Determinants of Serum Sclerostin in Healthy Pre- andPostmenopausal Women

Mohammed-Salleh M Ardawi,1,2 Hanan A Al-Kadi,1,3 Abdulrahim A Rouzi,1,4 and Mohammed H Qari1,5

1Center of Excellence for Osteoporosis Research, Faculty of Medicine and King Abdulaziz University Hospital, King Abdulaziz University,Jeddah, Saudi Arabia

2Department of Clinical Biochemistry, Faculty of Medicine and King Abdulaziz University Hospital, King Abdulaziz University,Jeddah, Saudi Arabia

3Department of Medical Physiology, Faculty of Medicine and King Abdulaziz University Hospital, King Abdulaziz University,Jeddah, Saudi Arabia

4Department of Obstetrics & Gynecology, Faculty of Medicine and King Abdulaziz University Hospital, King Abdulaziz University,Jeddah, Saudi Arabia

5Department of Hematology, Faculty of Medicine and King Abdulaziz University Hospital, King Abdulaziz University, Jeddah,Saudi Arabia

ABSTRACTSclerostin is a secreted Wnt antagonist produced almost exclusively by osteocytes that regulates bone mass. However, there is currently

limited information on the determinants of sclerostin in a large population-based study. The main objectives of the present study were

to: (1) establish reference normative interval values for serum sclerostin in randomly selected healthy premenopausal women; (2) study

the changes in serum sclerostin in relation to age in premenopausal and postmenopausal women and the factors that may influence

bone turnover; and (3) determine the effect of menopausal status on serum sclerostin. A total of 1803 women were studied (including

[n¼ 1235] premenopausal, and [n¼ 568] postmenopausal women, respectively, aged 20 to 79 years). A total of 443 healthy

premenopausal women (aged 35 to 45 years) were used to establish reference normative intervals for serum sclerostin. All women

studied were medically examined and had their bone mineral density values obtained for the lumbar spine (L1–L4) and femoral neck

according to a detailed inclusion criteria. In all women, values of serum sclerostin increased with increasing age up to the age of 45 years,

and remained increased in postmenopausal women. Significant increases were evident in serum sclerostin in postmenopausal women

with increasing years since menopause. Using stepwise multiple linear regression analysis, several variables were identified as

determinants of serum sclerostin, including age, parathyroid hormone, estradiol (E2), and follicle-stimulating hormone (FSH) for

premenopausal women; age, FSH, and E2 for postmenopausal women; and age, serum osteocalcin, FSH, and E2 in the entire sample

studied. Further studies are needed to establish the potential role of this increase in mediating the known age-related impairment in

bone formation. � 2011 American Society for Bone and Mineral Research.

KEY WORDS: SCLEROSTIN; BONE TURNOVER MARKERS; BONE DENSITY; PRE- AND POST- MENOPAUSAL WOMEN

Introduction

Based on the work of the past decade, it is now well

established that the Wnt/b-catenin signaling pathway is a

major regulator of bone mass.(1,2) Activation of this pathway

results in increased proliferation and differentiation of osteo-

progenitor cells and reduced apoptosis of mature osteoblasts,

favoring higher bone formation and increased bone density.(3–5)

In addition, the Wnt signaling may inhibit osteoclastogenesis.(6)

Activation of the Wnt/b-catenin canonical signaling pathway in

osteoblasts is mediated via binding of any of multiple Wnt

ligands to a seven-transmembrane domain-spanning frizzled

receptor and either of two coreceptors, namely, low-density

lipoprotein receptor-related proteins (LRPs) 5 or 6.(7)

Sclerostin is a glycoprotein secreted almost exclusively by

osteocytes and to a lesser extent other cell types (kidney,

vascular),(1) which travels through osteocytic canaliculi to the

bone surface and binds to LRP-5 and LRP-6 to inhibit the Wnt/

b-catenin canonical signaling pathway,(8–10) thereby decreasing

osteoblastogenesis and bone formation. In humans, the

importance of sclerostin is highlighted by two genetic disorders

associated with significant progressive increases in bone

mass,(11–14) namely: sclerosteosis (caused by loss-of-function

mutation in the gene encoding sclerostin, resulting in an

ORIGINAL ARTICLE JJBMR

Received in original form May 29, 2011; revised form June 19, 2011; accepted July 7, 2011. Published online August 2, 2011.

Address correspondence to: Mohammed-Salleh M Ardawi, PhD, FRCPath, PO Box No. 20724, Jeddah 21465, Saudi Arabia. E-mail: ardawims@yahoo.com

Additional Supporting Information may be found in the online version of this article.

Journal of Bone and Mineral Research, Vol. 26, No. 12, December 2011, pp 2812–2822

DOI: 10.1002/jbmr.479

� 2011 American Society for Bone and Mineral Research

2812

improperly spliced SOST-mRNA)(11–12); and Van Buchem’s

disease (caused by a deletion of an enhancer element that is

normally downstream of the SOST-gene),(13,14) respectively.

Furthermore, SOST-null mice have a high-bone-mass pheno-

type,(15) and over-expression of normal human SOST-alleles in

mice causes osteopenia.(16) These observations suggested that

inhibition of sclerostin may have therapeutic potential for the

treatment of low-bone-mass disorders. Indeed, recent studies

showed that using anti-sclerostin neutralizing antibodies

in rats(17) and primates(18) increased bone density and bone

strength. Similarly, in rodent models of fracture healing,

sclerostin antibodies treatment resulted in increased callus

density and bone strength at fracture sites and accelerated bone

repair.(19) This increased bone formation and bone mass by

sclerostin antibody was not blunted in ovariectomized rats

pretreated with alendronate.(20) More recently, Padhi et al.

showed that treatment of postmenopausal women with anti-

sclerostin antibody resulted in dose-dependent increased bone

formation markers.(21)

There are very limited reports in the literature on the serum

levels of sclerostin in humans: Mirza et al. showed elevated

sclerostin levels in 20 postmenopausal women compared with

an equal number of premenopausal women.(22) Modder et al.

reported similar results in 152 postmenopausal women

compared with 123 premenopausal women and showed a

sex difference whereby men exhibited higher sclerostin levels

than corresponding women.(23) Moreover, Gaudio et al. showed

increased circulating sclerostin levels in 40 long-term immobi-

lized patients.(24) Given the availability of a commercially

validated immunoassay for sclerostin and no available informa-

tion on a large-population studies, the main objectives of the

present study are to: (1) establish age-specific normative interval

values of serum sclerostin in randomly selected premenopausal

women; (2) study the changes in sclerostin in relation to age,

bone mineral density (BMD), and bone turnover markers (BTMs)

(namely: serum osteocalcin [s-OC]; serum procollagen type 1 N-

terminal propeptide [s-PINP]; serum cross-linked C-terminal

telopeptide of type 1 collagen [s-CTX]; and urinary N-telopeptide

of type 1 collagen [u-NTX)]) in pre- and postmenopausal

women; and factors reported to influence bone turnover; and

(3) determine the effect of menopausal status on serum

sclerostin.

Subjects and Methods

Study Design

Over a period of 50 months (October 2005 to November 2009), a

total of 5850 Saudi women were prospectively recruited at

random during a health survey from 40 primary health care

centers (PHCCs) scattered around the city of Jeddah (divided into

7 geographical areas) to ensure that the average health status

of the studied group reflected a randomly selected adult

population. The sample size was calculated using the sample-size

determination option in Epi-Info Statistical Package (version 6)

(USD, Stone Mountain, GA, USA). A description of the study

design and rationale has been reported previously.(25) Women

who agreed to participate in the study were asked to visit a

special clinic at the CEOR, King Abdulaziz University, Jeddah,

Saudi Arabia, to be enrolled in the study. Age, body weight,

height, body mass index (BMI) calculated as the weight (kg)

divided by height (m) squared (kg/m2), and waist-to-hip ratio

(WHR) were recorded. All women were medically examined and

interviewed using a locally developed and validated standard-

ized questionnaire to collect information on lifestyle, smoking

habits, and level of physical activity in leisure time; coffee and tea

consumption and the use of vitamins and medications. Women

with chronic diseases, including osteoarthritis or established

osteoporosis; with evident endocrine disorders; on any form of

drug therapy with possible effect on bone metabolism (eg

selective estrogen receptor modulators, calcitonin, phytoestro-

gens, glucocorticoids, anticonvulsants thyroid hormones and/or

estrogen therapy); or with cancer were excluded from the final

analysis. Women who were pregnant or lactating, taking oral

contraceptives, vitamin-D deficient with serum 25-hydroxy-

vitamin D [25(OH)D] levels being< 50 nmol/L,(26) smokers, or

who reported a recent fracture (within two years) were also

excluded. In addition, all participants in the present study

showed: (1) normal blood counts; (2) normal values for

renal creatinine (serum creatinine in women< 105mmol/L);

and (3) normal hepatic function tests (serum aspartate

aminotransferase< 30U/L; alanine aminotransferase< 30U/L;

alkaline phosphatase between 80U/L and 280U/L; and

gamma-glutamyl transferase< 60U/L). Premenopausal women

were included if they were between 20 and 45 years of age, and

had serum FSH levels< 15mIU/L, and were cycling regularly.

Postmenopausal women were included if they had experienced

their last menstrual cycle at least one year before and were

not taking any medications that are known to affect bone

metabolism. The levels of FSH in postmenopausal women

were> 15mIU/L. Accordingly, a total of 1803 healthy Saudi

women (age range 20 to 79 years) (premenopausal, n¼ 1235;

postmenopausal, n¼ 568) living in the Jeddah area, participated

in the present study and were included in the final analysis

(see Fig. 1). Demographic characteristics, hormones, and BTMs

together with BMD values for these women are presented in

Table 1.

Bone Mineral Densitometry Measurements

BMD (g/cm2) was determined for the anteroposterior lumbar

spine (L1–L4) andmean of proximal right and left femur (total and

subregions) by dual-energy X-ray absorptiometry (DXA), using

LUNAR Prodigy Model (Lunar Corp., Madison, WI, USA) according

to standard protocol. Quality-control procedures were carried

out in accordance with the manufacturer’s recommendations as

described previously.(25) BMD values were classified according

to WHO criteria: a T-score between �1 and �2.5 is indicative

of osteopenia, whereas a T-score of �2.5 and below reflects

osteoporosis, and a T-score of �1.0 and above is considered

normal.(27)

Specimen Collection

Venous blood samples were collected in the morning under

standardized conditions. Second-void morning urine samples

were collected on the same day of blood sampling. Serum and

SCLEROSTIN IN RELATION TO AGE, BMD, AND BTMS IN WOMEN Journal of Bone and Mineral Research 2813

urine samples were stored at �858C within 30minutes after

centrifugation at 2500 g for 10minutes. All samples were

collected between 9:00 and 11:00 a.m., after an overnight fast.

The samples were stored until analyzed for the determinations

of serum sclerostin, s-OC, s-PINP, s-CTX, and u-NTX. Serum

sclerostin and all biochemical BTMs together with other

hormones, and other analytes were carried out at the same

time-point, according to the manufacturer’s instructions.

Measurements of Sclerostin and Biochemical BTMs

Serum sclerostin levels were measured on coded specimen by

enzyme-linked immunosorbent assay (ELISA) supplied by

Biomedica (Biomedica Gruppe, Biomedica Medizinprodukte

GmbH & Co KG, Wien, Austria). This assay uses a polyclonal

goat anti-human sclerostin antibody as a capture antibody and a

biotin-labeled mouse monoclonal anti-sclerostin antibody for

detection. The intra-assay and interassay coefficients of varia-

tions (CVs) were 4.5% and 5.6%, respectively. Further validation

studies were performed for this assay. Linearity was assessed

serially diluting serum samples with sample diluents (dilutions

1:2, 1:4, and 1:8) and comparing observed values with expected

values (observed recoveries ranged from 102% to 108%) (data

not shown). Also, recovery of spiked standards was tested by

adding different concentrations of human recombinant scler-

ostin (three different concentrations) into eight different human

serum samples presenting with various levels of endogenous

sclerostin. Spiked recovery ranged from 92% to 108% (data not

shown). Serum OC was measured using electrochemilumines-

cence immunoassay (ECLIA) Elecsys autoanalyzer (Roche

Diagnostics GmbH, D-68298 Mannheim, Germany). The intra-

assay and interassay CVs were 1.8% and 1.2%, respectively.

Serum PINP was measured using ECLIA Elecsys autoanalyzer

[Roche Diagnostics GmbH, D-68298 Mannheim, Germany]. The

intra-assay and interassay CVs were 2.3% and 2.1%, respectively

with a sensitivity of< 5mg/L. Serum CTX was measured by

Elecsys b-CrossLaps assay using ECLIA Elecsys autoanalyzer.

The intra-assay and interassay CVs were 4%. Urinary NTX was

determined by utilizing a competitive-inhibition ELISA using

Osteomark kits (Ostex International, Seattle, USA). The intra-assay

and interassay CVs were 7.8% and 4.5%, respectively. In all

manual assays, a standard curve was run simultaneously with the

samples, and the curve was fitted for serum sclerostin and u-NTX;

the results were calculated using MultCalc (Wallac, Turku,

Finland). The results of all u-NTX were corrected for creatinine

(creat) concentration (mmol creat/l).

Measurements of Hormones and Other Analytes

Serum FSH, luteinizing hormone (LH), E2, and intact-PTH

were measured by commercially available immunoassays using

Elecsys autoanalyzer (Roche Diagnostics GmbH, D-68298

Mannheim, Germany). The intra-assay and interassay CVs were

less than 4.0%. Serum 25(OH)D was measured by direct

competitive chemiluminescence immunoassay using LIASON

autoanalyzer (DiaSorin Inc, Stillwater, MN, USA). The intra-assay

and interassay CVs were 7.8% and 3.8%, respectively. Serum

creatinine (creat), calcium (Ca), phosphate (PO4), magnesium

(Mg), and other biochemical analytes were measured by kits

and reagents supplied by Ortho-Clinical Diagnostics, USA using

Vitros 250 Chemistry System Autoanalyzer (Ortho-Clinical

Diagnostics–Johnson & Johnson Co., USA).

Statistical Analysis

Results are presented as means or Geometric means (� SD) and

categorical variables are expressed as frequencies as appropri-

ate. Data were analyzed using SPSS-Statistical Package (version

15.0 for Windows Smart Viewer) supplied by SPSS Inc. 2000,

Mapinfo Corp. (Troy, NY, USA). Results that were not normally

distributed were log-transformed before analysis. A 95%

reference intervals for serum sclerostin together with that for

s-OC, s-PINP, s-CTX, and u-NTX measured were calculated for

each as mean� 1.96 SD, and the confidence intervals (CI) for the

lower and upper bounds of the reference intervals were

computed as boundary� 1.96 standard error (SE). Initially locally

weighted scatterplot smoothing (LOWESS) curve analysis was

used to establish the trend of serum sclerostin and measured

biochemical BTMs data among the various age groups (five-year

bands). The LOWESS curve analysis is used to fit a curve to data

without selecting a model and cannot be used to obtain the

best-fit values; however, it is very useful in providing a simple

approach to produce smoothing of the data.(28) Thus, in the

present analysis of serum sclerostin and the biochemical BTMs

measured, the LOWESS curve analysis was applied to establish

the general trend and distribution of the scatter of BTMs data

Fig. 1. Subjects screening and recruitment according to inclusion and

exclusion criteria. A total of 5850 women originally recruited, of which

only 1803 were included in the final analysis and 4047 women were

excluded accordingly.

2814 Journal of Bone and Mineral Research ARDAWI ET AL.

among the various age groups. This approach was followed by

segmental linear regression analysis to fit two regression lines to

serum sclerostin and BTMs data by using suitable critical cut-off

point. Accordingly, the gradient of each regression line

generated was used to compare differences between the lines.

Further, ANOVA testing (independent two-samples t-test) was

applied to examine whether serum sclerostin and measured

BTMs values differ among women aged 30 to 34 or younger

versus women who were 35- to 45-years-old. Based on the above

two approaches, women aged 35- to 45-years-old were used for

further analysis to examine other determinants of serum

sclerostin and measured BTMs values to remove the confound-

ing effects of age. All women were stratified by 10-years age and

also by quartiles of serum FSH, LH, PTH, and E2 to assess

variations in serum sclerostin among these variables using one-

way ANOVA testing. Associations between continuous variables

were examined by Pearson’s correlation coefficient. ANOVA was

used to examine differences among the groups for different

variables, and the Bonferroni criterion was used when signifi-

cance tests were made. Independent relationships between

serum sclerostin and that of measured biochemical BTMs and

other variables were assessed by multiple regression and partial

correlation analysis.

Results

Table 1 shows the basic demographic, anthropometric char-

acteristics, various hormones, BTMs, and BMD values of

the studied premenopausal (n¼ 1235) and postmenopausal

(n¼ 568) women with a mean age of 42.82� 15.69 years.

Of the women studied, 24.9%, 34.9%, and 40.2% were lean

(BMI< 25.0 kg/m2); overweight (BMI� 25.0 to< 30.0 kg/m2); and

obese (BMI � 30 kg/m2), respectively. Postmenopausal women

exhibited higher parity (p< 0.0001), BMI (p< 0.0001), and WHR

(p< 0.0001) values compared with premenopausal women

(Table 1). Postmenopausal women exhibited higher levels of

serum FSH (p< 0.0001), LH (p< 0.0001), and intact-PTH

(p< 0.0001), but lower E2 (p< 0.0001) compared with premen-

opausal women. All premenopausal women exhibited normal

BMD values for lumbar spine (L1–L4) and femoral neck with lower

values for postmenopausal women. However, postmenopausal

Table 1. Demographic, Anthropometric Characteristics, Various Hormones, 25(OH)D, BTMs, Minerals, and BMD Values of the Studied

Healthy Pre- and Postmenopausal Women

Variables

Menopausal Status

ANOVA (p-value)Premenopausal (n¼ 1235) Postmenopausal (n¼ 568)

Age (yrs) 33.83� 8.41 62.38� 8.34 < 0.0001

Age at menarche (yrs) 12.46� 3.82 14.20� 4.16 < 0.051

Age at menopause (yrs) — 47.51� 6.18 —

Years since menopause (yrs) — 15.62� 8.75 —

Parity 3.28� 2.61 4.18� 3.23 < 0.0001

BMI (kg/m2) 27.97� 5.30 31.63� 6.07 < 0.0001

Lean (BMI< 25) 30.4% 12.9% < 0.001

Overweight (BMI � 25 –< 30) 37.6% 29.0% < 0.001

Obese (BMI � 30) 32.0% 58.1% < 0.0001

WHR 0.817� 0.082 0.897� 0.082 < 0.001

FSH (IU/L) 4.57� 2.48 28.72� 7.47 < 0.0001

LH (IU/L) 8.07� 2.10 25.59� 7.34 < 0.0001

E2 (pmol/L) 369.2� 87.3 135.1� 56.6 < 0.0001

Intact-PTH (pmol/L) 2.72� 0.76 4.55� 0.96 < 0.0001

25(OH)D (nmol/L) 66.47� 13.02 55.74� 5.32 < 0.0001

s-OC (ng/mL) 13.30� 4.51 17.18� 3.61 < 0.0001

s-PINP (mg/L) 51.33� 22.73 47.38� 5.52 < 0.0001

s-CTX (pg/ml) 279.5� 67.8 338.9� 43.7 < 0.0001

u-NTX (nmol/mmol creat) 34.49� 7.81 57.69� 8.36 < 0.0001

Serum Ca (mmol/L) 2.38� 0.13 2.38� 0.09 0.145

Serum PO4 (mmol/L) 1.25� 0.16 1.27� 0.16 < 0.015

Serum Mg (mmol/L) 0.891� 0.09 0.791� 0.16 < 0.031

BMD (g/cm2)

Lumbar spine (L1–L4) 1.079� 0.279 0.991� 0.074 < 0.001

Neck femur 1.107� 0.144 0.928� 0.075 < 0.001

BMI¼body mass index; WHR¼waist-to-hip ratio; FSH¼ follicle-stimulating hormone; LH¼ luteinizing hormone; E2¼ estradiol; intact-PTH¼ intact

parathyroid hormone; 25(OH)D¼ 25-hydroxyvitamin D; s-OC¼ serum osteocalcin; s-PINP¼ serum procollagen type-1 N-terminal propeptide;

s-CTX¼ serum cross-linked C-terminal telopeptide of type 1 collagen; u-NTX¼urinary N-telopeptide of type 1 collagen; Creat¼ creatinine;

Ca¼ calcium; PO4¼phosphate; Mg¼magnesium; BMD¼bone mineral density.Mean� SD, or as percentage.

SCLEROSTIN IN RELATION TO AGE, BMD, AND BTMS IN WOMEN Journal of Bone and Mineral Research 2815

women showed significantly higher s-OC, s-PINP, s-CTX, and u-

NTX values and significantly lower s-PINP and serum 25(OH)D

levels compared with premenopausal women (Table 1).

Using LOWESS curve analysis, the sclerostin values exhibited a

general trend showing that women under the age of 35 years

showed decreased sclerostin values: accordingly, segmental

linear regression analysis was performed using the critical values

of 35 (Fig. 2). Supplemental Table 1 shows two types of analysis:

(1) calculation of gradients of segmental linear regression for

95% CIs for women in the age group 30 to 34 years compared

with that of 35 to 45 years; and (2) independent t-testing

between the age group 30 to 34 years compared with that of 35

to 39 years, 40 to 45 years, and 35 to 45 years. Using the gradient

of segmental regression line for women aged 30 to 34, it was

significantly different from zero compared with the same values

obtained for women in the age group 35 to 45 years (which was

not significantly different from zero), as indicated by the CI values

(Supplemental Table 1). The ANOVA testing showed significantly

lower values for serum sclerostin for the age group 30 to 34 years

compared with that observed in the age group 35 to 45 years

(see Supplemental Table 1). Similar trends with higher values

among the age group 30 to 34 years compared with that among

the age group 35 to 45 years were obtained for the BTMs: s-OC,

s-PINP, s-CTX, and u-NTX. Accordingly, a total of 443 premeno-

pausal women (age 35 to 45 years) were used to establish

sclerostin reference normative values, and their demographic

characteristics and other variables are given in Table 2.

In the present study, the levels of serum sclerostin were stable

among the age group 35 to 45 years, and the reference

normative intervals of serum sclerostin are given in Supplemen-

tal Table 2, including values for geometric mean; log10mean� SD; 95% reference interval; and lower and upper limits

of 95% CIs. The geometric mean and SD values for serum

sclerostin for the 10-year age grouping of women studied are

presented in Table 3, together with relevant BTMs, compared

with that of the reference normative intervals group. Serum

sclerostin showed a general linear increase with age (Fig. 3), with

serum sclerostin showing a steep increase until the age of 35,

and remaining stable in those between 35 and 45 years of age;

then, serum sclerostin exhibited an increase with increasing age.

Stratifying women according to BMI (kg/m2) showed that obese

women (� 30) had significantly higher serum sclerostin values by

26.4% over that for lean (< 25) women (p< 0.049) (data not

shown). However, stratifying women by quartiles of WHR

showed no significant differences among the groups (data

not shown). Supplemental Table 3 shows the mean values of

serum sclerostin in relation to years since menopause (YSM) in

postmenopausal women studied compared with the premeno-

pausal values. Significant increases were observed for serum

sclerostin among postmenopausal women according to YSM

periods. Similar trends were evident for the BTMs examined

(Supplemental Table 3).

In premenopausal women, serum sclerostin showed signifi-

cant positive correlations with the following variables:

age (r¼ 0.879; p< 0.0001); BMI (r¼ 0.223; p< 0.0001); parity

(r¼ 0.368; p< 0.0001); serum FSH (r¼ 0.595; p< 0.0001); and

serum LH (r¼ 0.538; p< 0.001), respectively. Negative correla-

tions were evident between serum sclerostin and the following

variables: s-OC (r¼�0.671; p< 0.0001); s-PINP (r¼�0.713;

p< 0.0001); s-CTX (r¼�0.767; p< 0.0001); u-NTX (r¼�0.432;

p< 0.0001); serum E2 (r¼�0.519; p< 0.0001); serum intact-PTH

Fig. 2. Correlation between age (years) and serum sclerostin levels

among all women (n¼ 1803) studied. Significant positive correlation

was observed (R2¼ 0.828; p< 0.0001).

Table 2. Anthropometric Characteristics and Other Variables

in Premenopausal Women Used for Reference Intervals of

Biochemical Bone Turnover Markers

Variables n Results

Age (years) 443 39.83� 3.25

BMI (kg/m2) 443 29.10� 4.99

WHR 443 0.830� 0.087

Parity

0 (nullipara) 88 19.9%

1–2 73 16.5%

3–4 192 43.3%

� 5 90 20.3%

Physical activity

Exercise: Yes 252 56.9%

No 191 43.1%

Regular: Yes 74 29.4%

No 178 70.6%

Total dietary: Ca intake (mg/day)

< 600 290 65.5%

> 600–1000 101 22.8%

> 1000 52 11.7%

Total caffeine intake (mg/day)

< 200 285 64.3%

200–400 91 20.5%

> 400 67 15.2%

Vitamin D intake (IU/day)

None 225 50.8%

< 400 178 40.2%

� 400 40 9.0%

25(OH)D (nmol/L) 443 66.08� 13.55

n¼number; BMI¼body mass index; WHR¼waist-to-hip ratio;25(OH)D¼ 25-hydroxyvitamin D.

Mean� SD, or as percentage.

2816 Journal of Bone and Mineral Research ARDAWI ET AL.

(r¼�0.123; p< 0.001); serum 25(OH)D (r¼�0.072; p< 0.011);

BMD lumbar spine (L1–L4) (r¼�0.168; p< 0.0001); and BMD

neck femur (r¼�0.545; P< 0.0001), respectively. In premeno-

pausal women age- and BMI-adjusted correlations persisted for

s-OC (r¼�0.095; p< 0.014); s-CTX (r¼�0.289; p< 0.0001);

u-NTX (r¼�0.359; p< 0.0001); intact-PTH (r¼�0.111; p< 0.01);

FSH (r¼�0.122; p< 0.002); and E2 (r¼ 0.091; p< 0.020),

respectively. In postmenopausal women, serum sclerostin

showed significantly positive correlations with age (r¼ 0.696;

p< 0.0001); BMI (r¼ 0.120; p< 0.039); WHR (r ¼0.131; p< 0.042);

parity (r¼ 0.153; p< 0.0001); YSM (r¼ 0.672; p< 0.0001) and

s-OC (r¼ 0.479; p< 0.0001), respectively. However, significant

negative correlations were observed between serum sclerostin

and various variables including: serum FSH (r¼�0.622;

p< 0.0001); LH (r¼�0.646; p< 0.0001); serum E2 (r¼�0.256;

p< 0.001); serum intact-PTH (r¼�0.172; p< 0.004); serum

25(OH)D (r¼� 0.303; p< 0.0001); BMD lumbar spine (L1–L4)

(r¼�0.354; p< 0.0001); and BMD neck femur (r¼�0.372;

p< 0.0001), respectively (data not shown). In postmenopausal

women, age-adjusted and BMI-adjusted correlations persisted

for FSH (r¼�0.101; p< 0.018); and E2 (r¼�0.122; p< 0.002),

respectively. Moreover, when combining both pre- and

postmenopausal women (n¼ 1803), serum sclerostin showed

significant correlations with various variables examined, includ-

ing: age (r¼ 0.893; p< 0.0001); BMI (r¼ 0.271; p< 0.0001); WHR

(r¼ 0.146; p< 0.012); parity (r¼ 0.299; p< 0.0001); YSM

(r¼ 0.731; p< 0.0001); FSH (r¼ 0.587; p< 0.0001); LH

(r¼ 0.520; p< 0.0001); intact-PTH (r¼ 0.679; p< 0.0001); and

s-OC (r¼ 0.076; P <0.001), respectively. Significant negative

correlations were evident between serum sclerostin and other

variables: s-PINP (r¼�0.441; p< 0.0001); s-CTX (r¼�0.080;

p< 0.001); u-NTX (r¼�0.121; p< 0.001); serum E2 (r¼�0.751;

p< 0.0001); serum 25(OH)D (r¼�0.350; p< 0.0001); serum

calcium (r¼�0.055; p< 0.018); BMD lumbar spine (L1–L4)

(r¼�0.670; p< 0.0001) and BMD neck femur (r¼�0.312;

p< 0.0001), respectively (data not shown). In all women, age-

adjusted and BMI-adjusted correlations persisted for s-OC

(r¼ 0.165; p< 0.0001); s-PINP (r¼ 0.115; p< 0.0001); s-CTX

(r¼ 0.087; p< 0.0001); u-NTX (r¼ 0.123; p< 0.0001); FSH

(r¼ 0.187; p< 0.001); LH (r 0.112; p< 0.001); and E2 (r¼ 0.109;

p< 0.0001), respectively.

Serum sclerostin is identified as an independent variable in the

multiple linear stepwise regression analysis, whereas variables

showing a significant correlations (age, BMI, parity, FSH, LH, E2,

intact-PTH, 25[OH]D, s-OC, s-PINP, s-CTX, u-NTX, BMD lumbar

spine [L1–L4] and BMD neck femur) are identified as dependent

variables; the best predictors in all women were age, s-OC, FSH,

and E2, accounting for about 84% of the variance in serum

sclerostin (Supplemental Table 4). We also analyzed predictors

of serum sclerostin separately for pre- and postmenopausal

women. In premenopausal women, age, intact-PTH, E2, and FSH

were the best predictors of serum sclerostin, accounting for

about 73% of the variance. Whereas, in postmenopausal women

only age, FSH, and E2 were found to be predictors of serum

sclerostin, accounting for about 62% of the variance in serum

sclerostin (Supplemental Table 4).

Discussion

This study provides the first and the largest population-based

study of pre- and postmenopausal women using a commercially

available validated immunoassay for serum sclerostin. We have

shown that serum sclerostin levels are stable between the ages

of 35 and 45 in this group of premenopausal women. In the

present study the reference normative intervals for serum

sclerostin were identified to be in the age group of 35 to 45 years.

Women under the age of 30 years were excluded from the

reference normative intervals group: this lowered the possibility

of including women with lowered serum sclerostin as a result

of skeletal immaturity, lowered osteocytes activity, or both.

Further, women over 45 years were also excluded, because

detailed analysis showed that serum sclerostin increased beyond

this cut-off age. Similar observations were evident for the

analysis of the measured BTMs confirming previous studies.(25,29)

The study shows that serum sclerostin levels increased

Table 3. Geometric Mean and 95% Confidence Interval (95% CI) Age-Specific Values of Sclerostin and Bone Turnover Markers Among

Women Studied

Age group n Sclerostin (pmol/L) s-OC (ng/mL) s-PINP (mg/L) s-CTX (pg/mL)

u-NTX

(nmol/mmol creat)

20–29 435 16.87a (16.54–17.20) 17.27a (17.11–17.43) 78.79a (77.49–80.09) 360.9a (358.3–363.5) 29.05 (28.62–29.48)

30–39 435 25.04 (24.68–25.40) 11.18a (10.81–11.55) 33.90 (33.28–34.52) 200.9a (197.4– 204.4) 18.74a (18.31–19.17)

40–49 365 30.80a (30.15–31.45) 9.66 (9.35–9.97) 35.30 (34.63–35.97) 198.0 (195.1–200.9) 25.09 (23.75–26.43)

50–59 246 40.29a (39.41–41.17) 14.45a (14.06–14.84) 46.63a (45.98–47.27) 334.6a (330.4–338.8) 51.78a (51.23–52.34)

60–69 196 51.01a (49.48–52.54) 19.34a (19.08–19.60) 49.37a (48.67–50.07) 373.2a (369.5–376.9) 63.86a (62.74–64.98)

70–79 126 61.95a (59.98–63.91) 19.16a (18.64–19.68) 45.75a (44.66–46.84) 294.4a (287.4–301.4) 59.65a (58.45–60.85)

Total (20–79) 1803 31.72 (31.03–32.41) 14.24 (14.02–14.46) 49.31 (48.40–50.22) 282.4 (278.6–286.2) 34.79 (33.95–35.63)

Reference

valueb (35–45)

443 26.68 (26.39–26.97) 9.09 (2.54–15.20) 31.8 (21.90–32.31) 181.7 (179.3–230.2) 30.84 (19.35–46.32)

CI¼ confidence interval; n¼ number of women; s-OC¼ serum osteocalcin; s-PINP¼ serum procollagen type-1 N-terminal propeptide; s-CTX¼ serumcross-linked C-terminal telopeptide of type 1 collagen; u-NTX¼urinary N-telopeptide of type 1 collagen; Creat¼ creatinine.

All values are presented as geometric means with (95% CI).aStatistically significant from reference values.bReference values were calculated for the age group 35 to 45 years as geometric mean with (95% CI).

SCLEROSTIN IN RELATION TO AGE, BMD, AND BTMS IN WOMEN Journal of Bone and Mineral Research 2817

significantly with age in women. Thus, serum sclerostin levels

increased over life by an average of 3.7-fold (p< 0.0001): this

suggests that increased sclerostin production by osteocytes may

be involved in the age-related impairment of bone formation.

Moreover, given that sclerostin is produced almost exclusively

by osteocytes,(1,30,31) this observation is concurrent with the

expected changes in bone mass associated with aging. However,

it is possible that the increase with age of serum sclerostin was

related, at least partly, to decreased clearance of the protein, but

the increased serum sclerostin with age was minimally affected

after adjustment for the expected age-related decreases in

creatinine clearance. The large number of women studied

ensured a small uncertainty around the upper- and lower-

reference limits and thus, providing reliable reference ranges for

serum sclerostin in studied women. Women using hormonal

contraceptives were excluded from the study, because several

previous studies showed that bone remodeling, bone turnover,

or both are markedly decreased in women receiving combined

oral contraception, particularly women over 35 years of age.(25,29)

The mean reference normative interval values obtained for

serum sclerostin in the present study were similar to that given

by the manufacturer and the ranges for the values reported here

are higher than that reported for premenopausal women

(n¼ 123) by Modder et al. (15.1� 1.2 pmol/L) using the same

immunoassay system.(23) Such differences are most likely

because of differences in the selection criteria of women

studied as well as the mean age of the group studied by Modder

et al., although no information was given on the mean of the age

of women studied.(23) It is possible, however, that environmental

or lifestyle and/or genetic influences on bone mass, and

consequently serum sclerostin, not accounted for in the present

study should be taken into consideration to account for any

differences in serum sclerostin levels among the women studied

or to be studied in the future.

In the present study, the pattern of age-related changes

in serum sclerostin among women is shown by the increase in

bone turnover at menopause. Postmenopausal women showed

significantly higher serum sclerostin levels (48.79� 12.68 pmol/L;

n¼ 568) than premenopausal women (26.90� 9.71 pmol/L;

n¼ 1235). The higher serum sclerostin levels in postmenopausal

women maybe a cause, an effect, or both of the increased bone

turnover occurring in the postmenopausal state. These observa-

tions are consistent with previously reported studies by Modder

et al.(23) and by Polyzos et al.(32) Similar changes were obtained

for the BTMs studied confirming previous studies in different

populations.(25,29) However, it is uncertain whether the onset

of the changes in serum sclerostin takes place early in the

menopausal transition or before the cessation of menstruation.

In addition, the results of the present study showed that serum

sclerostin continued to increase with increasing YSM and

remained elevated in the elderly (Supplemental Table 3). This

is possibly related to the expected increased bone turnover (and

possibly osteocytic activities) associated with estrogen imbal-

ance,(33) with the rate of bone turnover slowing down after 4 to

8 years for most women,(34) whereas serum sclerostin continued

to increase with increasing YSM in the present study. To our

knowledge, there is no information in the literature on the

changes of serum sclerostin in relation to YSM.

Fig. 3. Scatterplots showing segmental regression analysis for age

and serum sclerostin (A), together with that for serum osteocalcin (B);

serum procollagen type 1 N-terminal propeptide (s-PINP) (C); serum

cross-linked C-terminal telopeptide of type 1 collagen (s-CTX) (D); urinary

N-telopeptide of type 1 collagen (u-NTX) (E), respectively, among

668 women aged 30 to 45 years.

2818 Journal of Bone and Mineral Research ARDAWI ET AL.

Previous studies have shown that BMI is an important

determinant of bone turnover and BMI plays a major role in bone

mass in women regardless of gonadal status and is a key risk

factor for osteoporotic fractures.(35,36) In the present study,

women with higher BMI exhibited significantly higher serum

sclerostin: thus serum sclerostin was 26.4% higher in obese

versus lean women (p< 0.0001). Conversely, women with higher

BMI exhibited significantly lower s-CTX and u-NTX values and a

trend toward lower s-OC and s-PINP values (data not shown).

Based on the previously known effects of sclerostin in inhibiting

bone formation,(1,30) bone-formation markers would be

expected to be inversely related to serum sclerostin levels.

Indeed, in the present study, premenopausal women exhibited

significantly negative correlations between serum sclerostin and

BTMs examined (namely, s-OC, s-PINP, s-CTX, and u-NTX);

however, such correlations disappeared after adjustment for age

and BMI. Conversely, in postmenopausal women, significant

positive correlations with s-OC, but negative weak correlations

with bone resorption markers (namely, s-CTX and u-NTX) with

serum sclerostin were observed, respectively; however, none

of these correlations persisted after adjustment for age and

BMI. These findings are consistent with that obtained for both

pre- and postmenopausal women reported previously(22) and

contrast with that reported by Modder et al. for postmenopausal

women studied.(23) However, these results indicate that in

healthy women serum sclerostin may provide information on

bone metabolism, ie, osteocyte functional activity, which is not

reflected and/or captured by measured BTMs.

In the present study, significant negative correlations were

evident between serum sclerostin and BMD for both lumbar

spine (L1–L4) and neck femur for both pre- and postmenopausal

women, which persisted when the entire population was

examined. However, these correlations disappeared after

adjustment for age and BMI, in contrast to what was reported

previously for elderly women, but concurrent with what was

reported in premenopausal women.(23)

In the present study, serum FSH and LH levels in postmeno-

pausal women are 6.3 and 3.2 times greater, respectively, than

those in premenopausal women (Table 1). Several studies

showed that serum FSH levels are not only related to age and

menopause but also to ethnic backgrounds.(37–39) Our results

showed that a decline in ovarian function, as indicated indirectly

by menopausal rise in serum FSH levels, is associated with higher

serum sclerostin and bone loss. Furthermore, serum FSH levels in

the studied women showed significantly positive correlations

with serum sclerostin. In addition, serum FSH exhibited positive

correlations with the BTMs examined (data not shown),

suggesting that bone turnover speed becomes more rapid in

women with menopausal rise in FSH; such finding for BTMs is

similar to those of other studies.(40) In contrast, however, Vural

et al.(41) found no correlation between serum FSH and that of

s-OC. Our results also show that serum sclerostin in quartile

groups with high serum FSH (Q3 and Q4) are higher than those in

the quartile groups with lower FSH levels (Q1 and Q2) (data not

shown). Moreover, we have examined and compared the effects

of serum FSH and LH on serum sclerostin and BTMs using

multiple linear regression analysis, and we have found that the

association between serum FSH and serum sclerostin and BTMs

is greater than that with serum LH. These phenomena indicate

that FSH in circulation is more significantly associated with

osteoblasts and possibly osteocytes than with osteoclasts, results

that contrast from those of previous in vitro cellular and

experimental animal studies.(42,43) However, the recent work by

Drake et al. showed that FSH did not directly regulate bone

resorption in postmenopausal women after suppression of their

FSH secretion.(44) The significant association between serum

sclerostin, FSH, and LH are considered to be the first reported in

the literature, and further studies in other populations should be

conducted to further our understanding of this association,

particularly at the cellular levels (ie, osteocytes).

Estradiol deficiency is considered to be partly responsible for

the rapid bone loss after menopause.(43) In the present study,

serum E2 showed significant negative correlations with serum

sclerostin in both pre- and postmenopausal women, which

persisted after adjustment for age and BMI. Further, serum E2 was

significantly associated with serum sclerostin in both pre- and

postmenopausal women. Modder et al., observed among

postmenopausal women that serum sclerostin levels were

significantly lower in women on estrogen therapy (ET) compared

with women not on ET.(45) These observations were concurrent

with the recent observations from the same group showing that

ET of postmenopausal women for 4 weeks resulted in a 27%

decrease in serum sclerostin levels.(47) Such observations are

consistent with the correlation analysis of the present study and

consistent with previous findings by both Mirza et al.(22) and

Modder et al.,(45) but contrast with that reported more recently

by Modder et al. for both pre- and postmenopausal women.(23)

Parathyroid hormone has been shown to decrease sclerostin

transcription in vitro,(46,47) and continuous or intermittent

chronic administration of PTH to rodents is associated with

decreased SOST-mRNA and sclerostin expression in osteo-

cytes.(48,49) Moreover, van Lierop et al. showed significantly

lowered serum sclerostin in patients with primary hyperpara-

thyroidism compared with euparathyroid controls.(48) The

present study showed an inverse relationship between serum

sclerostin and intact-PTH levels: such a relationship was stronger

for postmenopausal (r¼�0.172; p< 0.004) compared with that

of premenopausal (r¼�0.111; p< 0.01) women, respectively.

Also, stratifying women into quartiles of serum intact-PTH

showed that serum sclerostin was significantly lower in women

with the highest quartile (Q4) of intact-PTH compared with the

lowest quartile (Q1) (p< 0.001) (data not shown). Furthermore,

PTH contributed significantly to serum sclerostin variation

among the premenopausal women. Thus, our results are

consistent with the observations of Mirza et al.(22) for

postmenopausal women but contrast with that observed by

Modder et al. for both pre- and postmenopausal women.(23) Also,

our results are indirectly consistent with the recent report by

Drake and colleague, on the observed significant reduction in

circulating sclerostin levels after intermittent PTH 1-34 treatment

of postmenopausal women.(49) Interestingly, in this latter study,

bone marrow plasma and peripheral serum sclerostin levels

were significantly correlated (p< 0.0001), suggesting that the

circulating levels may be a good index of local bone production.

Taken all together, these observations including the results of the

present study, lend further support to the hypothesis that, in

SCLEROSTIN IN RELATION TO AGE, BMD, AND BTMS IN WOMEN Journal of Bone and Mineral Research 2819

humans and experimental animals, at least part of the anabolic

effect of PTH on bone may be mediated via an inhibition of

sclerostin production and/or stimulation of its clearance.

The present study has several strengths and limitations. The

strengths of our study include its large sample size and

acceptable sampling errors; its random selection of women from

the local population and covering a wide range of ages, thus,

avoiding sampling bias; and its very strict detailed inclusion

criteria and comprehensive details of lifestyle characteristics and

BMDmeasurements. In addition, another strength of our study is

that minimizing pre-analytical variations as a result of possible

circadian rhythm and/or food intake was ensured by standard-

ized sampling time and that all women studied were fasting

overnight. However, currently there is no published information

on the effects of various pre-analytical factors (eg, variations

related to food intake or fasting, seasonal changes, or circadian

rhythm) on serum sclerostin; such information requires further

study. The principal limitation of the present study was its cross-

sectional design, and thus the causative nature of the

associations between sclerostin and other variables cannot be

established. Moreover, another limitation of our study is the

single fasting measurement of serum sclerostin and other

analytes (eg, PTH, E2). The accuracy of self-reported data

concerning lifestyle practices may have been subject to report

bias, as can be the case with such type of studies; we

acknowledge that there may be unrecognized confounding.

Women who participated in the present study may have chosen

to take part because they are more aware of health issues and

healthier than average. Also, women in the present study

exhibited low dietary calcium intake (about 65.5% of women had

daily calcium intake estimated at< 600mg/day); that this will

affect the observations of the present study cannot be ruled out,

and further studies are needed in this regard. Furthermore, our

results were based on premenopausal women living in Jeddah,

Saudi Arabia, with quite rigorous inclusion criteria; thus, whether

the findings reported here will be completely applicable to other

populations remains to be determined in other populations.

Finally, we recognize that there is currently no information on the

metabolism or renal clearance of sclerostin and/or the stability of

sclerostin or its degradation in circulation and/or upon storage at

�858C; further studies are needed in this regard.

In summary, our study represents the first randomly selected,

large population-based assessment of circulating sclerostin

levels and provides reference normative interval values among

pre- and postmenopausal women over a wide age range using

a well-validated immunoassay. The study protocol specified

detailed inclusion criteria, which combined BMD measurement

and through medical examination, allowed a well-defined and

characterized study population to be examined. The results of

the present study point to the need for future further studies

examining the mechanisms for the age-related changes in

serum sclerostin levels described in pre- and postmenopausal

women, and also the contribution of sclerostin in mediating

the well-established age-related decrease in bone formation in

humans.(1,30) Serum FSH together with that of E2 showed

significant associations with serum sclerostin in both pre- and

postmenopausal women. The results have confirmed that the

levels of serum sclerostin increase in women with increasing

serum FSH and LH levels, and that FSH showed stronger

association with serum sclerostin than LH, particularly in

postmenopausal women. Serum sclerostin was poorly associated

with BTMs (except for s-OC), and based on its relationship with

measured BTMs in the present study indicate that serum

sclerostin measurements provide additional information on

bone metabolism that are not captured by currently available

BTMs, including the activity of osteocytes in vivo and the

modulation of their functions by mechanical loading and

therapeutic intervention in osteoporosis and other bone

diseases. However, measuring serum sclerostin and BTMs

simultaneously may prove helpful in evaluating and/or monitor-

ing the changes in the rate of bone turnover and ostecytic

function caused by aging or menopause in women or in bone-

diseased states in humans.

Disclosures

All the authors state that they have no conflicts of interest.

Acknowledgments

We are grateful to the Ministry of Higher Education for financial

support to the Center of Excellence for Osteoporosis Research

(CEOR) at King Abdulaziz University, Jeddah, Saudi Arabia. This

study was supported by grants from the Ministry of Higher

Education to the Center of Excellence for Osteoporosis Research

(CEOR) at King Abdulaziz University (grants # CEOR/001-08 and

CEOR/004-08), Jeddah, Saudi Arabia. It was approved by the

Human Research Ethical Committee of the Center of Excellence

for Osteoporosis Research, and the study protocol was in agree-

ment with King Abdulaziz University Hospital (KAUH) ethical

standards and the Helsinki Declaration of 1975, as revised in

1989. We thank all the subjects who participated in the study,

and we thank all the staff and colleagues at CEOR, King Abdulaziz

University Hospital, and the Primary Care Health Centers for their

invaluable assistance during the execution of the study. Special

thanks are due to Ms Veronica Orbacedo for her excellent help in

preparing the manuscript. Professor MSM Ardawi was awarded

grants CEOR/001-08 and CEOR/004-08 from Center of Excellence

for Osteoporosis Research, Supported by MOHE, Saudi Arabia.

Authors’ roles: All authors have contributed to the design of

the study, analysis and interpretation of data, the writing of the

article, and approval of the version to be published.

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