Introduction

Serum levels of 25-hydroxyvitamin D (25[OH]D) are directly related to bone mineral density (BMD) [1]. Calcium absorption decreases from digestive tract when 25(OH)D levels are inadequate (<75 nmol/l) [2]. Accordingly, low 25(OH)D leads to bone resorption and bone loss and thus increased risk of fracture [1, 3–5]. Vitamin D deficiency is common in the elderly since the capacity of the skin to synthesize provitamin decreases with age [6]. Low vitamin D status is prevalent in elderly women living in northern latitudes [7], but this deficiency can also be detected in young adults in northern Europe [8] and in the housebound elderly [9]. It is believed that in order to achieve an optimal vitamin D level of >75 nmol/l one should consume between 800 and 1,000 IU of vitamin D everyday [10].

In a recent meta-analysis, calcium alone and calcium with vitamin D were associated with reduced bone loss at the hip and spine [11]. In another study, the effect of vitamin D and calcium was nonsignificant at the spine and in the total body [12]. Dawson-Hughes et al. reported that the combined supplementation had no effect on proximal femur in a 3-year follow-up [13]. However, an increase of femoral BMD during a shorter 18-month follow-up has also been reported [5]. Chapuy et al. reported a lower rate of annual bone loss at the proximal femur with combined supplementation [14], but in a recent trial, vitamin D treatment alone had no bone effect [15]. These controversial findings could be partly explained by variations in dosages and different intervals between dosings [16]. In addition, different forms of vitamin D and the heterogeneity of study populations might confound the results.

There is still no definitive answer about how vitamin D and calcium supplementation can influence bone status, and in particular, there are no population-based trials to settle the issue. In this 3-year population-based Osteoporosis Risk Factor and Prevention Study-Fracture Prevention Study (OSTPRE-FPS) randomized trial, we hypothesized that vitamin D and calcium supplementation might be able to reduce bone loss in ambulatory postmenopausal women over the age of 65. The open population-based approach demonstrates the effect of the intervention in the general population, in comparison with the situation in women who simply continue their normal diet, perhaps using nonprescription sources of vitamin D and calcium supplementation.

Subjects and methods

Study design and participants

This study was based on the OSTPRE-FPS which began in 2003 in Kuopio, Finland. The primary aim of the study was to determine whether vitamin D and calcium supplementation would be effective in preventing falls and fractures in postmenopausal women. The Ethics Committee of Kuopio University Hospital approved the OSTPRE-FPS study in October 2001. Written informed consent was collected from the study participants at the baseline enquiry.

The targeted OSTPRE-FPS population (n = 5,407) was selected from the population-based OSTPRE cohort (n = 13,100) of women born in 1932–1941 [17]. The inclusion criteria for OSTPRE-FPS were age at a minimum of 65 years at the end of November 2002, living in the Kuopio Province at the onset of the trial, and not belonging to the former OSTPRE bone densitometry sample. The willingness to participate in a calcium and vitamin D trial was enquired via a postal enquiry between August and December 2002. The 3,432 volunteers out of 5,407 (63.5%) were randomized into two groups of equal size without stratification or random allocation sequence by an independent statistician. The subjects were assigned to their groups by postal notification according to the randomization. OSTPRE-FPS was conducted as an open-label trial. The power analysis was done based on incidence of fractures. There was no a priori power analysis to calculate the size of the subsample.

In the entire trial population, the intervention and control groups had 1,718 and 1,714 subjects, respectively. A subsample of 750 women was randomly selected from the 3,432 women at the baseline. In the subsample, the supplementation and control groups had both 375 subjects. The subsample underwent a detailed measurement program including the BMD and serum 25(OH)D measurements. The baseline measurements took place between February 2003 and May 2004 and the follow-up measurement between January 2006 and May 2007 (mean (SD) follow-up time 2.8 (0.4) years). Between the randomization and the actual start of the intervention, 237 subjects withdrew, and ten were lost during the follow-up. At the end of the trial, 306 and 287 subjects in intervention and control groups of the subsample, respectively, completed the follow-up (Fig. 1).

Fig. 1
figure 1

Flowchart of the subsample of the OSTPRE-FPS trial

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Baseline characteristics

The baseline postal questionnaire included questions about health disorders, medication, duration of hormone therapy (HT; years), gynecological history, calcium questionnaire (milligram per day, the total dietary calcium was calculated as summing up calcium derived from dairy products (cheese, milk, sour milk, yogurt, sour whole milk), juices including calcium and supplements (both prescription and natural products)), physical activity (type of activity and duration in hours per week), alcohol consumption (one alcohol unit was based on calculation that one bottle of beer has been estimated to contain 12 g and one glass of wine (12 cl) and hard liquor (4 cl) 14 g of absolute alcohol), and smoking (present status). The time since menopause (years) was calculated using the self-reported beginning of amenorrhea at baseline. In Eastern Finland, 75% of dietary calcium has been estimated to come from dairy products [18]. The self-reported calcium intake has been validated, and it showed moderate correlation against the 4-day food record [18]. The medications possibly affecting bone metabolism included corticosteroids, diuretics, cytotoxic drugs, anticonvulsive drugs, anabolic steroids, calcitonin, bisphosphonates, and vitamin D. Weight was measured with a digital calibrated scale (Phillips, type HF 351/00) and height with a calibrated wall meter.

Intervention

The intervention group obtained daily cholecalciferol 800 IU and calcium carbonate 1,000-mg supplementation (Calcichew-D3 Forte, Leiras-Nycomed Ltd.) divided into two daily doses for 3 years, and the control group received no supplementation. The vitamin D and calcium was a combined formula that was chewable, and subjects took it twice a day. Both groups were asked to continue with their previous diet. The supplements were distributed via prescriptions which were written and supervised by one of the authors (MT), and local pharmacies arranged the distribution of the supplements. The prescriptions were written for 1-year period at the beginning of the trial. The pharmacies contacted one of the authors (MT) once a year to renew the prescriptions. The pharmacies provided the supplements for 3 months at a time for the subjects of the intervention group. Compliance was calculated as the percentage of the delivered tablets of predicted consumption during the follow-up period.

Main outcome measure

BMD measurements were performed at the lumbar spine (L2–L4), left proximal femur, and total body with dual X-ray absorptiometry (DXA; Lunar Prodigy, GE, Madison, WI, USA) twice, at the baseline and at the end of the trial. The regions of interest at the proximal femur included femoral neck, trochanter, Ward’s triangle, and total proximal femur. The four vertebrae between L1 and L4 (n = 395) were used in the analyses, and whenever it was not possible (e.g., degenerative changes), we substituted it with any two or three consecutive vertebrae between L1 and L4 (n = 85). The phantom control of the DXA instruments was run daily. The in vitro long-term reproducibility (CV) for total femur region was 0.3%. The technical quality of each DXA measurement and analysis was carefully checked, and those with measurement errors were not included in the statistical analyses.

Serum 25(OH)D measurements

Blood samples (n = 593) were obtained in a nonfasting state at the baseline and the 3-year follow-up visits. Serum was frozen, and all 25(OH)D samples were analyzed promptly after the study closure. Serum 25(OH)D was determined by radioimmunoassay method using 25(OH)D RIA kit from DiaSorin (Stillwater, MN, USA). Total variation (CV%) for the Diasorin method was from 8.2% to 11.0% according to the manufacturer.

Statistical analysis

Data were analyzed on an intention-to-treat basis by retaining groups according to randomization (Fig. 1; SPSS, version 14.0.1; SPSS, Chicago, IL, USA). Differences between the intervention and control groups were analyzed with independent-samples t test for normally varied and with the Mann–Whitney test for nonnormally varied variables. Pearson chi-square test was used for the univariate analyses of category variables. The change in BMD within the group was defined with paired-samples t test. The difference of change of BMD between the groups was defined with general linear repeated-measures model. The results are presented time × treatment interaction from the repeated-measures analysis of variance. The differences of vitamin D levels and change between the groups were analyzed with independent-samples t test. All analyses were unadjusted. The secondary analysis was performed in adherent women (i.e., those who took at least 80% of their supplementation). Associations between serum 25(OH)D level, total calcium intake at the baseline, and the annual BMD change were analyzed with linear regression model.

Results

The characteristics of the study population at the baseline are shown in Table 1. All differences between the intervention and control groups were nonsignificant. There were no statistically significant differences between the entire study population and the subsample (data not shown).

Table 1 Baseline characteristics of the study population (n = 593)
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Main outcome results

Table 2 shows the change in BMD during the trial. Total body BMD increased significantly more in the intervention group than in the control group (0.84% vs. 0.19%, p = 0.011). The BMD decrease at Ward’s triangle was lower in the intervention group (−2.69% vs. −2.83%, p = 0.003). The BMD changes at the spine (p = 0.372), femoral neck (p = 0.188), trochanter (p = 0.085), and total proximal femur (p = 0.070) were statistically nonsignificant between the groups.

Table 2 Change in BMD in the intervention and control groups during the 3-year trial (general linear repeated-measures model)
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Secondary outcome results

The mean compliance was 79%. The proportions for 70%, 80%, and 90% compliance were 71.8%, 63.8%, and 47.4% of the intervention group, respectively.

The women who were adherent (i.e., those who took at least 80% of their supplementation) showed significantly lower bone loss in femoral neck (−1.26% vs. −1.73%, p = 0.002), Ward’s triangle (−1.63% vs. −2.83%, p < 0.0001), trochanter (0.25% vs. −0.88%, p = 0.001), and total proximal femur (−0.84% vs. −1.47%, p < 0.0001) than in the control group. Further, total body BMD increased more in the intervention group (+1.31% vs. +0.19%, p = 0.002). In contrast, lumbar spine bone loss was greater in the intervention group than in the control group (+0.67% vs. +0.76%, p = 0.033; Table 3).

Table 3 Change in BMD in the intervention with the adherent subjects (those who took at least 80% of their supplementation) and in the control group during the 3-year trial (general linear repeated-measures model)
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Baseline serum 25(OH)D associated with Ward’s triangle annual BMD change (p = 0.033), but it did not associate with trochanter (p = 0.726), total hip (p = 0.845), proximal femur (p = 0.099), lumbar spine (p = 0.060), or total body (p = 0.343) annual BMD change.

The total intake of calcium at the baseline did not predict annual BMD change at Ward’s triangle (p = 0.497), trochanter (p = 0.665), total hip (p = 0.720), proximal femur (p = 0.339), lumbar spine (p = 0.729), or total body (p = 0.668).

Serum 25(OH)D level

The mean (SD) baseline serum 25(OH)D levels were 50.1 (18.8) nmol/l and 49.2 (17.7) nmol/l in the intervention and control groups (p = 0.544), respectively. The absolute changes in 25(OH)D levels were 24.7 (24.1) nmol/l and 6.8 (19.3) nmol/l in the intervention and control groups (p < 0.001), respectively. At the end of the trial, the mean levels were 74.6 (21.9) nmol/l and 55.9 (21.8) nmol/l (p < 0.001), respectively.

Adverse effects of the supplementation

In total, 40 out of 290 women discontinued the intervention: 17 due to adverse effects, 22 due to other reasons, and one due to no specific reason. The adverse effects responsible for discontinuation were gastrointestinal symptoms (nine), exacerbation of diseases (two), and mouth irritation (one), skin symptoms (one), nausea (one), cough (one), backache (one), and weight increase (one).

Discussion

The daily cholecalciferol 800 IU and calcium carbonate 1,000-mg supplementation increased the total body BMD in women aged 66 to 71 years in this randomized 3-year population-based trial. However, no site-specific changes at the femoral region or at the lumbar spine were detected in the main analyses. In further analyses, the intervention effect appeared to be greater in total body and femoral regions among women who adhered to the supplementation.

It has been reported earlier that there is a progress of about 1% annual bone loss in the femoral neck in the elderly [19]. Furthermore, inadequate intakes of vitamin D and calcium lead to reduced calcium absorption, higher bone remodeling rates, and increased bone loss [3, 14]. The combination of these factors increases the risk of osteoporosis and consequently the risk for osteoporotic fractures. There is evidence to suggest that adequate sunlight exposure and the use of supplements are needed to meet human vitamin D needs [20]. Vitamin D supplementation has been shown to have long-term effects on the skeleton as a whole [13]. Similarly, the positive effect on total body BMD that was seen in this study may reflect the general skeletal effect. However, we cannot exclude the possibility that total body and spinal BMD increase could be confounded because of age-related degenerative changes, such as osteoarthritis and aortic calcification [21, 22]. On the other hand, vitamin D and calcium supplementation may have stronger effect on cortical bone [23]. Consequently, the positive effect seen in the total body BMD might be explained by higher proportion of cortical bone in the skeleton.

In a large RCT trial, 1,000 mg daily calcium alone showed 1.6% smaller bone loss in hip over 5-year follow-up in elderly women [24]. The relatively small changes detected in BMD between the supplemented and control groups in the present and earlier studies raise the question of the clinical relevance of the positive results. At the population level, the supplementation would not be sufficient to prevent osteoporosis, but it could at least decelerate the age-related bone loss to some extent. Additional nonpharmacological and pharmacological interventions should be considered, especially in subjects at a high risk of suffering osteoporotic fractures [25]. Dawson-Hughes et al. have reported that 1.1% net increase in total body BMD during the 3-year vitamin D (700 IU) and calcium (500 mg) trial was associated with relative risk of 0.4 of first osteoporotic fracture [13]. Accordingly, in our study, BMD increase of 0.9% but with slightly greater dose of vitamin D (800 IU) and calcium (1,000 mg) might point out for similar relative risk reduction.

Daily oral dosing with vitamin D seems to be a more effective way of administration than weekly or monthly ones [16]. However, recent study demonstrated the effect of large vitamin D loading and intermittent dosages to normalize serum levels [26]. Furthermore, vitamin D supplementation is effective in preventing the seasonal-related vitamin D level decrease and subsequent bone loss [27, 28]. In fact, vitamin D sufficiency might be more important than high calcium intake to maintain desirable serum parathyroid hormone levels [29]. BMD improvements in the vitamin D and calcium-supplemented subjects are largely lost if the supplementation is discontinued [30]. This is in agreement with the present study where women who were adherent had the most benefit from the supplementation. These findings argue in favor of the importance of regularity in the supplementation. Further, vitamin D supplementation supports other therapies, since optimal vitamin D repletion is necessary for achieving the maximal benefits from antiresorptive pharmacological therapies [31].

There is evidence of an age-related intestinal resistance to the actions of 1,25-dihydroxyvitamin D [32]. Accordingly, this has led to the question about the sufficiency of daily 1,000-mg calcium intake even in the old age. It is known that calcium absorption increases with higher vitamin D levels [2], and accordingly optimal calcium intake in people with adequate vitamin D status might be lower [33]. Nonetheless, it has been suggested that the vitamin D requirement is not greatly dependent on calcium intake [34], and a daily intake of 1,200 mg calcium in the general population should cover calcium needs [11, 34]. In the present study, the mean daily intake of calcium was 980 mg at baseline, which is nearly at the current Finnish recommendation (1,000 mg/day). Thus, the high nutritional calcium intake might have suppressed the positive effect of the supplementation. Further, recent findings suggest that calcium supplementation is associated with adverse cardiovascular events [35, 36]. Thus, the recommendation for supplying additional calcium for those subjects with a nutritional supply of over 1,000 mg needs to be evaluated in further studies. More studies are needed to study whether daily vitamin D confer additional health benefits.

Mean serum 25(OH)D levels were 50 nmol/l, which suggests that subjects were slightly deficient in terms of vitamin D [20]. In fact, it has recently been reported that only women with 25(OH)D levels below 50 nmol/l benefit from a higher calcium intake [37]. Therefore, we suggest that the positive effect of the supplementation might be attributable to greater extent to the adequate vitamin D intake than calcium intake. This is further supported by the earlier finding that BMD increases with higher 25(OH)D concentrations [37]. Since February 2003, following the recommendation by the Finnish Ministry of Social Affairs and Health, fluid milks (0.5 mg/100 ml), margarines, and spreads (10 mg/100 g) have been fortified by vitamin D in Finland. Indeed, this action in general has improved the mean vitamin D intake and status in Finland, but low 25(OH)D levels still occur [38].

The strengths of this study were its population-based design and the long follow-up. However, as only two thirds of individuals volunteered to participate (3,432 out of 5,407), the population-based benefit was diminished. The subsample of 593 women was randomly selected from those 3,432 subjects, and therefore it is a representative cohort. One limitation was that the compliance with the intended intervention was not complete (79% of the prescribed calcium–vitamin D tablets that were dispensed from the pharmacies). However, the present compliance was quite similar as in the previous study [12]. Further, the compliance was estimated based on prescriptions and not on exact count of tablets consumed. Since vitamin D and calcium supplementations are available over the counter and are routinely included in standard multivitamins, the subjects in the control group might well have increased their use of calcium and vitamin D supplementation. In fact, 3.8% of control subjects reported using prescribed vitamin D with or without calcium supplements at baseline, but this has risen to 16.1% by the end of the trial (data not shown). However, serum 25(OH)D levels increased significantly more in the intervention group (24.7 nmol/l) than in the control group (6.8 nmol/l). This confirms that women in the intervention group had used the study supplementation. The open population-based approach demonstrates the effect of the intervention in the general population, in comparison with the situation in women who simply continue their normal diet perhaps using nonprescription sources of vitamin D and calcium supplementation.

In conclusion, combined daily cholecalciferol 800 IU and calcium carbonate 1,000 mg has a positive effect on the skeleton in ambulatory postmenopausal women with adequate mean nutritional calcium intake. Whether there is effect without calcium supplementation needs to be confirmed in future studies.