Meta-Analysis of Long-Term Vitamin D Supplementation on Overall Mortality

Introduction It has been suggested that vitamin D is effective to prevent mortality. However, there is no consistent conclusion that the effects of vitamin D supplementation on all-cause mortality are associated with duration of treatment. We conducted a meta-analysis regarding this issue in an effort to provide a more robust answer. Methods A comprehensive search in a number of databases, including MEDLINE, Embase and The Cochrane Central Register of Controlled Trials, was conducted for collecting randomized controlled trials (RCTs) on vitamin D supplementation preventing mortality. Two investigators independently screened the literature according to the inclusive and exclusive criteria and the relative data were extracted. Data analysis was performed by using Review Manager 5.0 software. Results Data from forty-two RCT s were included. Vitamin D therapy significantly decreased all-cause mortality with a duration of follow-up longer than 3 years with a RR (95% CI) of 0.94 (0.90–0.98). No benefit was seen in a shorter follow-up periods with a RR (95% CI) of 1.04 (0.97–1.12). Results remain robust after sensitivity analysis. The following subgroups of long-term follow-up had significantly fewer deaths: female only, participants with a mean age younger than 80, daily dose of 800 IU or less, participants with vitamin D insufficiency (baseline 25-hydroxyvitamin D level less than 50 nmol/L) and cholecalciferol therapy. In addition, the combination of vitamin D and calcium significantly reduced mortality and vitamin D alone also had a trend to decrease mortality in a longer time follow up. Conclusions The data suggest that supplementation of vitamin D is effective in preventing overall mortality in a long-term treatment, whereas it is not significantly effective in a treatment duration shorter than 3 years. Future studies are needed to identify the efficacy of vitamin D on specific mortality, such as cancer and cardiovascular disease mortality in a long-term treatment duration.


Introduction
Vitamin D plays a key role in human health, while the prevalence of vitamin D insufficiency is high, especially among the elderly [1]. It has recently been identified to be associated with skeletal diseases such as osteoporosis [2] and nonskeletal diseases including cancer [3][4][5], cardiovascular disease [6], and kidney disease [7,8]. Meta-analysis has suggested that low vitamin D baseline levels are associated with increased risks of mortality [9]. This issue is becoming to be paramount importance given the high prevalence of vitamin D deficiency worldwide [10].
It has been documented that vitamin D supplementation prevents fractures and falls [11,12]. In recent years, several studies on meta-analysis of randomized controlled trials with regard to supplementation of vitamin D on total mortality have been published, which found that vitamin D supplementation reduced total mortality when given together with calcium, but not with vitamin D alone [13][14][15][16], and. A Cochrane systematic review found that vitamin D significantly decreased mortality in those with vitamin D insufficiency [15]. However, long-term health effects of vitamin D supplementation still remains unclear.
To investigate whether the effects of vitamin D supplementation on all-cause mortality are associated with duration of treatment, we undertook a comprehensive systematic database search and meta-analysis to access the effects of vitamin D supplementation on all-cause mortality.

Search strategy
A literature search was conducted on a number of databases, including Medline, Embase and The Cochrane Central Register of Controlled Trials for the period January 1960 to January 2013, to identify RCTs. Our core search terms were "randomized controlled trial", "vitamin D", "vitamin D 2 ", "vitamin D 3 ", "ergocalciferol", "cholecalciferol", "mortality", "death". We also searched for any additional studies in the reference lists of recent meta-analysis of vitamin D treatment for mortality. Our searches were limited to human trials, and no language or time restriction was applied.

Eligibility criteria
The preliminary search results were then examed on the basis of the following criteria.
Types of studies. Randomized controlled trials evaluating an intervention with vitamin D were identified as part of the review, while review articles, commentaries, letters, observational studies were excluded.
Interventions. The intervention group was restricted to vitamin D alone or in combination with calcium treatment; the control group was placebo, no treatment or calcium only therapy. Studies of patients receiving active vitamin D and intramuscular injection of vitamin D were excluded from the review.
Outcome. The number of deaths was reported separately for the vitamin D treatment group and the control group. For articles with a large sample size, if the number of deaths was not reported by treatment, we tried to contact the authors to obtain the missing data.

Data Extraction and Quality Assessment
Two statisticians independently extracted information from included trials using a standardized form., and then another statisticians verified them. The following information was subtracted from the study: first author, publishing year, sample size, duration, dwelling, intervention, serum 25 (OH) D levels at baseline, and main results (the number of participants who died). Quality assessment of included trials was conducted using the Cochrane Collaboration's tool [17]. Methodological features most relevant to the control of bias were examined, including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias [17]. Quality assessment was performed by two independent researchers.

Data Synthesis and Analysis
Meta-analysis were undertaken using Review Manager (Version 5.0). The primary outcome was the number of participants who died during follow-up. The pre-planned analysis was vitamin D arm (with or without calcium) versus control arm (placebo, calcium, or no treatment) according to duration of treatment. Mantel-Haenszel method was used to calculate risk ratios (RRs) and their 95% confidence intervals (CI). The I 2 statistic was used to assess the presence of heterogeneity, which ranges from 0% to 100% [18]. In case of lack of heterogeneity (I 2 < 50%), fixed-effects model was used to assess the overall estimate, or else random-effects model was chosen. The Begg test [19] and Egger test [20] were used to evaluate the presence of publication bias regarding our primary end points (RR of mortality). A 2-tailed P value of less than .05 was considered as statistically significant.

Sensitivity analysis and subgroup analysis
A sensitivity analysis was conducted by excluding studies with high risk of bias. Subgroup analysis was conducted only on trials with the duration of treatment at least 3 years or longer. The effect of vitamin D was assessed according to gender (male or female), age group (< 80 years or ≥ 80 years), dose of oral vitamin D daily (≤800 IU or >800 IU), baseline level of 25-hydroxyvitamin D (< 50 nmol per liter or ≥ 50 nmol per liter), type of vitamin D (ergocalciferol or cholecalciferol) and calcium co-administration status (Vitamin D + calcium vs. Calcium, Vitamin D + calcium vs. Placebo, or Vitamin D vs. Placebo), and specific mortality (cancer mortality, cardiovascular mortality)

Search Results
A total of 4,024 unique titles and abstracts were found from initial searches of the electronic database. With the inclusion/ exclusion criteria, 3,861 of which were excluded by scrutinizing the titles and abstracts, and 121 articles were further excluded after full text review. A total of 42 RCTs that met inclusion criteria were included in the final analysis . The details of study selection flow were explicitly described in Figure 1.

Study Characteristics
The main characteristics of the included studies are shown in Table 1 (1). Publishing year: The RCTs were published from 1992 to 2012 (2). Treatment duration: 29 RCTs have treatment durations less than 3 years, and the remaining 13 RCTs have treatment durations of 3 years or longer (3). Number of patients: A total of 85,466 patients (42,561 in the vitamin D group and 42,905 in the control group) were included in these 42 RCTs (4). Age of patients: The number of participants in each trial ranged from 46 to 36,282 and mean age of participants ranged from 37 to 89 years, with most participants older than 60 years (5). Vitamin D type and dose: Vitamin D 2 was used in 10 studies and vitamin D 3 was used in the remaining 32 studies. Vitamin D 2 or D 3 was given as daily doses ranging from 300 to 3,333 IU. Calcium supplementation was used in 26 trials (6). Baseline vitamin D status: 37 trials (80%) reported the baseline vitamin D status of participants based on serum 25-hydroxyvitamin D levels. Participants in 15 trials had baseline 25-hydroxyvitamin D levels at or above cutoff for vitamin D adequacy (50 nmol/l or 20 ng/ml). Participants in the remaining 22 trials had baseline 25hydroxyvitamin D levels in a range of vitamin D insufficiency (< 50 nmol/l or 20 ng/ml). The other 5 trials did not report the baseline vitamin D status of participants (7). Bias risk: 26 studies had a low risk of bias, and 16 had a high risk of bias ( Table 2).

Primary Analysis
Analysis was performed independently for two categories with follow-up duration either less than 3 years, or 3 years or more. In the category of 29 trials with follow-up less than 3 years, a total of 1,175 (13.3%) participants randomized to the vitamin D group and 1,118 (12.2%) participants randomized to the placebo or no intervention group died. Analysis showed that vitamin D did not significantly decrease all-cause mortality. The risk ratio of mortality for patients treated with vitamin D compared with that of control was 1.04 (95% CI: 0.97-1.12), which was statistically insignificant (P = 0.28), with insignificant heterogeneity (I 2 = 12%) ( Figure 2A).
In the second category of 13 trials with follow-up of 3 years or longer, a total of 3,693 (10.9%) participants randomized to the vitamin D group and 3,880 (11.5%) participants randomized to the placebo or no intervention group died. Data analysis showed that vitamin D significantly decreased all-cause mortality with a risk ratio of mortality 0.94 (95% CI: 0.90-0.98), which was statistically significant (P = 0.001), with insignificant heterogeneity (I 2 = 0%) ( Figure 2B).

Discussion
We conducted a systematic review and meta-analysis to evaluate the best available research evidence regarding vitamin D supplementation on overall mortality. A total of 42 RCTs were included in the present study, quality assessment suggested that the overall study quality was fair and no significant publication bias was detected. Our results demonstrates that vitamin D supplementation longer than 3 years leads to a significant reduction on overall mortality. When trials with a high risk of bias excluded in the sensitive analysis, the results remain robust. The effect of vitamin D on mortality reduction was significant in several subgroups of individuals: female patients, participants with a mean age younger than 80, dose of 800 IU or less, participants with vitamin D insufficiency (baseline 25-hydroxyvitamin D level less than 50 nmol/L) and cholecalciferol therapy. In addition, compared with placebo, vitamin D in combination with calcium significantly reduced mortality. Our findings confirmed those in an earlier Cochrane systematic review [15] on the effect of vitamin D treatment on overall mortality, which showed that participants with vitamin D insufficiency (25-hydroxyvitamin D level less than 20 ng/ml) decreased the overall mortality significantly, and indicated that cholecalciferol therapy was more favorable than ergocalciferol and that vitamin D as daily doses of 800 IU or less was more favorable than daily doses more than 800 IU. In contrast with two meta-analysis [13,16], which compared daily dose of 800 IU or greater with that less than 800 IU and suggested that daily dose of vitamin D did not differ in the effect on the outcome, our analysis indicated that the beneficial effect of vitamin D is clearly observed in the low daily dose. One explanation may be that several included trials [23,27,30] used intermittent and high dose of vitamin D, which has been suggested less likely to have a benefit, or to even have a negative effect among the elderly [23]. Consumption of intermittent and high dose of vitamin D leads to high concentrations of plasma 25-hydroxyvitamin D. Michaëlsson et al [63] concluded that both high and low concentrations of plasma 25-hydroxyvitamin D were associated with elevated risks of overall and cancer mortality.
A previous meta-analysis conducted by Autier et al [13] suggested that no relationship was found with duration of vitamin D supplement. In contrast, our results indicated that vitamin D supplementation significantly reduced the overall mortality when duration was longer than 3 years compared with that of control. However, no benefit was seen in those with durations less than 3 years. Additionally, two meta-analyses of RCTs with vitamin D treatment on falls also reported that patients benefit from vitamin D supplementation in a longer time duration [64,65].
Our results indicated that vitamin D was effective in reducing mortality among female patients. There was a lack of evidence to draw a conclusion of vitamin D's influence on male patients with only one identified trial collected death data by subgroup of gender. We concluded that vitamin D may decrease mortality in patients younger than 80 years old, but not in patients aged 80 years or older. However, no statistically significant difference was found for risk ratio of overall mortality between the two age groups (P= 0.59). This results support an early meta-analysis of vitamin D treatment on falls, which indicated that participants with a mean age younger than 80 benefited from vitamin D supplementation [64].
Several previous meta-analyses suggested that vitamin D supplementation reduced all-cause mortality when given together with calcium, but did not support an effect of vitamin D alone treatment [14][15][16]. In contrast, our results suggested that vitamin D combined with calcium reduced all-cause mortality significantly when compared with placebo (RR= 0.94; 95% CI: 0.88-0.99), but the effect was insignificantly when compared with calcium therapy (RR= 0.97; 95% CI: 0.91-1.03). Vitamin D alone had a trend to decrease mortality (RR= 0.93; 95% CI: 0.86-1.00) when administrated in a long time. It may indicate that calcium therapy does not increase risk of death [66]. Whether vitamin D given together with calcium is more There is not sufficient evidence to draw conclusions of the effect of vitamin D on specific mortality with only 3 trials collected mortality data in a rigorous fashion. Vitamin D may have a beneficial effect on cancer related mortality. But it needs more RCTs to better understand the effect of vitamin D on cancer. Meta-analyses of cohort studies have suggested that vitamin D intake was associated with reduced risk of colorectal cancer [67], breast cancer [68] but not prostate cancer [69]. Vitamin D had no significant effect on cardiovascular disease mortality. A growing number of literature suggest that low levels of vitamin D are associated with cardiovascular disease risk [70][71][72][73][74]. A limited number of interventional studies that investigated the effects of vitamin D supplementation on cardiovascular disease risk showed mixed results [75][76][77][78]. The effect of vitamin D on cardiovascular diseases remains to be identified.
The mechanism of vitamin D benefit on overall mortality is not clear. Both forms of vitamin D (D 2 and D 3 ) are converted to 25-hydroxyvitamin [25(OH)D] in the liver, and then hydroxylated to 1,25-dihydroxyvitamin D in the kidney [79]. 1,25(OH) 2 D is the only biologically active form of vitamin D, which increases calcium absorption and bone formation to maintain bone health, regulate blood pressure and insulin production, prevent heart disease, regulate immune function to prevent diabetes and autoimmune disease, regulate cell growth to prevent cancer [80,81].
Vitamin D insufficiency (< 50 nmol/L) has now been linked to a broad spectrum of human diseases from cancer to cardiovascular to autoimmune conditions [82]. Though vitamin D can acquire through cutaneous synthesis after sunlight exposure and nutrition [83], it is often not sufficient to reach the required levels of vitamin D, especially in patients with osteoporosis and fracture risk [84]. In that case, supplementation of vitamin D is required, in order to prevent vitamin D insufficiency and associated adverse outcomes.
Similar to other meta-analyses, our review has several limitations. First, though extensive searches were made, there were no data of Hispanic or Orientals. Second, most of the participants in the present study were older women, the effects of vitamin D on mortality in younger, healthy persons and in males are still inconclusive. Third, the overall RR (95% CI) effect was modest and could be the result of chance alone. Fourth, there were only 13 trials that have durations of follow up longer than 3 years.
In conclusion, our results implicated that long-term supplementation of vitamin D may have a beneficial effect on overall mortality, especially in patients with vitamin D insufficiency and younger than 80 years. Vitamin D in a dose of 800 IU daily or less was found to be more favorable than a dose greater than 800 IU and treatment with cholecalciferol was more favorable than ergocalciferol. Future studies are needed to test the efficacy of vitamin D on specific mortality, such as cancer and cardiovascular disease mortality in a longterm treatment duration.