Cell Biochem Biophys DOI 10.1007/s12013-014-0392-8
ORIGINAL PAPER
Low Bone Mass is Associated with Stroke in Chinese Postmenopausal Women: The Chongqing Osteoporosis Study Rui Zhou • Dong Liu • Rui Li • Shiming Zhou Min Cui • Lin Chen • Huadong Zhou
•
Ó Springer Science+Business Media New York 2014
Abstract The objective of the present study was to investigate the association of low bone mass with the risk of stroke and death in community residents of China. This study was based on the follow-up data acquired from 5,136 postmenopausal women aged 50 years or older between July 2006 and June 2011. Baseline and the follow-up bone mineral density (BMD) in these patients were measured by dual energy X-ray absorptiometry scanning. The association of BMD and risk of stroke and death was further evaluated by Cox proportional hazard analysis. During the follow-up, 148 subjects (2.9 %) sustained prospective stroke, and 261 subjects (5.1 %) died. After adjustments for age and BMI, our results indicated that neck BMD and osteoporosis were independent predictors of stroke (HR for neck BMD = 1.35, 95 % CI = 1.21–1.62; HR for osteoporosis = 2.24, 95 % CI = 1.47–3.58) and were also associated with increased risk of death (HR for neck BMD = 1.39, 95 % CI = 1.24–1.71; HR for osteoporosis = 1.97, 95 % CI = 1.21–2.97). Our results also suggest that low neck BMD and osteoporosis are associated with significantly elevated risk of stroke and death in Chinese postmenopausal women.
R. Zhou Department of Orthopedics, The Orthopedic Surgery Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, China D. Liu L. Chen Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China R. Li S. Zhou M. Cui H. Zhou (&) Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing, China e-mail: [email protected]
Keywords Bone mineral density Stroke Death Postmenopausal women
Introduction Osteoporosis and stroke were found to be strongly associated with aging and share common risk factors. They were often coexistent in the same patient, implying a possible relation between osteoporosis and risk of stroke. As the prevalence of osteoporosis and stroke is increasing dramatically in aging population across the globe, it could pose a major health problem [1–3]. China with its rapidly growing elderly population has high incidence of osteoporosis [4] and stroke as compared to Western societies [5, 6]. Recently, it has been observed that older women and men with low bone mineral density (BMD) have a higher incidence of stroke than older people with normal BMD [7–10]. However, there are contradictory reports suggesting no significant association between Low BMD and risk of stroke in men, whereas the risk was found to be only slightly higher among women with low BMD [11]. Therefore, larger prospective studies in elderly people are needed to clearly answer this question. Even though earlier prospective cohort studies of the elderly suggested that low BMD is associated with increased mortality [12, 13], the real relationship is still controversial. Therefore, the actual relation between lower BMD and individual mortality needs to be further investigated in randomized trials. In order to address the significance of baseline bone loss and its impact on subsequent stroke and death in Chinese elderly people, we conducted a 5-year prospective study in postmenopausal women in Southwest China and found that
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low neck BMD and osteoporosis significantly elevate the risk of stroke and death.
Materials and Methods Study Subjects The subjects enrolled in the current study were chosen from three randomly selected communities of Chongqing, one of the largest municipalities in Southwest China, with a population of 35 million. This study was approved by the Institutional Review Board of the Chongqing Daping Hospital and Southwest Hospital. Informed consent was obtained from all the subjects prior to the study. Baseline Screening The baseline screening of the present study was performed from January 2006 to June 2006. A total of 6,282 postmenopausal women aged 50 years and above were enrolled from the selected communities. The subjects were interviewed by organized meetings in the community centers, as part of selection process. For people who were absent at the meeting or who were unable to attend due to physical disabilities, our staff performed the interviews at the patients’ homes. At baseline, 6,282 individuals were screened while a total of 603 subjects, who were either not available at the time of screening (n = 284) or declined to participate (n = 319), were excluded from the present study. A total of 345 cases with stroke were diagnosed and excluded from the study. A total of 5,334 subjects without previous stroke were enrolled at baseline (Fig. 1). The following data were collected: demographic data, BMD, smoking and drinking status, and medical history. The trained interviewers including experienced neurologists and senior nurses administered these procedures. The reliability across the interviewers was measured by the Cohen’s Kappa statistical analysis. Based on the same subject sample, the agreement on data collection was found to be excellent, with a kappa score of 0.93. BMD and Osteoporosis Dual energy X-ray absorptiometry (DXA, Prodigy fan beam densitometer, Lunar Corp, GE Medical System, Madison, WI) was employed to determine BMD. BMD of femoral neck bone was further used as a measure of global bone health. BMD was measured at baseline. Osteoporosis, defined as BMD, that is 2.5 standard deviations (SD) below the mean normal BMD, which is obtained from 10,000 healthy women aged 20–29 years in Daping Hospital of Chongqing [14].
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Fig. 1 Flow chart of this study
Smoking and Drinking Status The smoking status was classified as past smokers (who had quit smoking for at least 6 months prior to enrollment), current smokers, or those who never smoked. Drinking status was classified as daily drinking, weekly drinking, monthly drinking, or occasional drinking. Clinical Assessment The medical history was collected from self-report, available medical records, or medication. The data included coronary heart diseases, chronic obstructive pulmonary disease, chronic hepatitis, chronic renal insufficiency, hypertension, diabetes mellitus, hypercholesterolemia, and Parkinson’s disease. Blood pressure measurement and electrocardiogram were performed on-site, and fasting blood samples were collected to measure glucose, total cholesterol, creatinine, uric acid, osteoprotegerin (OPG), and 25(OH) D. Body mass index (BMI) was also assessed. The subjects with abnormalities, suggestive of potential diseases that were not previously diagnosed, were admitted to Chongqing Daping hospital for further investigation. In addition, diagnosis of hypertension, diabetes mellitus,
Cell Biochem Biophys
hypercholesterolemia, ischemic heart disease, stroke, chronic renal insufficiency, and chronic hepatitis were based on International Classification of Diseases, 9th Revision (ICD-9). Assessment of Mortality Death of any of the studied subjects was confirmed through inquiry since their last visit. The date of death was confirmed on the basis of death certificates or by consulting the available hospital records. In some instances, death of the subjects was confirmed from consulting their immediate family. The subjects who lost contact were treated as missing cases. Follow-Up Subjects were followed up annually for 5 years from July 2006 to June 2011. Epidemiological questionnaires and telephone were used to obtain the follow-up information, such as stroke, changes of the diseases, and death every year during the 5-year follow-up period. Each self-reported stroke was confirmed by physicians on the basis of medical record and CT and MRI examination. The stroke in this study consisted of intracranial hemorrhage and cerebral infarction, and subjects with traumatic stroke were excluded. Statistical Analysis In univariate analyses, baseline variables between the subjects who developed stroke or died and those who did not were compared using Pearson v2 test, Fisher exact test, a t test, or the Mann–Whitney U test as deemed appropriate. Additionally, Cox proportional hazard models were used to assess the associations between BMD, osteoporosis and increased risk of stroke or death in two steps. The associations were first analyzed with adjustments for age and BMI. Then, the associations were analyzed with adjustments for other covariates, including age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus, former stroke, and ischemic heart disease. The statistical analyses were finally performed using SPSS 15.0 for Windows.
Baseline Characteristics in Subjects Who Did and Did Not Develop Stroke Table 1 summarizes the baseline characteristics of the subjects with and without prospective stroke. Compared with 4,988 subjects who did not experience a stroke, 148 subjects with prospective stroke were found to be more frequent in current smoking and daily drinking, and have more hypercholesterolemia, a lower neck BMD, and a higher prevalence of osteoporosis (p \ 0.01), more diabetes mellitus, and hypertension (p \ 0.001). These 148 subjects were also found to be older, and have higher BMI and serum levels of OPG than those subjects who did not sustain a stroke (p \ 0.05). Association Between the Bone Variables and Risk of Stroke The independent association between the bone variables and the future risk of stroke after adjustment for potential confounders is shown in Table 2. After adjustment for age and BMI, neck BMD was found to correlate with increased risk of stroke (HR 1.35, 95 % CI 1.21–1.26). After adjustment for the same variables, osteoporosis was also found to be an independent prospective risk factor for stroke (HR 2.24, 95 % CI 1.47–3.58). Further, it was found that neck BMD (HR 1.69, 95 % CI 1.42–1.93) and osteoporosis (HR 3.94, 95 % CI 1.92–9.68) were still found to be independent predictors of the risk of stroke after adjustments with age, BMI, smoking, drinking, hypertension, and diabetes mellitus. Baseline Characteristics in Subjects Who Died and Did Not Died The 261 subjects who died during the follow-up were found to be either frequent current smokers, or had hypertension, diabetes mellitus, a lower neck BMD, and a higher prevalence of osteoporosis than the remaining 4,875 subjects who did not die (p \ 0.01) during follow-up. These 261 subjects were also older, have a higher BMI, and more frequent in daily drinking than those subjects who did not die (p \ 0.05) during follow-up (Table 3). The Relations Between the Bone Variables and the Future Risk of Death
Results In this study, 5,334 people without stroke were enrolled at baseline. Later, during the 5-year follow-up period, 105 (2.4 %) patients declined, and another 93 (2.1 %) moved away with the remaining 5,136 subjects completing the follow-up. The subjects with a mean age of 62.8 years (range 50–83 years old) were included in this study.
We also established Cox proportional hazard models including age, BMI, smoking, drinking, hypertension, and diabetes mellitus to obtain insights into the independent predictors of death risk, and the results are shown in Table 4. After adjustments for age and BMI, neck BMD (HR 1.39, 95 % CI 1.24–171) was related to the increased risk of death. Additionally, after adjustment for the same variables, osteoporosis
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Cell Biochem Biophys Table 1 Baseline characteristics in 5,136 subjects who did and did not develop stroke
No strokes (n = 4,988)
Strokes (n = 148)
p value
Physical characteristics Age (year), ± SD
62.8 ± 9.1
64.5 ± 7.3
\0.05
BMI (kg/m2), ± SD
21.7 ± 4.2
23.1 ± 3.9
\0.05
Physical activity index, n (%)
1,265 (25.4)
36 (24.3)
NS
Never smoking, n (%)
1,940 (38.9)
62 (41.9)
NS
Past smoking, n (%)
1,581 (31.7)
50 (33.8)
NS
Current smoking, n (%)
856 (17.2)
44 (30.0)
\0.01
1,401 (28.1)
43 (28.9)
NS
Smoking status
Drinking status Occasional drinking, n (%) Daily drinking, n (%)
868 (17.4)
36 (24.3)
\0.01
Weekly drinking, n (%)
903 (18.1)
28 (18.9)
NS
Monthly drinking, n (%)
1,401 (28.1)
39 (26.4)
NS
Hypertension, n (%) Diabetes mellitus, n (%)
1,451 (29.1) 968 (19.4)
52 (35.1) 40 (26.8)
\0.001 \0.001
Hypercholesterolemia, n (%)
963 (19.3)
35 (23.5)
\0.01
Neck BMD (g/cm3), ± SD
0.838 ± 0.136
0.679 ± 0.121
\0.01
Osteoporosis n (%)
643 (12.9)
24 (16.2)
\0.01
Osteoprotegerin pmol/L, ± SD
3.08 ± 1.13
3.49 ± 1.64
\0.05
25(OH) D (ng/mL)
15.3 ± 6.7
17.8 ± 5.9
\0.05
Vascular risk factors
Bone mass measurements
NS not significant
Table 2 Independent relationship between the bone density and the risk of stroke HR
95 % CI
A: Bone variables adjusted for age and BMI Neck BMD
1.35
1.21–1.62
Osteoporosis
2.24
1.47–3.58
B: Bone variables adjusted for age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus, hypercholesterolemia Neck BMD Osteoporosis
1.69 3.94
1.42–1.93 1.92–9.68
Bone variables were analyzed per SD decrease, and osteoporosis was analyzed as yes/no
(HR 1.97, 95 % CI 1.21–2.97) was also found to be an independent risk factor for death. Further, the results demonstrated that both neck BMD (HR 1.36, 95 % CI 1.18–1.89) and osteoporosis (HR 3.63, 95 % CI 1.49–8.61) were independent predictors of the risk of death after adjustments for age, BMI, smoking, drinking, hypertension, and diabetes mellitus.
Discussion In this study, our results showed that femoral neck BMD is an independent risk factor for future stroke and death in
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Chinese postmenopausal women. From our data, we noticed a consistent association of low neck BMD and osteoporosis with the risk of stroke and death. The subjects were also found to have higher serum levels of OPG than those subjects who did not sustain a stroke. Low neck BMD increased the incident risk for stroke by 1.35-fold, while osteoporosis increased it by 2.24-fold. Additionally, low neck BMD increased the incident risk for death by 1.69-fold, and osteoporosis increased this risk by 3.94-fold. These results revealed the significance of altered bone metabolism on the incidence of stroke and death. Earlier studies showed that calcium loss from bone might directly correlate to arterial calcification [7, 10]. Other studies suggested that low BMD is related to atherosclerosis and aortic calcification. In particular, postmenopausal women with osteoporosis might be at a greater risk of developing coronary atherosclerosis [15, 16]. Framingham Heart Study also found that bone loss was directly related to aortic calcification in women but not men [17]. Low BMD may serve as a marker for stroke. In 2010, Nordstrom et al. [18] examined whether BMD was prospectively related to stroke, in a study of 4,302 men and women (with a mean age of 59 years old) at baseline. After a mean follow-up period of 5.6 years, 139 strokes were registered. The study indicated that decreased BMD as well
Cell Biochem Biophys Table 3 Baseline characteristics in 5,136 subjects who died during follow-up or not
No dead (n = 4,875)
dead (n = 261)
p value
Physical characteristics Age (year), ± SD
62.7 ± 9.3
65.4 ± 7.1
\0.05
BMI (kg/m2), ± SD
21.1 ± 4.2
23.8 ± 3.9
\0.05
Physical activity index, n (%)
1,243 (25.5)
65 (24.9)
NS
Smoking status Never smoking, n (%)
1,926 (39.5)
103 (39.4)
NS
Past smoking, n (%)
1,599 (32.8)
82 (31.5)
NS
Current smoking, n (%)
1,253 (25.7)
76 (29.3)
\0.01
1,530 (31.4)
79 (30.5)
NS
Drinking status Occasional drinking, n (%) Daily drinking, n (%)
1,112 (22.8)
68 (26.1)
\0.05
Weekly drinking, n (%)
941 (19.3)
48 (18.4)
NS
Monthly drinking, n (%)
1,292 (26.5)
65 (25.0)
NS
Hypertension, n (%) Diabetes mellitus, n (%)
1,472 (30.2) 1,014 (20.8)
94 (36.1) 70 (26.9)
\0.01 \0.01
Hypercholesterolemia, n (%)
994 (20.4)
56 (21.4)
NS
Neck BMD (g/cm3), ± SD
0.837 ± 0.136
0.712 ± 0.121
\0.01
Osteoporosis n (%)
626 (12.8)
41 (15.7)
\0.01
Osteoprotegerin pmol/L, ± SD
3.09 ± 1.13
3.11 ± 1.23
NS
25(OH) D (ng/mL)
15.9 ± 6.8
16.1 ± 4.7
NS
Vascular risk factors
Bone mass measurements
NS not significant
Table 4 Independent relationship between the bone density and the risk of death HR
95 % CI
A: Bone variables adjusted for age and BMI Neck BMD
1.39
1.24–1.71
Osteoporosis
1.97
1.21–2.97
B: Bone variables adjusted for age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus Neck BMD
1.36
1.18–1.89
Osteoporosis
3.63
1.49–8.61
Bone variables were analyzed per SD decrease, and osteoporosis was analyzed as yes/no
as osteoporosis of the femoral neck independently correlated with stroke [18]. The relationships between BMD and acute stroke were also examined in women aged C60 years in another study; measurements of the stroke in patients were performed for 6 days after the onset of stroke. The results found that female, but not male, stroke patients have lower BMD than the corresponding population controls [19]. Another study examined acute ischemic stroke patients who underwent bone densitometry tests within 7 days of stroke symptom onset and noticed that patients with osteoporosis of the right femoral neck were more likely to have poor outcome in acute ischemic stroke than
those without [20]. In a study of 4,024 ambulatory women aged 65 years or older, 83 women suffered first stroke during a 1.98-year follow-up period, and osteoporosis was associated with an increased stroke risk and it was suggested that low bone density may not cause stroke [21]. The above studies have shown that the relation between BMD and stroke is not fully elucidated; our present study found the association of low neck BMD and osteoporosis with risk of stroke. Low neck BMD and osteoporosis increased the incident risk for stroke. Our study found that the subjects, who died during the follow-up period, had lower neck BMD at baseline than those subjects who did not die. Nevertheless, our results are consistent with some studies, while different from other. Therefore, there may be community/population samplebased differences among the results obtained in different countries and we cannot also rule out differences due to ethnicity and life-styles. For example, Trivedi et al. reported in a study that examined the association of bone density and cardiovascular mortality in elderly aged 65–76 years that low bone density was a strong and independent predictor of all-cause and cardiovascular mortality [22]. In addition, a total of 1,429 ambulatory postmenopausal female volunteers aged over 50 years old were enrolled in another study in Japan, in which severity of osteoporosis was found to be a significant independent risk
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factor for all-cause mortality [13]. However, in a Rotterdam Study that enrolled 5,819 men and women aged C55 years and measured BMD during an average followup of 5.4 years, no significant relationship between BMD and overall mortality was observed in women [23]. Additionally, a cohort of 3,402 white and black subjects of 45 to 74 years age at baseline was enrolled in a large national study and no significant associations of BMD and mortality for either whites or blacks were observed [24]. Michael et al. also reported that low BMD was not associated with risk of cardiovascular disease in men. It was found that risk of cardiovascular disease and stroke was elevated among women with low bone density, whereas no significant associations were found. After controlling for baseline age and other factors, proximal femoral BMD was still not associated with subsequent death [11]. Although there are several common possible mechanisms contributing to a relation between low bone density and atherosclerotic disease, the mechanism linking bone density and atherosclerotic disease remains unclear. Osteoprotegerin (OPG), a novel member of the TNF receptor superfamily, could function as a molecular link between bone resorption and arterial calcification. In this regard, OPG was found to block ovariectomy-associated bone loss in rats and to act as a soluble factor in the regulation of bone mass [25]. In addition, atherosclerosis has also been considered as an inflammatory disease [26]. Therefore, these preliminary results suggest that OPG levels are significantly correlated with vascular function contributing to the pathogenesis of atherosclerosis in cerebral vascular disease. In conclusion, the present study revealed that low BMD and osteoporosis are associated with significantly increased risk of stroke and death in Chinese postmenopausal women. Understanding and recognition of this risk association is important to address preventive measures for these two major causes of morbidity and mortality in the elderly women. Further studies are needed to determine the mechanisms responsible for low BMD and risk of stroke and death in postmenopausal women. Acknowledgments This study was supported by funding from Chongqing Educational Commission (KJTD201337). Conflict of interest
None.
References 1. Rachner, T. D., Khosla, S., & Hofbauer, L. C. (2011). Osteoporosis: now and the future. Lancet, 377, 1276–1287. 2. Cummings, S. R., Nevitt, M. C., Browner, W. S., Stone, K., Fox, K. M., Ensrud, K. E., et al. (1995). Risk factors for hip fracture in white women. Study of osteoporotic fractures research group. New England Journal of Medicine, 332, 767–773.
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3. Dobkin, B. H. (2005). Clinical practice rehabilitation after stroke. New England Journal of Medicine, 352, 1677–1684. 4. Wang, Y., Tao, Y., Hyman, M. E., Li, J., & Chen, Y. (2009). Osteoporosis in china. Osteoporosis International, 20, 1651–1662. 5. Zhao, D., Liu, J., Wang, W., Zeng, Z., Cheng, J., Liu, J., et al. (2008). Epidemiological transition of stroke in China: twentyone-year observational study from the Sino-MONICA-Beijing Project. Stroke, 39, 1668–1674. 6. Xingye, L., & Zengdong, M. (2011). Relationship between quantity of illumination and human vitamin D concentration and bone mineral density in different region. Chin J Osteoporos, 18, 183–187. 7. Aronow, W. S. (2011). Osteoporosis, osteopenia, and atherosclerotic vascular disease. Arch Med Sci, 7, 21–26. 8. Tanko, L. B., Christiansen, C., Cox, D. A., Geiger, M. J., McNabb, M. A., & Cummings, S. R. (2005). Relationship between osteoporosis and cardiovascular disease in postmenopausal women. Journal of Bone and Mineral Research, 20, 1912–1920. 9. Szulc, P., Samelson, E. J., Kiel, D. P., & Delmas, P. D. (2009). Increased bone resorption is associated with increased risk of cardiovascular events in men: the MINOS study. Journal of Bone and Mineral Research, 24, 2023–2031. 10. Tamaki, J., Iki, M., Hirano, Y., Sato, Y., & Kajita, E. (2009). Low bone mass is associated with carotid atherosclerosis in postmenopausal women: the Japanese Population-based Osteoporosis (JPOS) Cohort Study. Osteoporosis International, 20, 53–60. 11. Mussolino, M. E., & Armenian, H. K. (2007). Low bone mineral density, coronary heart disease, and stroke mortality in men and women: the Third National Health and Nutrition Examination Survey. Annals of Epidemiology, 17, 841–846. 12. Suzuki, T., & Yoshida, H. (2010). Low bone mineral density at femoral neck is a predictor of increased mortality in elderly Japanese women. Osteoporosis International, 21, 71–79. 13. Shiraki, M., Kuroda, T., & Tanaka, S. (2011). Established osteoporosis associated with high mortality after adjustment for age and co-mobidities in postmenopausal Japanese women. Internal Medicine, 50, 397–404. 14. Kauppila, L. I., Polak, J. F., & Cupples, L. A. (1998). Guidelines for preclinical evaluation and clinical trials in osteoporosis. Geneva: World Health Organization. 15. Uyama, O., Yoshimoto, Y., Yamamoto, Y., & Kawai, A. (1997). Bone changes and carotid atherosclerosis in postmenopausal women. Stroke, 28, 1730–1732. 16. Barengolts, E. I., Berman, M., Kukreja, S. C., Kouznetsova, T., Lin, C., & Chomka, E. V. (1998). Osteoporosis and coronary atherosclerosis in asymptomatic postmenopausal women. Calcified Tissue International, 62, 209–213. 17. Kiel, D. P., Kauppila, L. I., Cupples, L. A., Hannan, M. T., O’Donnell, C. J., & Wilson, P. W. (2001). Bone loss and the progression of abdominal aortic calcification over a 25 year period: the Framingham Heart Study. Calcified Tissue International, 68, 271–276. 18. Nordstrom, A., Eriksson, M., Stegmayr, B., Gustafson, Y., & Nordstrom, P. (2010). Low bone mineral density is an independent risk factor for stroke and death. Cerebrovascular Diseases, 29, 130–136. 19. Jorgensen, L., Engstad, T., & Jacobsen, B. K. (2001). Bone mineral density in acute stroke patients: low bone mineral density may predict first stroke in women. Stroke, 32, 47–51. 20. Lee, S. B., Cho, A. H., Butcher, K. S., Kim, T. W., Ryu, S. Y., & Kim, Y. I. (2013). Low bone mineral density is associated with poor clinical outcome in acute ischemic stroke. International Journal of Stroke, 8, 68–72. 21. Browner, W. S., Pressman, A. R., Nevitt, M. C., Cauley, J. A., & Cummings, S. R. (1993). Association between low bone density
Cell Biochem Biophys and stroke in elderly women. The study of osteoporotic fractures. Stroke, 24, 940–946. 22. Trivedi, D. P., & Khaw, K. T. (2001). Bone mineral density at the hip predicts mortality in elderly men. Osteoporosis International, 12, 259–265. 23. Van Der Klift, M., Pols, H. A., Geleijnse, J. M., Van Der Kuip, D. A., Hofman, A., & De Laet, C. E. (2002). Bone mineral density and mortality in elderly men and women: the Rotterdam Study. Bone, 30, 643–648.
24. Mussolino, M. E., Madans, J. H., & Gillum, R. F. (2003). Bone mineral density and stroke. Stroke, 34, e20–e22. 25. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Lu¨thy, R., et al. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell, 89, 309–319. 26. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 362, 801–809.
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ORIGINAL PAPER
Low Bone Mass is Associated with Stroke in Chinese Postmenopausal Women: The Chongqing Osteoporosis Study Rui Zhou • Dong Liu • Rui Li • Shiming Zhou Min Cui • Lin Chen • Huadong Zhou
•
Ó Springer Science+Business Media New York 2014
Abstract The objective of the present study was to investigate the association of low bone mass with the risk of stroke and death in community residents of China. This study was based on the follow-up data acquired from 5,136 postmenopausal women aged 50 years or older between July 2006 and June 2011. Baseline and the follow-up bone mineral density (BMD) in these patients were measured by dual energy X-ray absorptiometry scanning. The association of BMD and risk of stroke and death was further evaluated by Cox proportional hazard analysis. During the follow-up, 148 subjects (2.9 %) sustained prospective stroke, and 261 subjects (5.1 %) died. After adjustments for age and BMI, our results indicated that neck BMD and osteoporosis were independent predictors of stroke (HR for neck BMD = 1.35, 95 % CI = 1.21–1.62; HR for osteoporosis = 2.24, 95 % CI = 1.47–3.58) and were also associated with increased risk of death (HR for neck BMD = 1.39, 95 % CI = 1.24–1.71; HR for osteoporosis = 1.97, 95 % CI = 1.21–2.97). Our results also suggest that low neck BMD and osteoporosis are associated with significantly elevated risk of stroke and death in Chinese postmenopausal women.
R. Zhou Department of Orthopedics, The Orthopedic Surgery Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, China D. Liu L. Chen Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China R. Li S. Zhou M. Cui H. Zhou (&) Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing, China e-mail: [email protected]
Keywords Bone mineral density Stroke Death Postmenopausal women
Introduction Osteoporosis and stroke were found to be strongly associated with aging and share common risk factors. They were often coexistent in the same patient, implying a possible relation between osteoporosis and risk of stroke. As the prevalence of osteoporosis and stroke is increasing dramatically in aging population across the globe, it could pose a major health problem [1–3]. China with its rapidly growing elderly population has high incidence of osteoporosis [4] and stroke as compared to Western societies [5, 6]. Recently, it has been observed that older women and men with low bone mineral density (BMD) have a higher incidence of stroke than older people with normal BMD [7–10]. However, there are contradictory reports suggesting no significant association between Low BMD and risk of stroke in men, whereas the risk was found to be only slightly higher among women with low BMD [11]. Therefore, larger prospective studies in elderly people are needed to clearly answer this question. Even though earlier prospective cohort studies of the elderly suggested that low BMD is associated with increased mortality [12, 13], the real relationship is still controversial. Therefore, the actual relation between lower BMD and individual mortality needs to be further investigated in randomized trials. In order to address the significance of baseline bone loss and its impact on subsequent stroke and death in Chinese elderly people, we conducted a 5-year prospective study in postmenopausal women in Southwest China and found that
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Cell Biochem Biophys
low neck BMD and osteoporosis significantly elevate the risk of stroke and death.
Materials and Methods Study Subjects The subjects enrolled in the current study were chosen from three randomly selected communities of Chongqing, one of the largest municipalities in Southwest China, with a population of 35 million. This study was approved by the Institutional Review Board of the Chongqing Daping Hospital and Southwest Hospital. Informed consent was obtained from all the subjects prior to the study. Baseline Screening The baseline screening of the present study was performed from January 2006 to June 2006. A total of 6,282 postmenopausal women aged 50 years and above were enrolled from the selected communities. The subjects were interviewed by organized meetings in the community centers, as part of selection process. For people who were absent at the meeting or who were unable to attend due to physical disabilities, our staff performed the interviews at the patients’ homes. At baseline, 6,282 individuals were screened while a total of 603 subjects, who were either not available at the time of screening (n = 284) or declined to participate (n = 319), were excluded from the present study. A total of 345 cases with stroke were diagnosed and excluded from the study. A total of 5,334 subjects without previous stroke were enrolled at baseline (Fig. 1). The following data were collected: demographic data, BMD, smoking and drinking status, and medical history. The trained interviewers including experienced neurologists and senior nurses administered these procedures. The reliability across the interviewers was measured by the Cohen’s Kappa statistical analysis. Based on the same subject sample, the agreement on data collection was found to be excellent, with a kappa score of 0.93. BMD and Osteoporosis Dual energy X-ray absorptiometry (DXA, Prodigy fan beam densitometer, Lunar Corp, GE Medical System, Madison, WI) was employed to determine BMD. BMD of femoral neck bone was further used as a measure of global bone health. BMD was measured at baseline. Osteoporosis, defined as BMD, that is 2.5 standard deviations (SD) below the mean normal BMD, which is obtained from 10,000 healthy women aged 20–29 years in Daping Hospital of Chongqing [14].
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Fig. 1 Flow chart of this study
Smoking and Drinking Status The smoking status was classified as past smokers (who had quit smoking for at least 6 months prior to enrollment), current smokers, or those who never smoked. Drinking status was classified as daily drinking, weekly drinking, monthly drinking, or occasional drinking. Clinical Assessment The medical history was collected from self-report, available medical records, or medication. The data included coronary heart diseases, chronic obstructive pulmonary disease, chronic hepatitis, chronic renal insufficiency, hypertension, diabetes mellitus, hypercholesterolemia, and Parkinson’s disease. Blood pressure measurement and electrocardiogram were performed on-site, and fasting blood samples were collected to measure glucose, total cholesterol, creatinine, uric acid, osteoprotegerin (OPG), and 25(OH) D. Body mass index (BMI) was also assessed. The subjects with abnormalities, suggestive of potential diseases that were not previously diagnosed, were admitted to Chongqing Daping hospital for further investigation. In addition, diagnosis of hypertension, diabetes mellitus,
Cell Biochem Biophys
hypercholesterolemia, ischemic heart disease, stroke, chronic renal insufficiency, and chronic hepatitis were based on International Classification of Diseases, 9th Revision (ICD-9). Assessment of Mortality Death of any of the studied subjects was confirmed through inquiry since their last visit. The date of death was confirmed on the basis of death certificates or by consulting the available hospital records. In some instances, death of the subjects was confirmed from consulting their immediate family. The subjects who lost contact were treated as missing cases. Follow-Up Subjects were followed up annually for 5 years from July 2006 to June 2011. Epidemiological questionnaires and telephone were used to obtain the follow-up information, such as stroke, changes of the diseases, and death every year during the 5-year follow-up period. Each self-reported stroke was confirmed by physicians on the basis of medical record and CT and MRI examination. The stroke in this study consisted of intracranial hemorrhage and cerebral infarction, and subjects with traumatic stroke were excluded. Statistical Analysis In univariate analyses, baseline variables between the subjects who developed stroke or died and those who did not were compared using Pearson v2 test, Fisher exact test, a t test, or the Mann–Whitney U test as deemed appropriate. Additionally, Cox proportional hazard models were used to assess the associations between BMD, osteoporosis and increased risk of stroke or death in two steps. The associations were first analyzed with adjustments for age and BMI. Then, the associations were analyzed with adjustments for other covariates, including age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus, former stroke, and ischemic heart disease. The statistical analyses were finally performed using SPSS 15.0 for Windows.
Baseline Characteristics in Subjects Who Did and Did Not Develop Stroke Table 1 summarizes the baseline characteristics of the subjects with and without prospective stroke. Compared with 4,988 subjects who did not experience a stroke, 148 subjects with prospective stroke were found to be more frequent in current smoking and daily drinking, and have more hypercholesterolemia, a lower neck BMD, and a higher prevalence of osteoporosis (p \ 0.01), more diabetes mellitus, and hypertension (p \ 0.001). These 148 subjects were also found to be older, and have higher BMI and serum levels of OPG than those subjects who did not sustain a stroke (p \ 0.05). Association Between the Bone Variables and Risk of Stroke The independent association between the bone variables and the future risk of stroke after adjustment for potential confounders is shown in Table 2. After adjustment for age and BMI, neck BMD was found to correlate with increased risk of stroke (HR 1.35, 95 % CI 1.21–1.26). After adjustment for the same variables, osteoporosis was also found to be an independent prospective risk factor for stroke (HR 2.24, 95 % CI 1.47–3.58). Further, it was found that neck BMD (HR 1.69, 95 % CI 1.42–1.93) and osteoporosis (HR 3.94, 95 % CI 1.92–9.68) were still found to be independent predictors of the risk of stroke after adjustments with age, BMI, smoking, drinking, hypertension, and diabetes mellitus. Baseline Characteristics in Subjects Who Died and Did Not Died The 261 subjects who died during the follow-up were found to be either frequent current smokers, or had hypertension, diabetes mellitus, a lower neck BMD, and a higher prevalence of osteoporosis than the remaining 4,875 subjects who did not die (p \ 0.01) during follow-up. These 261 subjects were also older, have a higher BMI, and more frequent in daily drinking than those subjects who did not die (p \ 0.05) during follow-up (Table 3). The Relations Between the Bone Variables and the Future Risk of Death
Results In this study, 5,334 people without stroke were enrolled at baseline. Later, during the 5-year follow-up period, 105 (2.4 %) patients declined, and another 93 (2.1 %) moved away with the remaining 5,136 subjects completing the follow-up. The subjects with a mean age of 62.8 years (range 50–83 years old) were included in this study.
We also established Cox proportional hazard models including age, BMI, smoking, drinking, hypertension, and diabetes mellitus to obtain insights into the independent predictors of death risk, and the results are shown in Table 4. After adjustments for age and BMI, neck BMD (HR 1.39, 95 % CI 1.24–171) was related to the increased risk of death. Additionally, after adjustment for the same variables, osteoporosis
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Cell Biochem Biophys Table 1 Baseline characteristics in 5,136 subjects who did and did not develop stroke
No strokes (n = 4,988)
Strokes (n = 148)
p value
Physical characteristics Age (year), ± SD
62.8 ± 9.1
64.5 ± 7.3
\0.05
BMI (kg/m2), ± SD
21.7 ± 4.2
23.1 ± 3.9
\0.05
Physical activity index, n (%)
1,265 (25.4)
36 (24.3)
NS
Never smoking, n (%)
1,940 (38.9)
62 (41.9)
NS
Past smoking, n (%)
1,581 (31.7)
50 (33.8)
NS
Current smoking, n (%)
856 (17.2)
44 (30.0)
\0.01
1,401 (28.1)
43 (28.9)
NS
Smoking status
Drinking status Occasional drinking, n (%) Daily drinking, n (%)
868 (17.4)
36 (24.3)
\0.01
Weekly drinking, n (%)
903 (18.1)
28 (18.9)
NS
Monthly drinking, n (%)
1,401 (28.1)
39 (26.4)
NS
Hypertension, n (%) Diabetes mellitus, n (%)
1,451 (29.1) 968 (19.4)
52 (35.1) 40 (26.8)
\0.001 \0.001
Hypercholesterolemia, n (%)
963 (19.3)
35 (23.5)
\0.01
Neck BMD (g/cm3), ± SD
0.838 ± 0.136
0.679 ± 0.121
\0.01
Osteoporosis n (%)
643 (12.9)
24 (16.2)
\0.01
Osteoprotegerin pmol/L, ± SD
3.08 ± 1.13
3.49 ± 1.64
\0.05
25(OH) D (ng/mL)
15.3 ± 6.7
17.8 ± 5.9
\0.05
Vascular risk factors
Bone mass measurements
NS not significant
Table 2 Independent relationship between the bone density and the risk of stroke HR
95 % CI
A: Bone variables adjusted for age and BMI Neck BMD
1.35
1.21–1.62
Osteoporosis
2.24
1.47–3.58
B: Bone variables adjusted for age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus, hypercholesterolemia Neck BMD Osteoporosis
1.69 3.94
1.42–1.93 1.92–9.68
Bone variables were analyzed per SD decrease, and osteoporosis was analyzed as yes/no
(HR 1.97, 95 % CI 1.21–2.97) was also found to be an independent risk factor for death. Further, the results demonstrated that both neck BMD (HR 1.36, 95 % CI 1.18–1.89) and osteoporosis (HR 3.63, 95 % CI 1.49–8.61) were independent predictors of the risk of death after adjustments for age, BMI, smoking, drinking, hypertension, and diabetes mellitus.
Discussion In this study, our results showed that femoral neck BMD is an independent risk factor for future stroke and death in
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Chinese postmenopausal women. From our data, we noticed a consistent association of low neck BMD and osteoporosis with the risk of stroke and death. The subjects were also found to have higher serum levels of OPG than those subjects who did not sustain a stroke. Low neck BMD increased the incident risk for stroke by 1.35-fold, while osteoporosis increased it by 2.24-fold. Additionally, low neck BMD increased the incident risk for death by 1.69-fold, and osteoporosis increased this risk by 3.94-fold. These results revealed the significance of altered bone metabolism on the incidence of stroke and death. Earlier studies showed that calcium loss from bone might directly correlate to arterial calcification [7, 10]. Other studies suggested that low BMD is related to atherosclerosis and aortic calcification. In particular, postmenopausal women with osteoporosis might be at a greater risk of developing coronary atherosclerosis [15, 16]. Framingham Heart Study also found that bone loss was directly related to aortic calcification in women but not men [17]. Low BMD may serve as a marker for stroke. In 2010, Nordstrom et al. [18] examined whether BMD was prospectively related to stroke, in a study of 4,302 men and women (with a mean age of 59 years old) at baseline. After a mean follow-up period of 5.6 years, 139 strokes were registered. The study indicated that decreased BMD as well
Cell Biochem Biophys Table 3 Baseline characteristics in 5,136 subjects who died during follow-up or not
No dead (n = 4,875)
dead (n = 261)
p value
Physical characteristics Age (year), ± SD
62.7 ± 9.3
65.4 ± 7.1
\0.05
BMI (kg/m2), ± SD
21.1 ± 4.2
23.8 ± 3.9
\0.05
Physical activity index, n (%)
1,243 (25.5)
65 (24.9)
NS
Smoking status Never smoking, n (%)
1,926 (39.5)
103 (39.4)
NS
Past smoking, n (%)
1,599 (32.8)
82 (31.5)
NS
Current smoking, n (%)
1,253 (25.7)
76 (29.3)
\0.01
1,530 (31.4)
79 (30.5)
NS
Drinking status Occasional drinking, n (%) Daily drinking, n (%)
1,112 (22.8)
68 (26.1)
\0.05
Weekly drinking, n (%)
941 (19.3)
48 (18.4)
NS
Monthly drinking, n (%)
1,292 (26.5)
65 (25.0)
NS
Hypertension, n (%) Diabetes mellitus, n (%)
1,472 (30.2) 1,014 (20.8)
94 (36.1) 70 (26.9)
\0.01 \0.01
Hypercholesterolemia, n (%)
994 (20.4)
56 (21.4)
NS
Neck BMD (g/cm3), ± SD
0.837 ± 0.136
0.712 ± 0.121
\0.01
Osteoporosis n (%)
626 (12.8)
41 (15.7)
\0.01
Osteoprotegerin pmol/L, ± SD
3.09 ± 1.13
3.11 ± 1.23
NS
25(OH) D (ng/mL)
15.9 ± 6.8
16.1 ± 4.7
NS
Vascular risk factors
Bone mass measurements
NS not significant
Table 4 Independent relationship between the bone density and the risk of death HR
95 % CI
A: Bone variables adjusted for age and BMI Neck BMD
1.39
1.24–1.71
Osteoporosis
1.97
1.21–2.97
B: Bone variables adjusted for age, BMI, current smokers, daily drinking, hypertension, diabetes mellitus Neck BMD
1.36
1.18–1.89
Osteoporosis
3.63
1.49–8.61
Bone variables were analyzed per SD decrease, and osteoporosis was analyzed as yes/no
as osteoporosis of the femoral neck independently correlated with stroke [18]. The relationships between BMD and acute stroke were also examined in women aged C60 years in another study; measurements of the stroke in patients were performed for 6 days after the onset of stroke. The results found that female, but not male, stroke patients have lower BMD than the corresponding population controls [19]. Another study examined acute ischemic stroke patients who underwent bone densitometry tests within 7 days of stroke symptom onset and noticed that patients with osteoporosis of the right femoral neck were more likely to have poor outcome in acute ischemic stroke than
those without [20]. In a study of 4,024 ambulatory women aged 65 years or older, 83 women suffered first stroke during a 1.98-year follow-up period, and osteoporosis was associated with an increased stroke risk and it was suggested that low bone density may not cause stroke [21]. The above studies have shown that the relation between BMD and stroke is not fully elucidated; our present study found the association of low neck BMD and osteoporosis with risk of stroke. Low neck BMD and osteoporosis increased the incident risk for stroke. Our study found that the subjects, who died during the follow-up period, had lower neck BMD at baseline than those subjects who did not die. Nevertheless, our results are consistent with some studies, while different from other. Therefore, there may be community/population samplebased differences among the results obtained in different countries and we cannot also rule out differences due to ethnicity and life-styles. For example, Trivedi et al. reported in a study that examined the association of bone density and cardiovascular mortality in elderly aged 65–76 years that low bone density was a strong and independent predictor of all-cause and cardiovascular mortality [22]. In addition, a total of 1,429 ambulatory postmenopausal female volunteers aged over 50 years old were enrolled in another study in Japan, in which severity of osteoporosis was found to be a significant independent risk
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Cell Biochem Biophys
factor for all-cause mortality [13]. However, in a Rotterdam Study that enrolled 5,819 men and women aged C55 years and measured BMD during an average followup of 5.4 years, no significant relationship between BMD and overall mortality was observed in women [23]. Additionally, a cohort of 3,402 white and black subjects of 45 to 74 years age at baseline was enrolled in a large national study and no significant associations of BMD and mortality for either whites or blacks were observed [24]. Michael et al. also reported that low BMD was not associated with risk of cardiovascular disease in men. It was found that risk of cardiovascular disease and stroke was elevated among women with low bone density, whereas no significant associations were found. After controlling for baseline age and other factors, proximal femoral BMD was still not associated with subsequent death [11]. Although there are several common possible mechanisms contributing to a relation between low bone density and atherosclerotic disease, the mechanism linking bone density and atherosclerotic disease remains unclear. Osteoprotegerin (OPG), a novel member of the TNF receptor superfamily, could function as a molecular link between bone resorption and arterial calcification. In this regard, OPG was found to block ovariectomy-associated bone loss in rats and to act as a soluble factor in the regulation of bone mass [25]. In addition, atherosclerosis has also been considered as an inflammatory disease [26]. Therefore, these preliminary results suggest that OPG levels are significantly correlated with vascular function contributing to the pathogenesis of atherosclerosis in cerebral vascular disease. In conclusion, the present study revealed that low BMD and osteoporosis are associated with significantly increased risk of stroke and death in Chinese postmenopausal women. Understanding and recognition of this risk association is important to address preventive measures for these two major causes of morbidity and mortality in the elderly women. Further studies are needed to determine the mechanisms responsible for low BMD and risk of stroke and death in postmenopausal women. Acknowledgments This study was supported by funding from Chongqing Educational Commission (KJTD201337). Conflict of interest
None.
References 1. Rachner, T. D., Khosla, S., & Hofbauer, L. C. (2011). Osteoporosis: now and the future. Lancet, 377, 1276–1287. 2. Cummings, S. R., Nevitt, M. C., Browner, W. S., Stone, K., Fox, K. M., Ensrud, K. E., et al. (1995). Risk factors for hip fracture in white women. Study of osteoporotic fractures research group. New England Journal of Medicine, 332, 767–773.
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3. Dobkin, B. H. (2005). Clinical practice rehabilitation after stroke. New England Journal of Medicine, 352, 1677–1684. 4. Wang, Y., Tao, Y., Hyman, M. E., Li, J., & Chen, Y. (2009). Osteoporosis in china. Osteoporosis International, 20, 1651–1662. 5. Zhao, D., Liu, J., Wang, W., Zeng, Z., Cheng, J., Liu, J., et al. (2008). Epidemiological transition of stroke in China: twentyone-year observational study from the Sino-MONICA-Beijing Project. Stroke, 39, 1668–1674. 6. Xingye, L., & Zengdong, M. (2011). Relationship between quantity of illumination and human vitamin D concentration and bone mineral density in different region. Chin J Osteoporos, 18, 183–187. 7. Aronow, W. S. (2011). Osteoporosis, osteopenia, and atherosclerotic vascular disease. Arch Med Sci, 7, 21–26. 8. Tanko, L. B., Christiansen, C., Cox, D. A., Geiger, M. J., McNabb, M. A., & Cummings, S. R. (2005). Relationship between osteoporosis and cardiovascular disease in postmenopausal women. Journal of Bone and Mineral Research, 20, 1912–1920. 9. Szulc, P., Samelson, E. J., Kiel, D. P., & Delmas, P. D. (2009). Increased bone resorption is associated with increased risk of cardiovascular events in men: the MINOS study. Journal of Bone and Mineral Research, 24, 2023–2031. 10. Tamaki, J., Iki, M., Hirano, Y., Sato, Y., & Kajita, E. (2009). Low bone mass is associated with carotid atherosclerosis in postmenopausal women: the Japanese Population-based Osteoporosis (JPOS) Cohort Study. Osteoporosis International, 20, 53–60. 11. Mussolino, M. E., & Armenian, H. K. (2007). Low bone mineral density, coronary heart disease, and stroke mortality in men and women: the Third National Health and Nutrition Examination Survey. Annals of Epidemiology, 17, 841–846. 12. Suzuki, T., & Yoshida, H. (2010). Low bone mineral density at femoral neck is a predictor of increased mortality in elderly Japanese women. Osteoporosis International, 21, 71–79. 13. Shiraki, M., Kuroda, T., & Tanaka, S. (2011). Established osteoporosis associated with high mortality after adjustment for age and co-mobidities in postmenopausal Japanese women. Internal Medicine, 50, 397–404. 14. Kauppila, L. I., Polak, J. F., & Cupples, L. A. (1998). Guidelines for preclinical evaluation and clinical trials in osteoporosis. Geneva: World Health Organization. 15. Uyama, O., Yoshimoto, Y., Yamamoto, Y., & Kawai, A. (1997). Bone changes and carotid atherosclerosis in postmenopausal women. Stroke, 28, 1730–1732. 16. Barengolts, E. I., Berman, M., Kukreja, S. C., Kouznetsova, T., Lin, C., & Chomka, E. V. (1998). Osteoporosis and coronary atherosclerosis in asymptomatic postmenopausal women. Calcified Tissue International, 62, 209–213. 17. Kiel, D. P., Kauppila, L. I., Cupples, L. A., Hannan, M. T., O’Donnell, C. J., & Wilson, P. W. (2001). Bone loss and the progression of abdominal aortic calcification over a 25 year period: the Framingham Heart Study. Calcified Tissue International, 68, 271–276. 18. Nordstrom, A., Eriksson, M., Stegmayr, B., Gustafson, Y., & Nordstrom, P. (2010). Low bone mineral density is an independent risk factor for stroke and death. Cerebrovascular Diseases, 29, 130–136. 19. Jorgensen, L., Engstad, T., & Jacobsen, B. K. (2001). Bone mineral density in acute stroke patients: low bone mineral density may predict first stroke in women. Stroke, 32, 47–51. 20. Lee, S. B., Cho, A. H., Butcher, K. S., Kim, T. W., Ryu, S. Y., & Kim, Y. I. (2013). Low bone mineral density is associated with poor clinical outcome in acute ischemic stroke. International Journal of Stroke, 8, 68–72. 21. Browner, W. S., Pressman, A. R., Nevitt, M. C., Cauley, J. A., & Cummings, S. R. (1993). Association between low bone density
Cell Biochem Biophys and stroke in elderly women. The study of osteoporotic fractures. Stroke, 24, 940–946. 22. Trivedi, D. P., & Khaw, K. T. (2001). Bone mineral density at the hip predicts mortality in elderly men. Osteoporosis International, 12, 259–265. 23. Van Der Klift, M., Pols, H. A., Geleijnse, J. M., Van Der Kuip, D. A., Hofman, A., & De Laet, C. E. (2002). Bone mineral density and mortality in elderly men and women: the Rotterdam Study. Bone, 30, 643–648.
24. Mussolino, M. E., Madans, J. H., & Gillum, R. F. (2003). Bone mineral density and stroke. Stroke, 34, e20–e22. 25. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Lu¨thy, R., et al. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell, 89, 309–319. 26. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 362, 801–809.
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