine (r = 0.526; p < 0.001), eGFR (r = −0.501; p < 0.001), P1NP (r = 0.170; p = 0.020) and osteocalcin (r = 0.162; p = 0.028); in middle-aged men, plasma cystatin C correlated with creatinine (r = 0.457; p < 0.001), eGFR (r = −0.455; p < 0.001), P1NP (r = 0.412; p < 0.001), osteocalcin (r = 0.471; p < 0.001) and CTx (r = 0.253; p = 0.016); and in the elderly men, plasma cystatin C correlated with creatinine (r = 0.567; p < 0.001) and eGFR (r = −0.583; p < 0.001).

Men with normal (T score >−1.0) LS BMD (n = 125) had significantly higher serum sclerostin levels as compared to those with low (T score ≤−1.0) LS BMD (n = 69) (67.5 ± 16.3 vs 62.2 ± 12.2 pmol/L; p = 0.027). There was no difference in sclerostin levels when comparing men with normal (T score >−1.0) FN BMD (n = 109) to those with low (T score ≤−1.0) FN BMD (n = 85) (66.8 ± 14.6 vs 64.1 ± 15.7 pmol/L; p = 0.181).

Among individuals with normal FN BMD, the elderly men had significantly higher serum sclerostin levels as compared to middle-aged men (70.4 ± 17 vs 63.9 ± 11.5 pmol/L; p = 0.019). Among men with low FN BMD, the elderly had higher, but statistically non-significant serum sclerostin levels as compared to middle-aged men (65.1 ± 14.3 vs 62.9 ± 17.5 pmol/L; p = 0.540).

Among individuals with normal LS BMD, there was no statistically significant difference between serum sclerostin levels in the elderly and the middle-aged men (70 ± 17.1 vs 64.8 ± 15 pmol/L; p = 0.071). Similarly, among individuals with low LS BMD, no statistically significant difference was found in serum sclerostin levels between the elderly and the middle-aged men (63.1 ± 12 vs 61.5 ± 12.4 pmol/L; p = 0.575).

Discussion

This is the first study where serum sclerostin levels were determined in a systematically selected healthy male cohort >50 years of age. Although a previous study has reported findings in a population-based male cohort, the individuals included in the study did not have to adhere to selection criteria defined at recruitment [11].

Our results demonstrate, perhaps confirm, that serum sclerostin levels increase with age in healthy men >50 years of age. We also found that among men with normal FN and/or LS T scores, the elderly men had higher sclerostin levels as compared to the middle-aged men. Mödder et al. [11] reported such dissimilarity between middle-aged and elderly men and, furthermore, suggested that the association of sclerostin levels with BMD in the elderly may be due to increased skeletal sclerostin production with aging. Although our study verifies these findings, further studies are necessary.

Mödder et al. [11] could not rule out the role of decreased protein clearance as a potential cause of age-related sclerostin elevation, and we found that cystatin C was a strong predictor of serum sclerostin levels in the elderly. Furthermore, Cejka et al. [19] proposed that due to its molecular mass of 22 kDa, sclerostin may be cleared via the kidneys and as such may accumulate in the presence of kidney failure. As such, it appears that the age-dependent deterioration of kidney function may also be an important contributor to increased serum sclerostin levels in elderly men. This is the first study on sclerostin levels, where cystatin C determination was used to assess kidney function. Compared to creatinine, cystatin C possesses the advantages of being independent of gender, muscle mass and age [20].

Knowing that sclerostin is produced almost exclusively by osteocytes and is a potent inhibitor of bone formation, an inverse association between sclerostin and BMD would be expected [1]. Our results, in agreement with at least two others, suggest the contrary, i.e., higher BMD is associated with likewise serum sclerostin levels [11, 19]. Although the cause is not know, it is proposed by Cejka et al. [19] that a higher bone mass would result in more osteocytes and as such higher sclerostin.

Noting the effect of sclerostin in inhibiting bone formation, one would expect that the markers of bone formation to be inversely associated with sclerostin. On the contrary, we have, in agreement to the findings of Mödder et al. [11] found no such association. Although the argument for this non-association by Mödder et al. [11] seems quite plausible, where they suggest that the higher sclerostin levels in the elderly men may be beyond a biologic saturation point for sclerostin effects on bone formation, additional studies are necessary to test this hypothesis.

Upon evaluating the scatter plots published by Mödder et al. [11] it may well be presumed that our serum sclerostin values are higher. This dissimilarity is most likely because of differences in the selection criteria between the two studies. Furthermore, environmental and genetic influences on bone mass, and as a consequence serum sclerostin levels, may contribute to the differences in the sclerostin levels.

Although data on physical activity was not collected in the present study, it may be presumed that elderly men are physically less active as compare to middle-aged men. Low physical activity has also been speculated as a cause of increased sclerostin levels [21].

There are limitations to our study. The HunMen study was not designed primarily for the aim of this study, and only 194 samples were available for serum sclerostin determination from a total of 206 participants [16]. The relatively small sample size of our study necessitated the pooling of individuals with osteopenia and osteoporosis into a single group of low BMD; as such we are not in a position to extrapolate our findings to those with osteopenia or osteoporosis per se. Furthermore, the sample size inhibited us from executing statistical analysis between smaller, more compact, age groups.

In summary, this is the first study to determine serum sclerostin levels in a systematically selected healthy male cohort >50 years of age using a validated immunoassay and assessing kidney function by determining cystatin C levels. Our findings necessitate further studies to examine the mechanisms for age-dependent increases in serum sclerostin levels.

Acknowledgments

This work is supported by the OTKA 105073 Research Grant (HPB) and the TÁMOP 4.2.1./B-09/1/KONV-2010-0007 Project (HPB, JW, EK, RF, AB, PAS), which is implemented through the New Hungary Development Plan, co-financed by the European Social Fund.

Conflict of interest

All authors state that they have no conflicts of interest.

Copyright information

© The Japanese Society for Bone and Mineral Research and Springer Japan 2013