alvina 155 ABSTRACT *Department of Occupational Medicine, Faculty of Medicine, Trisakti University Correspondence Lie T. Merijanti Susanto, dr, MKK Department of Occupational Medicine, Faculty of Medicine, Trisakti University Jl. Kyai Tapa No. 260 Grogol Jakarta 11440 Phone: 021-5672731 ext.2802 Email: lie_mery@hotmail.com Univ Med 2011;30:155-61. Serum osteocalcin and bone mineral density in postmenopausal women Lie T. Merijanti Susanto* September-December, 2011September-December, 2011September-December, 2011September-December, 2011September-December, 2011 Vol.30 - No.3 Vol.30 - No.3 Vol.30 - No.3 Vol.30 - No.3 Vol.30 - No.3 UNIVERSA MEDICINA Since high bone turnover is associated with decreased bone mass, biochemical markers of bone remodeling, such as serum osteocalcin, may be used to assess osteoporosis and to predict fractures in elderly women, particulary those involving trabecular bone, and use of a combination of bone mineral density (BMD) and biochemical markers may improve fracture prediction. The serum levels of osteocalcin constitute a specific biochemical parameter of bone formation. Compared to imaging techniques, assays for osteocalcin are safe, noninvasive and easily performed. The aim of this study was to determine the relationship of serum osteocalcin and BMD in postmenopausal women. A cross sectional study was performed on 53 postmenopausal women in South Jakarta from February to April 2010. The subjects were assessed for anthropometric characteristics, serum osteocalcin levels and BMD. BMD was measured at the lumbar spine, right femoral neck and at the left distal radius by dual energy X-ray absorptiometry (DXA). Mean serum osteocalcin was 28.99 ± 10.02 ng/ml. The Pearson correlation test on all subjects indicated a significant inverse correlation between serum osteocalcin and femoral neck BMD (r = - 0.29; p=0.034). By arranging the data into tertiles, a significant association was found in non-obese subjects between mean femoral neck BMD and serum osteocalcin (p=0.036). The Tukey posthoc multiple comparison test showed a significant mean difference in femoral neck BMD between the lowest and the highest tertiles of osteocalcin serum concentrations (p=0.028). Maintenance of body weight is important for maintaining BMD in postmenopausal women. Key words: Serum osteocalcin, BMD, body weight, postmenopausal women INTRODUCTION The increased knowledge of society on healthy living has resulted in an increase in the numbers of older persons worldwide, including Indonesia. The 2010 population census indicates that older persons constitute approximately 10 percent of the Indonesian population, whilst the 15.5 million menopausal women in the year 2000 are predicted to 156 Susanto Osteocalcin and bone mineral density increase to 24 million in 2015.(1) Osteoporosis is one of the health problems arising in women entering menopausal age, and will presumably seriously affect the lives of postmenopausal women. Osteoporosis and the potentially serious consequences of osteoporotic fracture increase with advancing age of the population, making skeletal health assessment an important component of routine care in postmenopausal women.(2) Osteoporosis-related fracture risk is usually assessed by bone mineral density (BMD) as a measure of bone mass and a predictor of fracture, since bone mass affects bone strength or the ability of a bone to withstand trauma. It is well established that 75- 90% of the variance in bone strength is related to BMD. The risk of fracture is known to be higher in women with low BMD, with the risk d o u b l i n g f o r a r e d u c t i o n o f 1 s t a n d a r d deviation in BMD.(3) However, since bone strength is also determined by other factors, such as bone geometry, microarchitecture, and size, BMD by itself is not a good predictor of fracture risk. Epidemiological data indicate that high body weight or high body mass index (BMI) are related to high bone mass, while a reduction in body weight may result in loss of bone mass.(4- 6) On the other hand, some are of the opinion that fat mass is negatively related to BMD.(7,8) Hsu et al. showed that postmenopausal women with a higher percentage of body fat may have a higher risk for osteoporosis, osteopenia, and nonspine fractures.(9) Decreased estrogen concentrations at m e n o p a u s e a g e l e a d t o l o w e r i n t e s t i n a l absorption of calcium, resulting in low serum c a l c i u m c o n c e n t r a t i o n s a n d i n c r e a s e d osteoclastic resorption of bone. Both increase bone turnover and constitute risk factors for the development of osteoporosis.(9) Menopause and aging are associated with accelerated loss of cortical bone. Bone loss is the result a negative remodeling balance due to impaired b o n e f o r m a t i o n a n d / o r i n c r e a s e d b o n e resorption.(10,11) Fractures may be predicted from bone turnover markers either via BMD, because low BMD is associated with high bone turnover,(12) or independently of BMD, since increased b o n e t u r n o v e r n e g a t i v e l y a ff e c t s b o n e microarchitecture and fragility.(13) Currently biochemical markers of bone turnover are being used for predicting the rate of bone loss and for assessing the risk of f r a c t u r e s i n p o s t m e n o p a u s a l w o m e n . Estimation of bone turnover rates may be obtained through determination of the serum or urinary concentrations of certain proteins that are representative of the bone remodeling process. These proteins may be divided into bone formation markers and bone resorption markers. The most specific and sensitive bone formation markers include osteocalcin and b o n e a l k a l i n e p h o s p h a t a s e , w h i c h a r e indicative of osteoblastic activity, whereas bone resorption markers, such as tartrate resistant acid phosphatase and cross-linked telopeptides, reflect osteoclastic activity.(14) Osteocalcin (OC), also termed Gla (ã carboxyglutamic acid) protein, is a bone matrix protein synthesized by mature osteoblasts, and c o n s t i t u t e s a p p r o x i m a t e l y 1 5 % o f noncollagenous bone matrix proteins.(15) Most of the osteocalcin (80-90%) is adsorbed to bone hydroxyapatite, with a minor percentage leaking into the circulation.(16) Serum OC may be detected by various tests, such as assays using monoclonal antibodies against OC N-mid and N terminal OC fragments. Osteoporotic females have significantly higher levels of OC, bone alkaline phosphatase, and crosslinked telopeptide-C (CTX ).(17) In general, serum OC concentrations increase with age, such that women aged over 65 years of age have an approximately 2 fold higher OC concentration in comparison with women under 44 years of age.(18) The aim of the present study was to investigate the relationship between serum o s t e o c a l c i n a n d B M D v a l u e s i n postmenopausal women. 157 METHODS Study design From February to April 2010 a cross- sectional study was carried out at the Mampang Prapatan Community Health Center, South Jakarta. Study subjects The study sample was selected by cluster a n d s i m p l e r a n d o m s a m p l i n g f r o m postmenopausal women in the Mampang Prapatan area meeting the following inclusion criteria: postmenopausal women in the age range of 47-60 years, duration of menopause of more than one year, not consuming steroids and bisphosphonates, actively mobile, able to communicate, and willing to participate in this study. The exclusion criteria were: a past h i s t o r y o f h y s t e r e c t o m y, m a l i g n a n c i e s , h o r m o n a l t h e r a p y, d i a b e t e s m e l l i t u s , abnormality of liver and kidney functions, and acute infections. A total of 53 female subjects were included in this study. All participants gave written informed consent. Questionnaire The respondents filled out a questionnaire relating to demographic data, including age, education, employment, number of pregnancies, and duration of menopause. Anthropometric assessments Height was measured to the nearest 0.1 cm using a portable microtoise and weight to the nearest 0.1 kg using Sage portable scales. Body mass index (BMI) was calculated as the weight (kg) divided by the square of the height (m). For Asian populations, BMI classified into the following categories: underweight (<18.5 kg/m2), normal ( 18.5-22.9 kg/m2), overweight (23.0 -25 kg/m2), and obese (>25 kg/m2).(19) BMD measurements B M D m e a s u r e m e n t s ( g / c m 2) for the lumbar spine (L1-L4), femoral neck and distal radius were obtained by dual energy X ray absorptiometry (DXA) using a Lunar DPX Bravo Nomusa densitometer (GE Medical Systems). The BMD measurements were performed at Budi Jaya Hospital, South Jakarta. Osteocalcin measurements Measurement of OC was performed on fasting serum samples collected before 10 a.m. and stored at a temperature below –70 ºC before a n a l y s i s . D e t e r m i n a t i o n o f s e r u m O C c o n c e n t r a t i o n w a s p e r f o r m e d a t P r o d i a Laboratories, Jakarta, using the Elecsys N mid o s t e o c a l c i n a s s a y r e a g e n t k i t ( R o c h e Diagnostics, Cat No. 12 149 133). The Elecsys N mid osteocalcin assay utilizes two monoclonal antibodies, one specific for epitopes on the N mid OC fragment and the other specific for the N terminal OC fragment, and therefore detects the intact OC as well as the N-mid fragment. The detection range of the kit was 0.5 – 300 ng/ ml, while the coefficient of variation was 2.28%. Serum OC concentrations were divided into tertiles, the lowest tertile ranging from 12 up to 24.39 ng/ml, while the middle and highest tertiles were 24.4 – 32.45 ng/ml and 32.46 – 70.59 ng/ml, respectively. Ethical clearance The protocol for this study was approved by the Research Ethics Committee, Medical Faculty, Trisakti University. Statistical analysis Data were entered to SPSS format using version 17.0. Categorical variables were s u m m a r i z e d u s i n g f r e q u e n c i e s a n d percentages, while continuous variables were s u m m a r i z e d u s i n g m e a n a n d s t a n d a r d deviation. One-way analysis of variance (ANOVA) was used to compare the mean d i ff e r e n c e i n B M D i n t h e t e r t i l e s e r u m osteocalcin levels. A significant ANOVA test was followed by a Tukey post hoc multiple comparison test to determine which specific Univ Med Vol.30 No.3 158 Susanto Osteocalcin and bone mineral density groups were significantly different. Statistical significance was set at p < 0.05. RESULTS I n t h i s s t u d y t h e r e w e r e 5 3 w o m e n meeting the inclusion criteria, for whom the major characteristics are shown in Table 1. The mean age of the respondents was 53.49 ± 3.36 years, while the duration of menopause was 2 - 7 years. A total of 41 subjects (77.4 %) was non-obese and the mean of femoral neck BMD was 0.78 ± 0.12 g/cm2. In non-obese postmenopausal women, femoral bone mass was found to be significantly higher (0.82 ± 0.89 g/cm2) in the lowest serum OC tertile, in comparison with that in the mid (0.77 ± 0.11 g/cm2) and higher tertiles and (0.71 ± 0.13 g/cm2) respectively (Table 2). However, for the lumbar and radial bones, the mass was not significantly different between lower serum OC tertile and the other two tertiles. Chara cteristic n (%) Age (yrs) 53.49 ± 3.36 a Duration of menopause (yrs) 4.55 ± 2.3 8 a Education Did not finish senior high school (SMA) 4 5 (84.9) Senior high school graduate 7 (13.2) Academy/university 1 (1.9) Employment Unemployed 3 1 (58.5) Self-em ployed (informal sector) 2 2 (41.5) Number of pregnancies 0-3 pregn ancies 1 9 (35.8) > 3 pregnancies 3 4 (64.2) Osteocalcin (n g/ml) 28.99 ± 10.02 a Tertiles of serum osteocalcin (ng/m l) Lowest 1 7 (32.1) Mid 18 (34) Highest 18 (34) Nutritional status Non obese 4 1 (77.4) Obese 1 2 (22.6) BMD values Lumbar BMD (g/cm2) 0.90 0.12 a Femoral neck BMD (g/cm2) 0.78 ± 0.1 2 a Radial BMD (g/cm2) 0.53 ± 0.0 8 a Table 1 Characteristics of the subjects (n=53) a Mean ± SD a Mean ± SD Tertiles of serum osteoca lcin (ng/ml ) p Lowest (n=13) Mid (n=14) H ighest (n=14) 12 – 24.39 24.4 – 32.45 32.46 – 70.59 BMD (g/cm 2)a Lumbar 0.91 ± 0.11 a 0.86 ± 0.10 0.8 5 ± 0.11 0.364 Femoral neck 0.82 ± 0.89 0.77 ± 0.11 0.7 1 ± 0.13 0.036 Radial 0.56 ± 0.09 0.51 ± 0.08 0.5 1 ± 0.05 0.271 Table 2. Mean of bone marrow density by tertiles of serum osteocalcin in non-obese subjects 159 The results of the Tukey post hoc multiple comparison test indicated a significant mean difference in femoral neck BMD between the lowest and highest teriles of serum osteocalcin concentrations (p=0.028) (Table 3). The results of the Pearson correlation test showed a significant inverse correlation between serum osteocalcin concentrations and femoral neck BMD (r = -0.29; p = 0.034) (Table 4). DISCUSSION In the present study, femoral neck BMD had a significant negative correlation with serum osteocalcin. Since there is a complex relationship between bone turnover and bone m a s s , s u c h t h a t h i g h b o n e t u r n o v e r i s associated with decreased bone mass,(21) it has been suggested that bone markers can predict fractures in elderly women, particularly those involving trabecular bone,(22) and that the use of a combination of BMD and bone markers can improve fracture prediction.(23) A case control study of 90 postmenopausal women with mean age of 66 ± 8 years showed results that were consistent with the results of the present study, in that there is a significant inverse correlation of proximal femur BMD with osteocalcin. (24) However, a study in Pakistan, involving 50 postmenopausal women with mean age of 54.36 ± 0.81, obtained inconclusive results indicating lack of a significant association between BMD and s e r u m o s t e o c a l c i n c o n c e n t r a t i o n s . ( 2 5 ) Biochemical markers of bone turnover, such as osteocalcin, may also be used for early e v a l u a t i o n o f t h e e f f e c t s o f t r e a t m e n t . Additionally, serial measurements of bone markers may assist in evaluating the patients’ responses to specific antiresorptive therapy.(26) Serum OC is considered a specific marker of osteoblast function, as its levels have been shown to correlate with bone formation rates. However, since it is also released from bone matrix during bone resorption, it reflects the overall turnover of bone and is considered as a bone turnover marker. OC has a high affinity for calcium and has a compact á helical conformation that is calcium dependent. The ã carboxyglutamic acid (Gla) residues of OC a r e c a p a b l e o f b i n d i n g t o b o n e m a t r i x h y d r o x y a p a t i t e , t h u s l e a d i n g t o b o n e mineralization. Calcium- and phosphorus- deficient osteoporotic women may have a decreased rate of bone mineralization due to a reduction in hydroxyapatite crystal formation. In this condition, free OC may be present in the circulation, thus explaining the increased serum OC concentration in osteoporotic post- menopausal women.(2,12,18) Tertile of serum osteocalcin Femoral neck BMDa p Lowest Mid 0.051 ± 0.041 0.444 Highest 0.112 ± 0.041 0.028 Mid Lowest -0.051 ± 0.041 0.444 Highest 0.061 ± 0.041 0.305 Table 3. Mean differences and SEs of femoral neck BMD in non-obese subjects (Tukey multiple comparisons procedures) a Mean ± SD BMD Serum osteocalcin p r values Lum bar - 0.079 0.572 Femoral neck -.0.290 0.034 Radi al -.0.093 0.502 Table 4. Correlation between serum osteocalcin and BMD in all subjects (n=53) Univ Med Vol.30 No.3 160 Susanto Osteocalcin and bone mineral density The current study showed that non-obese postmenopausal women in the high osteocalcin tertile had the lowest mean femoral neck BMD values. These results are similar to those of a population survey that included women in the age range of 20–69 years, indicating that women with high OC concentrations had the lowest mean BMD values.(27) Other studies on elderly women have shown low body weight to be a major predictor of BMD and bone loss, and this association may be mediated by increased bone turnover.(28,29) In the study by Zhao et al.(8) it was found that fat mass is negatively related to bone mass when adjusted for body weight.(8) In another study, there was a significant risk of osteoporosis, osteopenia, and non spine fractures in subjects with high weight-adjusted body fat percentages.(12) BMD in postmenopausal women is determined by peak bone mass and the amount of bone lost since menopause. In several studies, body weight or BMI was shown to be inversely associated with bone turnover markers, where persons with a low body weight have high bone turnover, whereas overweight and obese persons had a lower serum OC than those of normal weight.(30-32) Apparently, the effect of o b e s i t y o n f r a c t u r e r i s k d e p e n d s o n i t s definition. If defined on the basis of BMI or body weight, obesity may be protective against b o n e m i n e r a l l o s s o r v e r t e b r a l f r a c t u r e . However, if obesity is based on the percentage of body fat, obesity may be a risk factor for o s t e o p o r o s i s . I n t h i s c o n n e c t i o n , b o t h metabolic syndrome and hyperglycemia could be risk factors for osteoporosis. High HDL protects against osteoporosis, increasing BMD and lowering fracture risk.(33) While biochemical markers of bone turnover may be able to predict bone loss and thus fracture risk, they may also predict fracture risk independently of BMD. High bone turnover can disrupt the trabecular architecture by i n c r e a s i n g t h e i n c i d e n c e o f t r a b e c u l a r perforation and buckling, thus reducing bone strength, without necessarily appreciably affecting BMD. Because of the overlap in BMD distribution of normal and fracture populations, the BMD criteria used are useful in population- based measurements but less useful in assessing i n d i v i d u a l r i s k . ( 3 4 ) S i m i l a r l y, a s i n g l e measurement of a biochemical marker of bone turnover may be unable to predict even short- term individual fracture risk. However, there is increasing evidence that a combination of a biochemical marker and BMD may be a better p r e d i c t o r o f f r a c t u r e t h a n B M D a l o n e . Biochemical markers of bone turnover cannot substitute for serial BMD measurements, but may be useful when considered in conjuction with BMD measurement. The present study admittedly has several limitations. Firstly, this study performed only investigations on bone formation without investigating bone resorption. Secondly, the study design was cross-sectional and therefore cannot depict a causal relationship between OC concentrations and BMD. Lastly, the age of the respondents was relatively homogenous in the 50–60 year range, while the duration of menopause was 2-7 years, such that this study can only describe bone changes occurring during early menopause. CONCLUSIONS This study demonstrates a significant inverse correlation between serum osteocalcin and femoral neck BMD. In non-obese subjects there is a significant difference in femoral neck BMD values between the lowest and the h i g h e s t t e r t i l e s o f o s t e o c a l c i n s e r u m concentrations. ACKNOWLEDGEMENTS The author wishes to thank the Faculty of Medicine, Trisakti University, for funding this study and the respondents who were willing to participate in this study. Special thanks are notably due to Prof.Dr.dr. Adi Hidayat, MS for his helpful comments and suggestions on the manuscript. 161 REFERENCES 1. Badan Perencanaan Pembangunan Nasional. Proyeksi penduduk Indonesia 2000-2025. Jakarta: BPS, Bappeneas, dan UNFPA Indonesia; 2005. 2. Jagtap VR, Ganu JV, Nagane NS. BMD and serum intact osteocalcin in postmenopausal osteoporosis women. Ind J Clin Biochem 2011; 26:70-3. 3. Cummings SR, Black DM, Nevin MC, Browner W, Cauley J, Ensrud K, et al. Bone density at various sites for prediction of hip fractures. The study of osteoporotic fractures research group. Lancet 1993;341:72-5. 4. Douchi T, Yamamoto S, Oki T, Maruta K, Kuwahata R, Yamasaki H, et al. Difference in the effect of adiposity on bone density between pre and postmenopausal women. Maturitas 2000;34: 261-6 5. Guney E, Kisakol G, Ozgen G, Yilmaz C, Yilmaz R, Kabalak T. Effect of weight loss on bone metabolism: comparison of vertical banded gastroplasty and medical intervention. Obes Surg 2003;13:383-8. 6. Radak TL. Caloric restriction and calcium’s effect on bone metabolism and body composition in overweight and obese premenopausal women. Nutr Rev 2004;62:468-81. 7. Fillip R, Raszewski G. Bone mineral density and bone turnover in relation to serum leptin, alpha ketoglutarate and sex steroids in overweight and obese postmenopausal women. Clinical Endocrinol 2009;70:214-20. 8. Zhao LJ, Liu YJ, Liu PY, Hamilton J, Recker RR, Deng HW. Relationship of obesity with osteoporosis. J Clin Endocrinol Metab 2007;92: 1640-6. 9. Arifin Z, Hestiantoro A, Baziad A. Pemberian susu yang difortifikasi kalsium kadar tinggi dan vitamin D dalam memperbaiki turnover tulang perempuan pascamenopause. Maj Obstet Ginekol Indones 2010;34:31-8. 10. Garnero P, Delmas PD. Contribution of bone mineral density and bone turnover markers to the estimation of risk of osteoporotic fracture in post menopausal women. J Musculoskelet Neuronal Interact 2004;4:50-63. 11. Leeming DJ, Alexandersen P, Karsdal MA, Qvist P, Schaller S, Tanko LB. An update on biomarkers of bone turnover and their utility in biomedical research and clinical practice. Eur J Clin Pharmacol 2006;62:781-92. 12. Ravn P, Fledelius C, Rosenquist C, Overgaard K, Christiansen C. High bone turnover is associated with low bone mass in both pre and postmenopausal women. Bone 1996;19:291-8. 13. Seeman E, Delmas PD. Bone quality the material and structural basis of bone strength and fragility. N Engl J Med 2006;354:2250-61. 14. Eastell R, Hannon RA. Biomarkers of bone health and osteoporosis risk. Proceedings of the Nutrition Society; 2007 July 16-19; Ulster: Coleraine;2007. 15. Civitelli R, Armamento-Villareal R, Napoli N. Bone turnover markers: understanding their value in clinical trials and clinical practice. Osteoporosis Int 2009;20:853-51. 16. Lee AJ, Hodges S, Eastell R. Measurement of osteocalcin. Ann Clin Biochem 2000;37:432-46. 17. Iki M, Akiba T, Matsumoto T, Nishino H, Kagamimori S, Kagama Y, et al. and JPOS Study Group. Reference database of biochemical markers of bone turnover for the Japanese female population. Osteoporos Int 2004;15:981–91. 18. Filip RS, Zagorski J. Age and BMD related differences in biochemical markers of bone metabolism in rural and urban women from Lublin region, Poland. Ann Agric Environ Med 2004;11:255-9. 19. World Health Organization. Appropriate body mass index for Asian populations and its implications for policy and intervention strategies. Lancet 2004;363:157-63 21. Hsu YH, Venners SA, Terwedow HA, Feng Y, Niu T, Li Z, et al. Relation of body composition, fat mass, and serum lipids to osteoporotic fractures and bone mineral density in Chinese men and women. Am J Clin Nutr 2006;83:146-54. 22. Gerdhem P, Ivaska KK, Alatalo SL. Biochemical markers of bone metabolism and prediction of fracture in elderly women. J Bone Miner Res 2004;19:386-93. 23. Kanis JA. Diagnosis of osteoporosis and assessment of fracture risk. Lancet 2002;359: 1929-36. 24. Pirro M, Leli C, Fabbriciani G, Manfredelli MR, Callarelli L, Bagaglia F, et al. Association between circulating osteoprogenitor cell numbers and bone mineral density in postmenopausal osteoporosis. Osteoporos Int 2010;21:297–306. DOI 10.1007/ s00198-009-0968-0. 25. Lateef M, Baig M, Azhar A. Estimation of serum osteocalcin and telopeptide-C in postmenopausal osteoporotic females. Osteoporos Int 2010; 21:751–5. DOI 10.1007/s00198-009-1001-3. Univ Med Vol.30 No.3