Effects of vitamin D supplementation on 25(OH)D concentrations and blood pressure in the elderly: a systematic review and meta-analysis

Background: Hypertension and vitamin D deficiency are prevalent among the elderly. This study evaluated the effects of vitamin D supplementation on changes in serum 25-hydroxyvitamin D (25(OH)D) concentrations and blood pressure (BP) in the elderly (age > 60 years). Methods: Randomized controlled trials from electronic databases on the elderly taking oral vitamin D, until the end of March 2019, were selected. Two reviewers independently screened the literature on the basis of specific inclusion criteria. The primary outcomes were serum 25(OH)D level, systolic BP (SBP), and diastolic BP (DBP) changes. Results: Our analysis revealed significant differences in serum 25(OH)D concentrations changes between the vitamin D and control groups (mean difference [MD] = 13.84; 95% confidence interval [CI] = 10.21–17.47; P < 0.000). There were no significant differences in SBP and DBP changes between the vitamin D and control groups. Subgroup analysis revealed significant differences in SBP changes between the hypertensive and vitamin D-deficient subgroups (MD = –4.01; 95% CI = –7.45 to –0.57; P = 0.02 and MD = –1.91; 95% CI = –3.48 to –0.34; P = 0.02, respectively), and DBP changes only in the hypertensive subgroup (MD = –2.22; 95% CI = –4.1 to –0.34; P = 0.02). Conclusions: Vitamin D supplementation significantly increases 25(OH)D concentrations and seems beneficial in lowering BP, specifically in the elderly with elevated BP and vitamin D deficiency.


Introduction
High blood pressure (BP), or hypertension, is still regarded as one of the most influential factors for cardiovascular diseases, especially in the elderly. An increasingly aging population and the increasing prevalence of hypertension emphasize the importance of proper treatment of hypertension. Nutrient supplementation is an alternative treatment since the elderly have multiple chronic diseases and take multiple drugs 1,2 . Vitamin D is one kind of steroid hormone and micronutrient synthesized in the skin by exposure to ultraviolet B rays and also obtained through dietary intake or supplementation 3 . Most vitamin D is distributed in the human body in the form of serum 25(OH)D 4 .
Vitamin D deficiency has become an important public health concern because it could take place at any age, and most countries report deficiency as high in the elderly 3,5 . Vitamin D has an essential part in metabolism regulation and has a significant role in the pathogenesis of hypertension 4 . Since Vitamin D can inhibit renin transcription, maintain parathyroid hormone balance, vasodilation blood vessels, and reduce sympathetic nerve activity 3 , it is reasonable that hypovitaminosis D is strongly associated with arterial hypertension. However, the result of meta-analyses has revealed that the relationship between serum 25(OH)D concentrations and a decrease in BP is inconsistent. Qi et al. (2017) reported that low serum 25(OH)D concentrations are not significantly associated with a risk of hypertension 6 . In contrast, other studies demonstrated a significant relationship between low serum 25(OH)D concentrations and hypertension 3,7 . Another meta-analysis also proved that the serum level of 25(OH)D was significantly associated with the risk of incident hypertension on the general population 3 .
The elderly is an age group susceptible to deficiency of this fat-soluble vitamin. Skin aging reduces 7-dehydrocholesterol production to 75%, which is known to play a key role as the main source of vitamin D in the human body 8 . The impaired eating ability in the elderly may also contribute to low concentrations of vitamin D. Therefore, vitamin D deficiency is often associated with various geriatric syndromes. Low concentrations of vitamin D affects the activity of endocrine hormones as in sufferers of diabetes mellitus type 2 (T2DM) and cardiovascular functions, such as coronary artery disease, heart failure, stroke, and hypertension 9 . A recent meta-analysis in individuals with vitamin D deficiency showed oral vitamin D3 reduces both systolic BP (SBP) and diastolic BP (DBP) in individuals with hypertension and decreases SBP in individuals above 50 years. In contrast, another study has revealed that in younger women, there is a strong association between high serum 25(OH)D concentrations and the risk of hypertension 3 . Since the elderly, defined as individuals of more than 60 years of age, have a high risk of vitamin D deficiency and suffer hypertension 1,2,10 , it is important to provide a meta-analysis of randomized controlled trials gathering the evidence of the effects of vitamin D supplementation compared to placebo on serum 25(OH)D concentrations and BP, specifically in the elderly population.

Methods
Data source and study selection A comprehensive search was performed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement 11 . All authors searched independently correlated studies in multiple electronic database including, PubMed, ClinicalTrials.gov, and the Cochrane Library from inception until 29 th March 2019 using a combination of keywords and subject headings. The search strategies used on PubMed and Cochrane Library using the following keywords: (vitamin D) AND ((blood pressure) OR (hypertension)) AND (elderly). While in clinicaltrials.gov, the terms used were "hypertension", "blood pressure", and "vitamin D". Further relevant articles were then obtained using manually searching the references of retrieved articles.

Eligibility criteria
All titles and abstracts were screened using Mendeley reference software, and duplications were removed manually. Full text of relevant articles were examined for eligibility criteria. The inclusion criteria were as follows: randomized controlled trial (RCT) design; participants with average age > 60 years; primary outcomes SBP change, DBP change, and serum 25(OH)D level change; and only vitamin D (cholecalciferol) intervention. The exclusion criteria were as follows: nonrandomized study; no full text available; the study control group was not placebo; outcomes relevant to our interest not reported; intervention with combined vitamin D and other nutrients supplementation; and non-English full text.

Data synthesis and analysis
Data from each included article were extracted by three investigators (F.F., C.F., N.Y.) by utilizing a piloted form. If there is any disagreement between the authors, the final decision was made by discussion and majority vote. The following data were extracted: year of publication, year of study, geographic location, sample size, health status of participants, mean age, intervention dose, duration of the study, mean and standard deviation (SD) of serum 25(OH)D concentrations, SBP, and DBP in both intervention and placebo groups at the baseline and at the end of study, and changes from the baseline.

Amendments from Version 2
These are the major differences between this version of our article and the previously published version 1. Since lab data reports ' concentrations' not 'levels', we have replaced term 'levels' with ' concentrations', except for cut-off and definitions 2. We have added information and citations about clinical implications of this study, provided evidence for the use of vitamin D for hypertensive elderly people, and completed the analysis of blood pressure reduction which has significant clinical relevance compared to other interventions. This is discussed in at the third and second to last paragraph of discussion section.
3. For better readability, we have reorganized the final paragraph of discussion focusing on the strengths and limitations of study Any further responses from the reviewers can be found at the end of the article REVISED Each RCT's quality was evaluated using the risk of bias tools developed by Cochrane collaboration evaluating six domains. We assessed the selection bias by evaluating the study description on the method of the randomization, the method of allocation concealment, and evaluated if there is difference in baseline between the two groups. Performance and detection bias were evaluated by finding a description about the blinding method. Attrition bias was assessed by calculating the number of participants that withdrew from the study. Reporting bias and other bias were then evaluated if there found any concern not addressed in the other domain 12 .
The continuous data were presented as mean difference (MD) and SD. Where the change in mean (Δ Mean) was not available, we calculate the change by subtracting post intervention outcome with the baseline data. When a study did not report enough information of the change on SD (ΔSD), we calculated the data imputation applying the formula for imputing SD from baseline 13 : ΔSD was then calculated as: To calculate the estimated effect size on MD, we used randomeffect model if there was heterogeneity found using X 2 test and I 2 test 14 . p value of <0.10 dan I 2 > 50% were considered high. Otherwise, the fixed-effects Mantel-Haenszel model was used. We performed univariate meta-regression analyses to evaluate differences in the continuous outcome variable. Analyses of subgroups were conducted to assess predefined sources of heterogeneity. Dose of supplementation, duration of the study, treatment regimen, hypertension, and vitamin D status were considered as sources of heterogeneity. We assessed publication bias by visual assessment on graphical funnel plots with Egger's regression test of asymmetry 15 .
All statistical analyses were performed using STATA 16.0 (STATA Corporation). P value < 0.05 was considered statistically significant. Figure 1 presents the flowchart of this study. We screened 980 articles. Of those, 28 were excluded because of duplicate publication, and 42 articles were assessed for eligibility criteria. Of those, 30 were not eligible to be included. Finally, 12 RCTs 16-27 were included in the quantitative synthesis. The quality assessment demonstrated that almost all of the included studies has a low risk of bias. The results for quality assessment was summarized in Extended data: Figure S1 28 .  Table 1 shows that most participants revealed baseline data of mean 25(OH)D concentrations <20 ng/mL, and the maximum was 30 ng/mL 17 . Eight RCTs evaluated changes of 25(OH)D concentrations after giving vitamin D intervention. The 1293 participants were divided into treatment (n = 641) and control groups (n = 652). Pooling data revealed that the vitamin D group had a significant higher serum 25(OH)D concentrations compared to the control group (MD = 13.84; 95% CI = 10.21-17.47; P < 0.0001). We observed heterogeneity among the RCTs (I 2 = 93%), so we selected a random-effects model ( Figure 2).

BP change
The change of SBP and DBP was synthetized from 12 RCTS. We did not observe heterogeneity among the RCTs (I 2 < 50%), so we selected a fixed-effects Mantel-Haenszel model. Pooled analysis revealed that overall, the SBP change (MD = -0.83; 95% CI = -1.88 to 0.23; P = 0.12) and DBP changes (MD = 0.40; 95% CI = -1.00-0.19; P = 0.18) in vitamin D group were not significant compared with the control group. The effects of vitamin D on SBP and DBP were summarized as forest plots presented in Figure 3 and Figure 4. The funnel plots of SBP change and DBP change are summarized in Figure 5. Our analysis showed that there was no publication bias when examining funnel plots and the result of Egger's test of asymmetry for SBP change and DBP change (p = 0.158; p= 0.069; respectively). Table 2 and Table 3 present the pooled estimated effect size of vitamin D on the change of SBP and DBP, on the basis of BP baseline, vitamin D status baseline, intervention dose, treatment duration, and treatment regimen. Our analysis indicate that vitamin D supplementation had no significant influence on SBP and DBP changes on the basis of dose, duration, and treatment regimen. However, subgroup analysis revealed a marginal trend toward significance in terms of DBP changes with treatment duration ≤ 6 months (MD = -0.82; 95% CI = -1.66 to -0.02; P = 0.05). Subgroup analysis by hypertensive and deficiency of vitamin D status indicated that vitamin D supplementation could significantly reduce SBP (MD = -4.01; 95% CI = -7.45 to -0.57; P = 0.02 and MD = -1.91; 95% CI = -3.48 to -0.34; P = 0.02, respectively). However, we found a significant difference in DBP changes only in the hypertensive subgroup (MD = -2.22; 95% CI = -4.1 to -0.34; P = 0.02) ( Table 2 and  Table 3). The forest plot of each subgroup analysis is available as Extended data: Figure S2 28 . Our study provided enough observations to conduct univariate meta-regression, summarized in Table 2 and Table 3. The result for SBP change was presented as bubble plots in Figure 6.

Effects of vitamin D on serum 25(OH)D concentrations
Serum 25(OH)D concentrations has a major role as a marker for determining vitamin D status in humans. As mentioned before,   In addition, the average normal value for serum 25(OH)D concentrations for all ages is 30 ng/mL, whereas in the elderly it is >20 ng/mL or 50 nmol/L 30 .
Deficiency in vitamin D could be caused by physiological and pathological factors in the elderly. One of the most common  physiological factors is decreasing pre-vitamin D production in the skin. The reasons are that compared with young adults, the skin's capacity to produce vitamin D decreases by 75% at 70 years 29,31 . In addition, the elderly have a tendency to wear closed clothing for fear of flu, thus causing minimal exposure to ultraviolet B rays 9 . Their food intake decreases because of a decrease in chewing ability and financial conditions 30 . Also, decreased calcium absorption results in impaired vitamin D metabolism and decreased kidney function [29][30][31][32][33][34] . Pathological factors are related to organs that play a role in the digestion and metabolism of vitamin D. Decreased bioavailability in the digestive tract (malabsorption due to disease) inhibits  (2008), which has revealed a significant difference between the treatment and control groups with same doses and duration 24 . Another study has reported increasing serum 25(OH)D concentrations at follow-up in the vitamin D group, with no change in the placebo group 18 . The relationship between serum 25(OH)D concentrations and a decrease in BP is still debatable. A meta-analysis of observational cross-sectional and prospective studies on general populations has proven the relationship between serum 25(OH)D concentrations and the risk of incident hypertension 3 . However, a newer meta-analysis showed oral vitamin D3 has no significant effect on blood pressure in individuals with vitamin D deficiency 5 .

Effects of vitamin D on BP
This study included research from four different continents. However, characteristically there are no specific differences for each continent. The results were random and more relevant to the baseline data and effect of the RCT itself. The present study provides evidence that although the supplementation could increase serum 25(OH)D concentrations, there was no significant difference in SBP and DBP changes compared with the control group. It means that the increasing serum 25(OH)D concentrations were not followed by decreasing BP among elderly. Several studies have revealed not only a relationship between an increase in serum 25(OH)D concentrations and a decrease in BP after vitamin D supplementation but also a significant change in other conditions, such as parathyroid hormone, serum calcium, renin, and angiotensin II concentrations 16,20,23    30 mg/dL nifedipine in patients with grade I or II essential hypertension can reduce SBP or DBP 23 . They were the first to look at the effectiveness of the interaction between vitamin D and specific drugs in contrast to other studies that only provide inclusion criteria in the form of no change in medication consumption.
Several meta-analyses have been conducted to evaluate the association between vitamin D and BP. The findings of the meta-analysis of observational studies on general populations have confirmed the association between vitamin D status and the risk of incident hypertension 3,7,8 . However, the associations are not proof of causality, so placebo-controlled RCTs are required in order to prove the effects of vitamin D on BP. Previous RCTs and meta-analyses have revealed that vitamin D might be beneficial in lowering BP, especially in vitamin D-deficient patients with hypertension, which is similar to our results 4,18 . In contrast to our findings, Ke et al. (2015) reported no increased risk of hypertension in the elderly or in vitamin D-deficient participants; however, their research involved a prospective study design 3 . The other important finding in our study was significant differences in SBP and DBP changes among the hypertensive subgroup. Previous meta-analyses have also revealed a significant effect of vitamin D on BP in patients with hypertension at the baseline and no significant decrease in normotensive patients at the baseline 5,36 . In contrast to our findings, a meta-analysis by Golzarand et al. (2016) showed that vitamin D showed hypotensive effects in both healthy and hypertensive subjects 35 . To the best of our knowledge, there has been no research that suggests that there are differences in the metabolism of vitamin D in the elderly with hypertension and norm tension, except secondary hypertension associated with kidney organs 37 . Increased BP also occurs in arterial hypertension patients who have vitamin D deficiency. Patients with deficiency in vitamin D may acquire the effects of vitamin D supplementation 20 . However, administering vitamin D to participants who meet the same criteria can give zero results with regard to a decrease in BP because of the shorter time of administration (only 8 weeks with almost the same dose) 25 . In addition, an updated meta-analysis's results were similar to our study in that subgroup analysis showed vitamin D supplementation may reduce SBP and DBP in patients with low vitamin D status and hypertension 5 .
In addition to hypertension patients, T2DM patients also exhibit a decrease in BP, although only SBP, after a high dose of vitamin D supplementation 21,26 . Again, serum 25(OH)D concentrations also contributed to the effects of vitamin D supplementation on lowering BP. Pre diabetic patients reveal absolutely no effect of the same dose of vitamin D on BP because they are not vitamin D deficient 24 . Most of the RCTs included in this study reveal an insignificant decrease in BP after given a dose supplementation of vitamin D. In the case of ISH, which is common among the elderly, vitamin D supplementation is less effective. The reason is probably because vitamin D cannot decrease blood vessel stiffness and is effective only during the early stages of the disease. Circulating renin concentrations are not raised in the elderly and not the other way around as renin raises the BP and is suppressed by vitamin D 22 . Other studies have revealed no effect of vitamin D supplementation on lowering BP in postmenopausal women 17 , chronic peripheral arterial disease patients 20 , and the elderly without certain medical conditions 16,18,19 . The actual conditions suffered by the elderly due to multiple chronic diseases and therefore multiple drugs should be considered as an important aspect that may influence the effects of vitamin D on BP.
Our study revealed that vitamin D only reduces blood pressure on hypertensive elderly. Similar to our findings, most studies identified lower BP reduction by dietary or drug interventions in hypertensive people rather than normotensive populations [38][39][40]  A meta-analysis of 34 RCTs demonstrated that SBP/DBP reduction by -10/-5 mmHg significantly reduces negative health outcomes 46 . Another study by Andersen et al. reported 9.2 mmHg reduction in SBP might represent improvements in treatment. Even though our findings showed a lower BP reduction than the aforementioned studies, this result might be due to the elderly were reported as being treated less successfully than young and middle-aged individuals 47 . Moreover, this assessment suggests that the 4.01 mmHg reduction in SBP might be considered as clinically meaningful since a meta-analysis on the effects of dietary interventions showed clinically significant BP reduction with the average net change in SBP and DBP of -3.31 mmHg and -2.24 mmHg, respectively 38 .
Searching articles until the end of March 2019, our study provides the most up-to-date meta-analysis and strongly supports that vitamin D supplementation significantly decreases BP in the elderly, specifically with elevated BP and deficiency in vitamin D. To the best our knowledge, this is the first study that analyses the effects of vitamin D on serum 25(OH)D concentrations, the gold standard to measure vitamin D status in humans.
The newest meta-analysis has revealed that vitamin D has no significant effect on BP in vitamin D-deficient people; it reduces SBP in vitamin D-deficient people older than 50 years and in people with both vitamin D deficiency and hypertension 5 . However, a previous meta-analysis involved subjects between 18 and 74 years old and serum 25-OHD are lower than 20ng/mL, and subgroup analysis used criteria older than 50 years old. Meanwhile, our study involved subjects with the mean age more than 60 years, according to WHO definition for elderly 10 . However, this study had a few limitations. First, although we conducted a systematic review of peer-reviewed research, we did not include agency reports, dissertations, and conference proceedings. Second, we included only English-language RCTs. Third, the RCTs were heterogeneous with respect to demographic characteristics of the participants, the duration, supplemental doses, and treatments for hypertension

Conclusions
Vitamin D deficiency is prevalent among the elderly, and vitamin D supplementation significantly increases serum 25(OH)D concentrations. The use of vitamin D supplementation appears to be beneficial in lowering BP, specifically in the elderly with hypertension and vitamin D deficiency. We recommend vitamin D supplementation for elderly individuals with hypertension and serum 25(OH) D concentrations below the target values. The actual conditions suffered by the elderly because of multiple chronic diseases and therefore multiple drugs should be considered as important factors that influence the effects of vitamin D on BP. Future studies with homogenous treatment duration, dose intervention, and treatment regimens need to be carried out to identify optimal treatment regimens for hypertension in the elderly that may need to include correction of vitamin D deficiency.

Data availability
Underlying data All data underlying the results are available as part of the article and no additional source data are required.

Extended data
Open Science Framework: Extended data for "Effects of vitamin D supplementation on 25(OH)D concentrations and blood pressure in the elderly: a systematic review and meta-analysis." http://doi.org/10.17605/OSF.IO/EXF26 28 .
This project contains the following extended data: • Spreadsheets in .sav format containing data for supplementation efficacy outcomes in 25(OH) concentrations, systolic blood pressure, and diastolic blood pressure.
• Supplementary figure in .doc format containing: Figure  S1: results for quality assessment, Figure S2: forest plot of each subgroup analysis, Figure S3: Funnel plot and egger test results.

Reporting guidelines
Open Science Framework: Extended data for "Effects of vitamin D supplementation on 25(OH)D concentrations and blood pressure in the elderly: a systematic review and meta-analysis." http://doi.org/10.17605/OSF.IO/EXF26 28 .
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

supplementation on 25 (OH)D levels and blood pressure in the elderly: a
Vitamin D was not measured in any studies analysed, but 25-hydroxyvitamin D [25OHD], and this term should be used throughout. Lab data reports 'concentrations' not 'levels' and this term should be used while 'levels' is good for cut-offs and definitions.

2.
Specific comments: Table 2, the heading 'vitamin D' would be clearer saying 'baseline vitamin D status'. ○ Page 10, column 1, line 2, do you mean decreases by 75% or decreases by 25% -please clarify; line 3, ..have..; 2 nd column, line 6, do you mean '…reduce renal activation, [though not that in other target tissues], but such problems were amongst the exclusion criteria'? ○ Page 10, 2 nd column, para 2, the last sentence reads as if this is the 1 st study to assess the effects of supplementation on serum 25OHD values but this cannot be true either for this submission or for the 'newer' meta-analysis mentioned in the previous sentence that needs a reference. These comments should be corrected -maybe you mean '...in elderly people for both vitamin D status and effects on blood pressure'? ○ Page 11, 1 st column, line 15, 'adjunctive' might be a better word than 'supportive'; Para 2, line 4, '....have confirmed the association' might be better than proven & ' ….however associations are not proof of causality …..; column 2, 1 st para, lines 6/7 are obscure, this section reads as if deficiency lowers blood pressure, which is not what is meant -maybe say, re. ref 24, ' ...because they were not vitamin D deficient'. Re ref 22 comment, it would be better to say that circulating renin concentrations are not raised in the elderly and not the other way around as renin raises the BP and is suppressed by vitamin D, [if you are sure that this is a general finding]. Para 3, do you mean that supplemental doses were heterogeneous or that treatments for hypertension were heterogeneous, or, as I suspect, that both of these were heterogeneous? Please clarify.

○
Page 12, para 1, is unclear, if you mean that studies homogeneous for duration, etc, need to be carried out, please rephrase for clarity, perhaps concluding, '...so as to identify optimal treatment regimes for hypertension in the elderly that may need to include correction of vitamin D deficiency'? ○ Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes

Are sufficient details of the methods and analysis provided to allow replication by others? Yes
Is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.

Are the conclusions drawn adequately supported by the results presented in the review? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: diabetes, metabolism, vitamin d and betel quid chewing.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com