Bone 42 (2008) 260 – 266 www.elsevier.com/locate/bone
Vitamin D deficiency as a risk factor for osteoporotic fractures N.M. van Schoor a,⁎, M. Visser a,b , S.M.F. Pluijm c , N. Kuchuk d , J.H. Smit e,f , P. Lips a,d a
b
EMGO Institute, VU University Medical Center, Amsterdam, The Netherlands Institute of Health Sciences, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, The Netherlands c Department of Public Health, Erasmus MC, University Medical Centre Rotterdam, The Netherlands d Department of Endocrinology, VU University Medical Center, Amsterdam, The Netherlands e Department of Sociology and Social Gerontology, Vrije Universiteit, Amsterdam, The Netherlands f Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands Received 18 June 2007; revised 26 September 2007; accepted 4 November 2007 Available online 17 November 2007
Abstract The evidence on the association between vitamin D deficiency and fracture incidence is contradictory. Therefore, the objective of this study was to examine whether low serum 25-hydroxyvitamin D (25(OH)D) levels are associated with osteoporotic fractures. The study was conducted among 1311 community-dwelling older men and women of the Longitudinal Aging Study Amsterdam (LASA), an ongoing multidisciplinary cohort study. Serum 25(OH)D was determined using a competitive protein binding assay. Fractures were assessed during six years of follow-up. The data were analyzed using Cox proportional hazards model. In total, 11.3% of the persons had a serum 25(OH)D below 10 ng/ml, 48.4% had a value below 20 ng/ml, and 82.4% had a value below 30 ng/ ml. Furthermore, 115 persons (8.5%) had one or more osteoporotic fractures. Different cut points of serum 25(OH)D were examined with a cut point of 12 ng/ml giving the best discrimination between persons with and without fractures (17.5% of the persons fell below this cut point). The lowest percentage of fractures (5.6%) was found above 30 ng/ml. Because an interaction effect with age was found (p = 0.04), further analyses were conducted separately for persons aged 65–75 years (n = 656) and for persons aged 75–89 years (n = 664) at baseline. After adjustment for age, sex, season of blood collection, body mass index, number of chronic diseases, serum creatinine, cognition, smoking and alcohol use, serum 25(OH)D below or equal to 12 ng/ml was associated with an increased fracture risk in the youngest age group (HR = 3.1; 95% CI: 1.4–6.9), but not in the oldest age group (HR = 1.3; 95% CI: 0.7–2.2). For commonly used cut points of serum 25(OH)D (b 10 ng/ml, 10–19.9 ng/ml, 20– 29.9 ng/ml, ≥ 30 ng/ml), no statistically significant associations were found after adjustment for confounding. Serum 25(OH)D levels below or equal to 12 ng/ml were associated with an increased fracture risk in persons aged 65–75 years. The relatively low cut point of serum 25(OH)D in our population is possibly caused by high calcium intake in the Netherlands. © 2007 Elsevier Inc. All rights reserved. Keywords: Vitamin D deficiency; Fractures; Risk factor; Osteoporosis; Elderly
Introduction Vitamin D deficiency is very common in the elderly. The prevalence varies strongly between different studies, depending on the population and definition used [1]. Vitamin D deficiency in older persons is caused by insufficient sunlight exposure, a decreased functional capacity of the skin to synthesize vitamin ⁎ Corresponding author. VU University Medical Center, EMGO Institute, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Fax. +31 20 4446775. E-mail address:
[email protected] (N.M. van Schoor). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.11.002
D3 and low dietary vitamin D intake [1]. Vitamin D plays an important role in calcium homeostasis [1]. In addition, it stimulates bone growth while protecting osteoblasts from apoptosis [2]. Fractures are one of the most severe consequences of osteoporosis. Osteoporotic fractures are associated with impaired functioning, decreased quality of life, increased nursing home admission, increased mortality, and high health care costs [3–9]. In epidemiological studies, vitamin D deficiency has been associated with several determinants of fractures, such as low bone mineral density, muscle weakness, increased body sway and falls [10–13]. Two meta-analyses show that the incidence
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of falls and fractures can be decreased by vitamin D supplementation [14,15]. The meta-analysis on fractures suggests that the anti-fracture effect only exists for a vitamin D dose of 700 to 800 IU daily. Since the publication of this meta-analysis, five large trials were published that did not find an effect of vitamin D supplementation on fractures [16–20]. One of these studies used a vitamin D dose of 400 IU daily [18]; two studies used 800 IU daily [16,17], one study used 100,000 IU four-monthly (equivalent to 822 IU daily) [20], and one study used 2.5 mg three-monthly (equivalent to 1100 IU daily) [19]. Two of these studies used ergocalciferol [19,20]. Prospective cohort studies are needed to examine the strength of the association between different levels of serum 25(OH)D deficiency and fractures. Up till now, the association between low serum 25(OH)D levels and fracture risk was only examined in three large cohort studies [21–23]. The first study did find an association between serum 1,25-dihydroxyvitamin D and risk for hip fracture, but not for serum 25(OH)D. In addition, no association with vertebral fractures was found [21]. The second study found an increased fracture risk for a subgroup of women with 25(OH)D levels below 20 ng/ml, but not for 30 ng/ ml [22]. The third study found an increased fracture risk for women with 25(OH)D levels below 12 ng/ml and 20 ng/ml, but these were explained by age [23]. In the latter study, no significant associations with fracture risk were found for women having 25(OH)D levels below 30 ng/ml. Because the evidence on the association between vitamin D deficiency and fracture incidence is contradictory, the aim of the present study was to examine whether low serum 25(OH)D levels are associated with osteoporotic fractures. The study was performed using data of the LASA study, an ongoing multidisciplinary cohort study in a representative sample of older Dutch men and women. The analyses were performed using both commonly used cut points of serum 25(OH)D (b10 ng/ml, 10–19.9 ng/ml, 20–29.9 ng/ml, ≥ 30 ng/ml), as well as the optimal cut point found in our dataset. Materials and methods Subjects LASA is an ongoing multidisciplinary cohort study on predictors and consequences of changes in physical, cognitive, emotional and social functioning in older persons [24]. A random sample of men and women aged 55 years and over, stratified by age, sex, urbanization grade and expected 5-year mortality rate was drawn from the population registers of eleven municipalities, in three regions of the Netherlands. In total, 3107 persons were enrolled in the baseline examination in 1992/93. For the present study, persons who participated in the medical interview in 1995/96, and were born on or before 1930 (aged 65 years and older as of January 1, 1996), were selected (n = 1509). In 1352 of these persons, blood samples were drawn and valid serum 25(OH)D could be determined in 1320 samples. A six-year fracture follow-up was available for 1311 persons. The Medical Ethics Committee of the VU University Medical Center approved the study, and all persons gave informed consent.
Osteoporotic fractures Fractures were assessed using two different methods. In the first period (1995/96–1998/99), a fracture calendar was used. Participants were instructed to complete a set of questions regarding fractures every three months, and to send
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them to our institute [25]. In the second period (1998/99–2001/02), fractures were assessed by an interview at the end of the three-year period. For both periods, the exact date of the fracture was asked and verified with the general practitioner or hospital. More than 90% of the fractures included in the analyses could be verified. In case of persons who dropped out or were deceased, the general practitioner was interviewed about potential fractures and the circumstances of the fractures. All fractures were considered osteoporotic, except for head/hand/finger/foot/toe fractures and fractures caused by traffic accidents, which were excluded from the analyses. Nine out of 115 fractures (7.8%) were caused by a fall from more than standing height, for example a fall from a bike (without having a traffic accident).
Serum 25(OH)D and parathyroid hormone (PTH) Morning blood samples were obtained in 1995/96. Subjects were allowed to have tea and toast, but no dairy products. The blood samples were centrifuged and stored at −20 °C. Serum 25(OH)D was determined using a competitive protein binding assay in 1997/1998 (Nichols Diagnostics, San Juan Capistrano, CA, USA). The coefficient of variation was 10%. Serum PTH was determined by an immunoradiometric assay (Incstar Corp., Stillwater, MN, USA). All analyses were performed at the Endocrine Laboratory of the VU University Medical Center. In a subsample (n = 54), the Nichols assay was compared with the Diasorin assay (DiaSorin SpA, Saluggia, Italy). The intercept of the regression line was 0.36 indicating that the Diasorin gave values that were 0.36 ng/ml higher at low values. The regression and correlation coefficient were both 0.96 (P Lips, unpublished observations, March 2006).
Potential effect modifiers Potential effect modifiers were: age and sex. Data on age and sex were derived from population registries. We hypothesized that the effects of low serum 25(OH)D on fractures would be stronger in the oldest age group (≥75 years), while vitamin D deficiency is more prevalent and more severe in old age and the incidence of fractures increases with age [26,27]. The cut point of 75 years was chosen a priori, and selected because this was the median in our population. Furthermore, sex was examined as an effect modifier.
Potential confounders Potential confounders were: age, sex, season of blood collection, education, body mass index (BMI), number of chronic diseases, serum creatinine level, cognition, smoking, alcohol use, physical activity and physical performance. Education level was assessed by asking the respondent for the highest education level completed. This was converted into years of education. BMI was calculated as body weight in kilograms divided by height in meters squared. Body weight was measured without clothes and shoes using a calibrated bathroom balance scale; body height was measured using a stadiometer. Number of chronic diseases was assessed by self-report. Questions were asked about seven major diseases, i.e. chronic obstructive pulmonary disease, cardiac disease, peripheral arterial disease, diabetes mellitus, stroke, cancer, and joint disorders (osteoarthritis/rheumatoid arthritis). Serum creatinine level was measured using the Jaffe alkaline picrate reaction with a Hitachi 747 analyzer, and was included as a marker for renal function (Roche Diagnostics, The Netherlands). Cognitive impairment was assessed with the Mini-Mental State Examination [28]. A score below 24 indicates the presence of a cognitive impairment [29]. Smoking (never, former, current smoker) and alcohol use (does not drink, light, moderate, and excessive drinking) [30] were assessed by questionnaires [31]. Activity level was assessed in minutes per day by the LASA Physical Activity Questionnaire, a questionnaire covering the following areas: walking outside, bicycling, gardening, light household activities, heavy household activities, and a maximum of two sport activities during the previous two weeks [32]. Physical performance was assessed by three tests: time needed to walk 3 m along a rope, turn 180° and walk back; time needed to stand up from and sit down on a chair five times with arms folded across the chest; and the ability to perform the tandem stand (one foot placed behind the other on a straight line) for at least 10 s. Score zero was given to those respondents who could not complete a test; scores one to
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four were given according to the quartile of the distribution of time needed. The total sum score ranged from zero (low physical performance) to twelve (high physical performance).
Potential mediators Specific attention should be paid to the role of physical activity and physical performance. These variables could not only confound the association between vitamin D and fractures, they could also partly mediate it. Several studies have shown that vitamin D deficiency may lead to decreased physical activity and physical performance [11,22,33], which may lead to a subsequent increased risk for fractures [25].
Statistical analysis First, baseline differences between persons with and without fractures were tested. Depending on the distribution of the variable, differences in mean were tested using T-test, differences in median were tested using Mann–Whitney U Test, and differences in frequencies were tested using Pearson Chi-square Test. Second, adjusted analyses using Cox proportional hazards model with time to first fracture as the outcome were performed for different cut points of serum 25(OH)D. Based on the magnitude of the effect, the optimal cut point in our dataset was determined. Third, the following effect modifiers were tested: age (b75 years vs. ≥75 years) and sex. Fourth, the association of serum 25(OH)D with fractures was adjusted for confounding. Because of power limitations due to the higher number of vitamin D categories, the analyses using the commonly used cut points were only adjusted for age, sex and season. The analyses using the optimal cut point were adjusted for age, sex, season of blood collection, education, BMI, number of chronic diseases, serum creatinine level, cognition, smoking and alcohol use. Physical activity and physical performance were only included at the last step in a final model, because these variables could both confound and mediate the relationship under study. Linearity of potential confounders was checked by adding dummies to the model, and by adding a quadratic term. Multi-collinearity was checked by calculating correlation coefficients between the independent variables. These were sufficiently low to enter all variables (r b 0.4). The proportion of fractures among the population attributable to low serum 25(OH)D levels (population attributable fraction, PAF) was calculated as: PAF = [p(HR-1) / p(HR-1) + 1] ⁎ 100%, with p being the proportion of subjects having a low serum 25(OH)D level, and HR the corresponding hazard ratio adjusted for potential confounders. Finally, two sensitivity analyses were performed using the fully adjusted models. In the first sensitivity analysis, an additional confounder was added to the model, i.e. milk consumption from age 50 on. In LASA, calcium intake was not assessed by a validated food questionnaire, and therefore this question was used as a proxy for calcium intake [“high calcium intake”: milk products used every meal (3 or more glasses a day) or milk products used every day but not every meal (1–2 glasses a day) vs. “low calcium intake”: milk products used every week but not every day or milk products used less than once a week]. In the second sensitivity analysis, fractures that could not be verified (n = 10) were excluded.
Results In Table 1, the baseline characteristics are presented. In total, 115 persons had an osteoporotic fracture of which 31 (27.0%) were wrist fractures, 29 (25.2%) hip fractures, 14 (12.2%) rib fractures, 11 (9.6%) vertebral fractures and 30 (26.0%) other fractures. Persons with fractures were significantly older, were more often female, had a lower number of years of education, were more often cognitively impaired, and had a lower physical performance than persons without fractures. In addition, trends towards lower serum 25(OH)D levels (p = 0.07) and lower physical activity (p = 0.07) were observed. In Table 2, the number of fractures per vitamin D category is presented. The largest risk reduction (5.1%, p = 0.01) was found for a cut point of 12 ng/ml. In addition, the largest hazard ratio,
Table 1 Baseline characteristics
Age (years) a Sex (% female) 25(OH)D (ng/ml) a, b Season of 25(OH)D assessment % winter (October–March) PTH (pmol/l) c Education (years) (5–18) a Body mass index (kg/m2) a Number of chronic diseases (0–5) a Creatinine (μmol/l) c MMSE b 24 Smoking Never Former Current Alcohol use No Light Moderate Excessive/very excessive Physical activity (min/day) c Physical performance (0–12)
No fracture (n = 1196)
Fracture (n = 115)
p-value
75.4 ± 6.6 50.3% 21.5 ± 9.7
76.8 ± 6.6 60.9% 19.8 ± 9.7
0.03 0.03 0.07
55.0% 3.2 [2.5–4.3] 9.0 ± 3.3 26.8 ± 4.3 1.2 ± 1.0 90.0 [79.0–103.0] 10.7%
62.6% 3.0 [2.5–3.9] 8.2 ± 3.0 27.0 ± 3.8 1.4 ± 1.3 88.0 [76.0–100.0] 18.3%
0.12 0.31 0.01 0.76 0.20 0.31
35.7% 46.2% 18.1%
36.5% 44.3% 19.1%
0.93
24.2% 50.7% 18.8% 6.3% 136.4 [79.3–205.9] 7.5 ± 3.1
28.7% 44.3% 23.5% 3.5% 124.3 [71.4–191.8] 6.7 ± 3.2
0.24
0.02
0.07 0.01
Differences in mean were tested using T-test, differences in median were tested using Mann–Whitney U Test, and differences in frequencies were tested using Pearson Chi-square Test. MMSE = Mini-Mental State Examination. a Mean ± standard deviation are presented. b To convert to SI units, multiply serum 25-hydroxyvitamin D by 2.496 (nmol/l). c Median [interquartile range] is presented.
and the only borderline significant association with fractures was found for this cut point (HR = 1.5; 95% CI: 1.0–2.4 after adjustment for confounding variables). In total, 17.5% of the persons had a serum 25(OH)D below this cut point. In further analyses, the cut point of 12 ng/ml is referred to as the optimal cut point for serum 25(OH)D. Both for the commonly used cut points (b 10 ng/ml, 10– 19.9 ng/ml, 20–29.9 ng/ml, ≥ 30 ng/ml) as well as for the optimal cut point in this study, an interaction effect with age was found (p = 0.06 for serum 25(OH)D b 10 ng/ml vs. ≥ 30 ng/ml; p = 0.04 for serum 25(OH)D b 12 ng/ml vs. ≥ 12 ng/ml), suggesting a different relationship in the youngest age group and the oldest age group. Therefore, further analyses were performed for persons aged 65–75 years (n = 648) and persons aged 75 years and older (n = 663) at baseline. In the youngest age group, 45 persons had osteoporotic fractures of which 33.3% were wrist fractures, 22.2% hip fractures, 11.1% rib fractures, 11.1% vertebral fractures and 22.3% other fractures. In the oldest age group, 70 persons had osteoporotic fractures of which 22.9% were wrist fractures, 27.1% hip fractures, 12.9% rib fractures, 8.6% vertebral fractures and 28.5% other fractures. No interaction with sex was found. Seasonal differences in serum 25(OH)D and PTH were observed. In persons aged b 75 years, mean serum 25(OH)D was
N.M. van Schoor et al. / Bone 42 (2008) 260–266 Table 2 Optimal cut point serum 25(OH)D for fractures
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Table 3 Risk of fractures using commonly used cut points of serum 25(OH)D
Serum 25(OH)D
Number of fractures (%)
Risk difference p-value a
HR (95% CI) b
≤10 ng/ml (n = 150) N10 ng/ml (n = 1161) ≤12 ng/ml (n = 230) N12 ng/ml (n = 1081) ≤14 ng/ml (n = 312) N14 ng/ml (n = 999) ≤16 ng/ml (n = 411) N16 ng/ml (n = 900) ≤18 ng/ml (n = 526) N18 ng/ml (n = 785) ≤20 ng/ml (n = 632) N20 ng/ml (n = 679) ≤30 ng/ml (n = 1079) N30 ng/ml (n = 232) b10 ng/ml (n = 148) 10–19.9 ng/ml (n = 484) 20–29.9 ng/ml (n = 447) ≥30 ng/ml (n = 232)
15 (10.0%) 100 (8.6%) 30 (13.0%) 85 (7.9%) 37 (11.9%) 78 (7.8%) 43 (10.5%) 72 (8.0%) 53 (10.1%) 62 (7.9%) 63 (10.0%) 52 (7.7%) 102 (9.5%) 13 (5.6%) 15 (10.1%) 48 (9.9%) 39 (8.7%) 13 (5.6%)
1.4% p = 0.57 5.1% p = 0.01 4.1% p = 0.03 2.5% p = 0.14 2.2% p = 0.17 2.3% p = 0.14 3.9% p = 0.06 – p = 0.26
1.0 (0.5–1.7) 1.5 (1.0–2.4) 1.3 (0.8–2.0)
Number of Model 1 fractures (%)
Model 2
b75 years 25(OH)D b10 ng/ml (n = 30) 4 (13.3%) 10–19.9 ng/ml (n = 194) 17 (8.8%) 20–29.9 ng/ml (n = 257) 18 (7.0%) ≥30 ng/ml (n = 167) 6 (3.6%)
4.3 (1.2–15.2) 2.5 (1.0–6.3) 1.9 (0.8–4.9) Reference group
3.4 (0.9–12.1) 1.8 (0.7–4.7) 1.6 (0.7–4.2) Reference group
≥75 years 25(OH)D b10 ng/ml (n = 118) 11 (9.3%) 10–19.9 ng/ml (n = 290) 31 (10.7%) 20–29.9 ng/ml (n = 190) 21 (11.1%) ≥30 ng/ml (n = 65) 7 (10.8%)
0.9 (0.4–2.4) 1.0 (0.5–2.3) 1.0 (0.4–2.3) Reference group
0.8 (0.3–2.2) 1.0 (0.4–2.2) 1.0 (0.4–2.4) Reference group
1.1 (0.7–1.6) 1.0 (0.7–2.4) 1.1 (0.7–1.6) 1.3 (0.7–2.4) 1.4 (0.6–3.0) c 1.4 (0.7–2.7) c 1.4 (0.7–2.6) c Reference group
a
Differences in frequencies were tested using Pearson Chi-square Test. The hazard ratio (HR) and 95% CI (confidence interval) were calculated using Cox proportional hazards model after adjustment for age, sex, season, education, body mass index, number of chronic diseases, creatinine level, cognition, smoking, alcohol use. c Because of power limitations adjusted for age, sex, season. b
23.0 ng/ml (standard deviation = 9.1) in winter and 26.0 ng/ml (SD = 9.4) in summer (p b 0.01); in persons aged ≥ 75 years, mean serum 25(OH)D was 17.5 ng/ml (SD = 8.8) in winter and 19.7 ng/ ml (SD = 9.2) in summer (p b 0.01). In persons aged b75 years, median serum PTH was 2.8 pmol/l [interquartile range = 2.2–3.7] in winter and 3.0 pmol/l [IQR = 2.5–3.9] in summer (p b 0.01); in persons aged ≥75 years, median serum PTH was 3.5 pmol/l [IQR = 2.6–4.5] in winter and 3.5 pmol/l [IQR = 2.6–4.7] in summer (p = 0.45). No significant differences in physical activity and fractures were observed. In persons aged b75 years, the incidence of fractures was 8.2% in winter and 5.3% in summer (p = 0.15); in persons aged ≥75 years, the incidence of fractures was 11.5% in winter and 9.4% in summer (p = 0.37). In Table 3, the effect of serum 25(OH)D on fractures is presented for age b 75 and age ≥ 75 years. In this table, the commonly used cut points of serum 25(OH)D were used. As compared with the reference group of 30 ng/ml or higher, a significantly increased risk for fractures was observed in the youngest age group for persons having a serum 25(OH)D level below 10 ng/ml (HR = 4.3; 95% CI: 1.2–15.2). However, this significant effect disappeared after adjusting for age, sex and season. No associations were observed in the oldest age group. Because there appeared to be a trend in the youngest age group, we also calculated the p-value for trend. The p-value for trend was statistically significant in the unadjusted analyses (p = 0.02), but not in the adjusted analyses (p = 0.09). In Fig. 1, the association between vitamin D status and fractures using the optimal cut point of 12 ng/ml is presented. Significant effects were found in the youngest age group. In contrast to the analyses using the commonly used cut points, the effects remained statistically significant after adjustment for
Presented are the hazard ratio and 95% confidence intervals. Model 1: univariate. Model 2: adjusted for age, sex, season.
confounding (HR = 3.1; 95% CI: 1.4–6.9). In addition, after adjustment for physical activity and physical performance, two variables that could both be a confounder and a mediator, the effect of serum 25(OH)D on fracture incidence also remained statistically significant (HR = 2.7; 95% CI: 1.1–6.4). The proportion of fractures attributable to serum 25(OH)D levels below 12 ng/ml in the youngest age group was 14.2% in the third model and 11.9% in the fourth model. Sensitivity analysis Adding calcium intake as a potential confounder (HR = 1.5; 95% CI: 0.9–2.4), or excluding non-verified fractures (HR = 1.4; 95% CI: 0.9–2.4), resulted in the same cut point, although not statistically significant. Adding calcium intake to
Fig. 1. Risk of fractures using the optimal cut point of serum 25(OH)D. Presented are the hazard ratios and 95% confidence intervals for fractures comparing serum 25(OH)D b=12 ng/ml vs. serum 25(OH)D N12 ng/ml in the age groups b75 years (a) and ≥75 years (b). In the youngest age group, 53 persons had a serum 25(OH)D ≤12 ng/ml and 595 persons had a serum 25(OH)D N12 ng/ml. In the oldest age group, 177 persons had a serum 25(OH)D level ≤12 ng/ml and 486 persons had a serum 25(OH)D N12 ng/ml. Model 1: univariate. Model 2: adjusted for age, sex, season. Model 3: Model 2 + education, body mass index, number of chronic diseases, creatinine level, cognition, smoking, alcohol use. Model 4: Model 3 + physical activity, physical performance. These variables can both act as a confounder and as a mediator.
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the analyses using the commonly used cut points of serum 25 (OH)D resulted in similar associations (data not shown). Excluding non-verified fractures (n = 10) also resulted in similar associations, although the associations as observed in models 3a and 3b of Fig. 1 were slightly weaker and no longer statistically significant (model 3a: HR = 2.5; 95% CI: 1.0–6.1; model 4a: HR = 2.1; 95% CI: 0.8–5.5). Discussion In this study, a statistically significant and independent relationship between serum 25(OH)D levels lower than or equal to 12 ng/ml and fractures was found in persons aged 65– 75 years, while no association was observed for older persons. When using the commonly used cut points of serum 25(OH)D, significant associations in univariate analyses were observed, but these disappeared after adjustment for confounding. One of the most striking results is that serum 25(OH)D was only associated with fracture risk in persons aged 65–75 years at baseline, but not in persons aged 75 years or older. Interestingly, a similar result was found in an earlier study of our department in which an association between serum 25(OH)D lower than 10 ng/ml and recurrent falling was found in persons aged 65–75 years at baseline, but not in persons aged 75 years or older, during one year of follow-up [34]. Our results on fractures might be explained in several ways. First, it could be that the causes of fracture become more multifactorial in the oldest age group, and that the effect of vitamin D deficiency becomes relatively less important for fracture risk. Second, the lack of effect in the oldest age group might be explained by the higher number of persons who deceased in the oldest age group during the six-year fracture follow-up (16.2% in youngest age group vs. 40.9% in oldest age group). In that case, the general practitioner was interviewed about the occurrence of fractures. This might have resulted in an underestimation of the fracture incidence, especially in the oldest age group. The latter explanation is supported by the fact that no interaction with age was found when looking at the first three years of fracture follow-up (data not shown). The optimal cut point in our study was 12 ng/ml. To examine whether 12 ng/ml was also the optimal cut point within both age groups, a post hoc analysis was performed. In the youngest age group, the largest hazard ratio was found for 12 ng/ml (HR = 3.1; 95% CI: 1.4–6.9 after adjustment for confounding variables). In the oldest age group, no significant associations between vitamin D deficiency and fractures were found. Interestingly, a trend towards significance was found for a cut point of 30 ng/ml, and the lowest percentage of fractures was found for persons having a serum 25(OH)D above 30 ng/ml. Although the aim of our study was not to examine the threshold for treatment, it is important to note that the threshold may differ between countries. In the USA and Northern European populations such as Norway, for example, much higher 25 (OH)D levels are found due to fortification of food products such as milk with vitamin D and the more frequent use of supplements and cod liver oil. In a round table discussion in 2003, the minimum desirable serum 25(OH)D concentration
was estimated to be between 50 and 80 nmol/l (20 and 32 ng/ml) [36]. Such a threshold should preferably be based on clinical endpoints but insufficient data are available. In the Netherlands, calcium intake is high. In a previous study of our department, performed in independently living elderly, apartment houses and homes for the elderly, the median calcium intake from dairy products was 859 mg/day in the placebo group and 876 mg/day in the vitamin D group [35]. Unfortunately, we did not assess calcium intake by a validated food questionnaire in our study. Adding a question about calcium intake since the age of 50 did not essentially change the results. However, we cannot exclude that calcium intake is a confounding variable, which might have influenced the optimal cut point. It may be that a lower serum 25(OH)D is tolerated if calcium intake is increased. Physical activity and physical performance were analyzed in a separate model in this study because these variables could both confound and mediate the relationship between vitamin D deficiency and fractures. Interestingly, the hazard ratio decreased after adding physical activity and physical performance, which suggests that these variables have a mediating effect. The associations were stronger for physical performance than for physical activity. As described in the Introduction, only three large cohort studies were found in the literature examining the association between low serum 25(OH)D levels and fracture risk [21–23]. The first study, which was performed in the USA, used a case– cohort approach and was able to include 133 women with a hip fracture and 138 women with a vertebral fracture. However, no associations with serum 25(OH)D were found. In the second study, which was a Swedish study, an association between serum 25(OH)D levels below 20 ng/ml and fractures was found (HR = 2.0; 95% CI: 1.0–4.0). The serum 25(OH)D levels in this study were much higher than in our study with only 4% of the women having levels below 20 ng/ml and 26% having levels below 30 ng/ml. The observed association was not adjusted for confounding variables. In the third study, which was a French study, unadjusted associations between serum 25(OH)D levels below 12 and 20 ng/ml and fractures were observed (p = 0.05 for both cut points), but these were explained by age. In the latter study, no association was found for a cut point of 30 ng/ml. All three studies were limited to women. Five recently published randomized controlled trials did not find an effect of vitamin D and calcium supplementation on the incidence of fractures [16–20]. One of the explanations might be that the participants of these studies had too high baseline values of serum 25(OH)D. In the Record Trial, the mean baseline serum 25(OH)D concentration was 15.2 ng/ml in a subsample of 60 persons [16]; in the study of Jackson et al., the mean baseline serum 25(OH)D concentration was 18.4 ng/ml in participants having a hip fracture and 19.4 ng/ml in controls [18]; in the study of Law et al., the median baseline serum 25(OH)D concentration was 18.8 ng/ml in a subsample of 18 persons [19]. In two earlier studies, which were performed in nursing home residents, very strong effects of vitamin D supplementation on fracture incidence were found [37,38]. The first study reported baseline values of 13 ng/ml in the placebo group and 16 ng/ml in the vitamin D3-calcium group, but from a cross-calibration study it
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appeared that the mean serum 25(OH)D value in the placebo group was 7.2 ng/ml after correction [39]. In the second study, the serum 25(OH)D concentrations were 8.5 ng/ml, 9.0 ng/ml and 9.1 ng/ml in the three different study arms. Strengths of our study are that LASA is a representative sample of the older Dutch population with a long fracture follow-up and the possibility to adjust for many different confounders. A limitation is that fractures in the second period were only recorded at the end of the follow-up period with the possibility of the underreporting of fractures by the participants or general practitioners of persons who died during the followup. Only few fractures occurred below 10 ng/ml and above 30 ng/ml, decreasing the power in these subgroups. Furthermore, serum 25(OH)D was only measured at a single time point. However, as expected, there was a strong negative correlation between serum 25(OH)D and serum PTH (r = − 0.315, p b 0.01). Furthermore, in persons having serum 25(OH)D below or equal to 12 ng/ml, serum PTH was significantly higher as compared to persons having serum 25(OH)D above 12 ng/ml (p b 0.01 for persons aged b75 years and for persons aged ≥ 75 years). Future studies should clarify, whether there is only an association between low serum 25(OH)D and fractures in the youngest age group, or also in the oldest age group. In addition, it would be interesting to examine whether the threshold for treatment differs between countries. In conclusion, serum 25(OH)D levels lower or equal to 12 ng/ml were associated with an increased fracture risk in persons aged 65–75 years. The relatively low cut point of serum 25(OH)D in our population is possibly caused by high calcium intake in the Netherlands. However, future studies need to confirm this. Conflict of interest Dr. Lips served as a consultant for Merck and received research grants from Merck. All other authors have no conflicts of interest.
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