Vitamin D (VitD) supplementation has been advocated for cardiovascular risk reduction; however, supporting data are sparse. The objective of this study was to determine whether VitD supplementation reduces cardiovascular risk. Subjects in this prospective, randomized, double-blind, placebo-controlled trial of post-menopausal women with serum 25-hydroxyvitamin D concentrations >10 and <60 ng/mL were randomized to Vitamin D3 2500 IU or placebo, daily for 4 months. Primary endpoints were changes in brachial artery flow-mediated vasodilation (FMD), carotid-femoral pulse wave velocity (PWV), and aortic augmentation index (AIx). The 114 subjects were mean (standard deviation) 63.9 (3.0) years old with a 25-hydroxyvitamin D level of 31.3 (10.6) ng/mL. Low VitD (<30 ng/mL) was present in 47% and was associated with higher body-mass index, systolic blood pressure, glucose, CRP, and lower FMD (all p<0.05). After 4 months, 25-hydroxyvitamin D levels increased by 15.7 (9.3) ng/mL on vitamin D3 vs. −0.2 (6.1) ng/mL on placebo (p<0.001). There were no significant differences between groups in changes in FMD (0.3 [3.4] vs. 0.3 [2.6] %, p = 0.77), PWV (0.00 [1.06] vs. 0.05 [0.92] m/s, p = 0.65), AIx (2.7 [6.3] vs. 0.9 [5.6] %, p = 0.10), or CRP (0.3 [1.9] vs. 0.3 [4.2] mg/L, p = 0.97). Multivariable models showed no significant interactions between treatment group and low VitD status (<30 ng/mL) for changes in FMD (p = 0.65), PWV (p = 0.93), AIx (p = 0.97), or CRP (p = 0.26).In conclusion, VitD supplementation did not improve endothelial function, arterial stiffness, or inflammation. These observations do not support use of VitD supplementation to reduce cardiovascular disease risk.
Citation: Gepner AD, Ramamurthy R, Krueger DC, Korcarz CE, Binkley N, Stein JH (2012) A Prospective Randomized Controlled Trial of the Effects of Vitamin D Supplementation on Cardiovascular Disease Risk. PLoS ONE 7(5): e36617. https://doi.org/10.1371/journal.pone.0036617
Editor: Jeffrey S. Berger, New York University School of Medicine, United States of America
Received: December 5, 2011; Accepted: April 3, 2012; Published: May 7, 2012
Copyright: © 2012 Gepner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has no commercial sponsor but was supported by the University of Wisconsin School of Medicine and Public Health Institute of Clinical and Translational Research and the University of Wisconsin Osteoporosis Clinical Center and Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Although the definition of “low” vitamin D (VitD) status is controversial, suboptimal VitD status is common worldwide . Circulating 25-hydroxyvitamin D [25(OH)D] values <30 ng/mL are present in up to 57% of healthy US adults and in up to 50% of postmenopausal women with osteoporosis , . A growing literature suggests that low levels of VitD are associated with increased total mortality  and cardiovascular disease (CVD) risk –. VitD inadequacy has been linked to hypertension, insulin resistance, metabolic syndrome, and congestive heart failure –; however, these associations mostly are derived from cross-sectional and observational studies –, –. Low VitD status could increase CVD risk by activating a pro-inflammatory cascade resulting in endothelial dysfunction and increased arterial stiffness, markers that contribute to hypertension and that are well-recognized surrogates of CVD risk –. The limited number of interventional studies that investigated the effects of VitD supplementation on CVD risk have had mixed results , –. To date, the only prospective randomized trial that evaluated the effects of VitD supplementation on CVD events was the Women's Health Initiative . No differences in CVD events or stroke over 7 years were observed in women treated with 400 IU daily of VitD compared to placebo; however, this study has been criticized for using an inadequate VitD dose . Based on currently available data, this dose of VitD only would be expected to raise 25(OH)D levels by 2–3 ng/mL . Moreover, the compliance in WHI only was about 60%, so this small increase in 25(OH)D levels would have even less of an observed effect . Observational data relating low VitD to CVD risk and other health conditions have led some medical providers to prescribe VitD supplements for CVD risk reduction, although data supporting this intervention are sparse. The aim of this study was to determine if VitD supplementation would improve flow-mediated vasodilation (FMD) of the brachial artery, a measure of endothelial function, and 2 measures of arterial stiffness, carotid-to-femoral pulse wave velocity (PWV) and aortic augmentation index (AIx). These markers, and their changes, are predictors of CVD risk , , .
The first 34 subjects were part of a pilot study performed under NCT 00690417. The remainder of the subjects were performed under NCT 01049048.
This study was approved by the University of Wisconsin Health Sciences Institutional Review Board. It was conducted according to the principles expressed in the Declaration of Helsinki. All subjects provided written consent.
Study Design and Population
This was a prospective, randomized, double-blind, placebo-controlled trial of post-menopausal women with serum 25(OH)D concentrations between 10 and 60 ng/mL, as this range of vitamin D levels was thought to be representative of the majority of the US population, exclusive of those for whom withholding supplementation would not meet current standards of care. Participants were healthy, community-dwelling, ambulatory women from Madison, Wisconsin who were recruited between March 2009 and June 2010. Following informed consent, a screening evaluation of demographic and laboratory assessments were performed. Qualifying volunteers returned for a baseline visit. Endothelial function was evaluated by measuring brachial artery FMD in a core ultrasound laboratory using a standardized protocol described below , , . Arterial stiffness was evaluated by determining carotid-to-femoral PWV and AIx using applanation tonometry, as described below , . Laboratory evaluations were performed in a CLIA-approved lab and included fasting glucose, lipids, calcium, parathyroid hormone, and high-sensitivity C-reactive protein (CRP). Laboratory tests, FMD, and arterial stiffness studies were performed prior to initiating therapy and after 4 months. All measurements were performed in the morning after fasting for ≥8 hours. The first 34 subjects were enrolled as part of a pilot study that did not include arterial stiffness measurements. Arterial tonometry measurements were recorded in the remainder of the subjects after the techniques became available and recruitment was demonstrated to be feasible (Figure 1).
Exclusion criteria included: history of CVD, serum calcium >10.5 mg/dL, untreated primary hyperparathyroidism, history of nephrolithiasis, hypercalciuria, malignancy, tuberculosis, sarcoidosis, Paget's disease, malabsorption syndromes, estimated glomerular filtration rate ≤25 mL/minute, use of medications that interfere with vitamin D metabolism or affect bone turnover (including hormone replacement therapy), use of active metabolites of vitamin D within 6 months of screening, use of tanning beds or salons, or unwillingness to utilize sunscreen during periods of sun exposure of >15 minutes. All subjects agreed to avoid use of cod-liver oil and non-study vitamin D supplementation and to utilize sunscreen of SPF-15 or higher when sun exposure for >15 minutes.
Randomization and Treatment Allocation
At the baseline study visit, volunteers were randomly assigned to receive either 2500 IU of oral D3 in a cookie or an identical tasting placebo cookie daily (D-Rich Foods, Inc., Manitowoc, Wisconsin). Randomization was carried out in a 1∶1 ratio without blocking using computer-generated random numbers. One individual in the Osteoporosis Research Center was assigned to randomize subjects and package vitamin D/placebo cookies with labels; this individual did not participate in recruitment, data collection, or analysis. All others including volunteers, study staff, investigators, and data analysts were blinded to the group assignment. There was no cross-over and all subjects were analyzed in an intention-to-treat manner. Enrollment was complete following recruitment of the pre-specified number of subjects.
The primary outcome measures were brachial artery FMD, PWV, and AIx. Secondary outcomes included brachial and central aortic blood pressures, serum glucose, fasting lipid, and CRP levels. All tests were performed with subjects fasting between 7:00 and 11:00 AM.
Measurement of Endothelial Function and Arterial Stiffness
Endothelial function was measured by ultrasound assessment of brachial artery FMD , , . Subjects were placed in a supine position in a temperature-controlled room for 10 minutes before imaging. A blood pressure cuff was placed on the widest part of the proximal right forearm. Using an 8-MHz linear array vascular ultrasound transducer and a state-of-the-art ultrasound system (Acuson Sequoia 512, Siemens Medical Solutions, Issaquah, Washington), the brachial artery was located above the elbow and scanned longitudinally. After recording B-mode ultrasound images of the brachial artery and spectral Doppler velocities, the cuff was inflated to 250 mmHg for 5 minutes to induce reactive hyperemia. Immediately after deflation, spectral Doppler images were obtained to verify hyperemia. Brachial artery B-mode images were obtained 60 and 90 seconds after cuff release. Studies were recorded digitally; brachial artery diameters were measured in triplicate with a digital border tracing tool (Access Point Web 3.0, Freeland Systems, Westfield, Indiana). Studies were read in subject pairs (baseline and 4 months), blinded to study treatment. Reproducibility of measurements from this lab is excellent and has been reported recently using the exact same techniques, including during the time this study was being conducted , .
Arterial stiffness was measured by arterial tonometry (AtCor SphygmoCor Px, AtCor Medical, Sydney, Australia) , . Tonometry recordings of the carotid and femoral arterial pulses were taken when a reproducible signal with a clear upstroke was obtained. The carotid-femoral PWV was determined by the intersecting tangents method , . PWV (m/s) was calculated as the distance-to-transit time ratio of the recorded pulse wave. The time delay (seconds) from the electrocardiogram R-wave to the foot of the arterial pulse waveform was measured at the proximal (carotid) and distal (femoral) sites, based on an analysis of 10 seconds of stable tonometry tracings. The difference in the proximal and distal delay was considered to be the carotid-femoral transit time. The PWV distance was calculated as the difference in the absolute distance between the suprasternal notch and the carotid and femoral tonometry sites, respectively. AIx and central aortic pressures were derived from radial tonometry using a validated, generalized transfer function and calibrated using oscillometric brachial artery blood pressures. All AIx measurements were read independently and standardized to a heart rate of 75 bpm. An operator index greater than 85% was required for all analyzed tracings. Reproducibility of these measurements from this lab is excellent. For PWV, mean (standard deviation) differences for repeated measurements is 0.035 (0.44) m/s with an intra-class correlation coefficient of 0.91. For AIx and central pulse pressure, mean differences are 0.02 (4.0)% and 0.20 (2.8) mmHg, respectively, with intra-class correlation coefficients of 0.90 and 0.97, respectively.
Fasting serum chemistry, glucose, lipids, and CRP levels and urinary calcium determinations were performed at General Medical Laboratories (Madison, WI). CRP was measured by nephelometry on a Siemens Vista analyzer using two control products to assess the daily performance of the instrument. The coefficient of variation was approximately 3.2%. Serum 25(OH)D was determined using reverse phase high-performance liquid chromatography as previously described , .
Statistical Analysis and Sample Size
All analyses were conducted using STATA (Stata Statistical Software: Release 11, College Station, TX). Between groups comparisons were performed using two-tailed Student's t-tests or Mann-Whitney rank sum comparisons (for variables with non-normal distributions). Pearson correlations were used to determine associations with FMD. General linear models with 2 factors including group and vitamin D status (>30 vs <30 ng/mL), plus their interaction, were used to determine independent associations. All models were adjusted for body-mass index. Similar analyses were performed for PWV, AIx, central aortic pressures, CRP, and their changes. Bonferroni-adjusted p-values were reported for these analyses. A priori power calculations estimated that a minimum of 37 subjects in each arm would enable us to detect a 3% change in FMD with 80% power (alpha = 0.05). The sample size was sufficient to detect an approximately 10% reduction in PWV with a standard deviation of approximately 15%.
Of 133 potential subjects that were screened, 114 were eligible (Figure 1). They were 63.9 (3.0) years old with a baseline 25(OH)D level of 31.3 (10.6) ng/mL (Table 1). The median baseline 25(OH)D level was 30.3 ng/mL. These subjects were randomized equally to each group; however, 2 subjects withdrew from each group and 1 subject in the placebo group did not have arterial stiffness measurements because of equipment failure. The final data analysis included 55 subjects in the treatment group and 54 in the placebo group. PWV and AIx were measured in 38 and 37 consecutive subjects in the treatment and placebo groups, respectively. Baseline characteristics were similar between groups with the exception of the treatment group having a slightly higher body-mass index (27.1 [4.7] vs. 25.3 [5.1] kg/m2, p = 0.022). There were no differences between groups in baseline brachial artery diameter (p = 0.66) or other measures of CVD risk including blood pressures, CRP, lipid profiles, or glucose level (all p>0.4) (Table 1).
Effects of Treatment with VitD or Placebo
After 4 months, serum 25(OH)D increased by 15.7 (9.3) ng/mL in the treatment group versus −0.2 (6.1) ng/mL in those taking placebo (p<0.001). Despite improved VitD status, there were no significant differences between groups in any of the pre-specified outcomes including change in absolute FMD (0.001 [0.009] vs. 0.001 [0.013] cm p = 0.729), maximum relative FMD (0.3 [3.4] vs. 0.3 [2.6] %, p = 0.77), PWV (0.00 [1.06] vs. 0.05 [0.92] m/s, p = 0.65), AIx (2.7 [6.3] vs. 0.9 [5.6] %, p = 0.10), or CRP (0.3 [1.9] vs. 0.3 [4.2] mg/L, p = 0.97) (Table 2, Figure 2). Additionally, there were no significant changes in central aortic or brachial blood pressures (p>0.4). There was a small, statistically significant increase in the total/high-density lipoprotein cholesterol ratio in the placebo group compared to the treatment group (0.17 [0.36] vs. 0.07 [0.39], p<0.001). No adverse events were reported.
Of those in the treatment group, 92% (N = 51) had vitamin D levels >30 ng/mL after 4 months of therapy. General linear models showed no significant interaction between treatment group and VitD status for changes in maximum relative FMD (p = 0.65), PWV (p = 0.93), AIx (p = 0.97), or CRP (p = 0.26). No differences were observed after adjustment for baseline body-mass index.
Low VitD status (<30 ng/mL) was present in 47% (n = 54) of subjects at baseline. Subjects with low VitD status had a mean serum 25(OH)D level of 22.3 (standard deviation 5.26, range 9.1–29.0) ng/mL; lower than those with normal VitD status who had a mean serum 25(OH)D level of 39.7 (6.8, range 30.2–57.0) ng/mL. Low VitD status was associated with higher body-mass index (28.2 [5.0] vs. 24.2 [4.2] kg/m2, p<0.001), brachial systolic blood pressure (126.5 [13.3] vs. 118.6 [10.8] mmHg, p = 0.004), glucose (104.6 [42.3] vs. 90.8 [7.8] mg/dL, p = 0.003), CRP (2.9 [3.4] vs 1.5 [2.1] mg/L, p = 0.001), and lower maximum relative FMD (4.1 [3.0] vs. 5.4 [3.6] %, p = 0.043). After 4 months, those with low baseline 25(OH)D levels had a nominally statistically significant greater increase in maximum relative FMD than those with 25(OH)D levels >30 ng/mL (1.1 [2.4] vs.−0.5 [3.4]%, p = 0.002); however, this difference was independent of treatment group (Figure 2, Table S1).
We evaluated the effects of a higher dose of vitamin D supplementation (2500 IU daily) than used in the Women's Health Initiative on several vascular parameters and markers of CVD risk. In our study, treated subjects had circulating 25(OH)D levels that increased, on average, by over 15 ng/mL and over 90% of treated subjects achieved 25(OH)D levels >30 ng/mL. Nevertheless, we did not observe an improvement in FMD or arterial stiffness measures. Similarly, we did not observe differential improvements in central or peripheral blood pressures or CRP levels between treatment groups.
Our prospective, randomized, blinded study findings did not show improvements in FMD or either arterial stiffness measure after treatment with VitD. The few randomized controlled trials that have evaluated the effects of vitamin D supplementation on endothelial function or arterial stiffness have had mixed results in regard to FMD , , , PWV , and AIx ; however, those studies were notably smaller than our study and predominantly were performed in adults with medical conditions such as kidney disease and/or type II diabetes mellitus. Similarly, there are mixed reports regarding changes in blood pressure following VitD supplementation , . Despite higher blood pressures in individuals with lower 25(OH)D levels, we found no change in blood pressure after VitD supplementation, in agreement with the largest of these trials . Despite higher CRP levels in individuals with lower 25(OH)D levels, we also found no change in CRP after supplementation, a finding consistent with previous reports –. The lack of an effect of VitD supplementation on these markers of CVD risk was confirmed in our subgroup analyses restricted to participants with low VitD status at baseline using multivariable linear models which showed that treatment group did not influence change in FMD, PWV, or AIx. Although individuals with low VitD status at baseline had a small increase in maximum relative FMD after 4 months, this increase was not influenced by treatment group. Our results challenge the hypothesis that VitD supplementation reduces CVD risk.
Vitamin D receptors are widely distributed throughout the body and have been isolated from vascular endothelial cells and cardiac myocytes . Mechanistically it is conceivable that low VitD levels could have deleterious CVD effects by dysregulation of systemic calcium metabolism, as cardiac myocyte contraction depends on calcium homeostasis and since coronary artery calcification is predictive of CVD risk , , . Also, VitD modulates lymphocytic cytokine production , potentially affecting growth and proliferation of vascular smooth muscle cells and cardiomyocytes , stimulating vascular tissue anticoagulant activity , and suppressing renin gene expression , which could lead to the clinical manifestations of hypertension, coronary artery disease, and congestive heart failure. However, mechanistic hypotheses do not necessarily imply that supplementing individuals with low 25(OH)D levels will reverse these adverse processes and reduce CVD risk . Blood pressure reductions have been observed with VitD supplementation , , but the majority of studies have shown no change , , . Furthermore, 2 randomized controlled trials showed no improvement in left ventricular function in patients with heart failure randomized to VitD therapy , .
Although plausible biological mechanisms and epidemiological data suggest that VitD deficiency may increase and VitD supplementation may reduce CVD risk, observational studies cannot account for unmeasured confounders. In our study, as in others, individuals with low VitD tended to have a greater CVD risk factor burden , , . It has been proposed that low VitD is responsible for these observations; however, it is possible that these CVD risk factors simply are associated with lower 25(OH)D levels. Indeed, previous studies have shown that the risk factors for VitD deficiency are similar to traditional CVD risk factors . In our study, despite an average increase in circulating 25(OH)D levels of nearly 16 ng/mL, we observed no improvements in FMD, PWV or AIx, surrogate CVD risk markers that predict initial and recurrent CVD events .
Our endpoints are surrogate markers for CVD risk; we did not evaluate CVD death, myocardial infarction, or stroke. Although our markers are predictive of CVD events and are well-established research tools, they are imperfect and an absence of change in these measures does not exclude the possibility that VitD supplementation may reduce CVD risk , , . Participants in this study were generally healthy post-menopausal women with typical 25(OH)D levels. It is possible that VitD supplementation in men or in individuals with higher baseline CVD risk or certain co-morbidities, including abnormal endothelial function or lower 25(OH)D levels, may reduce CVD risk. Additionally, research on the effects of VitD supplementation is challenged by the absence of a widely accepted definition of vitamin D “insufficiency.” Proposed “reasonable” 25(OH)D levels range from 20 ng/mL (50 nmol/L) to 32 ng/mL (80 nmol/L) ,  and some advocate VitD supplementation in individuals with 25(OH)D levels that are <50 ng/mL . Subjects in this study started with a wide range of 25(OH)D levels; those subjects with higher starting levels may have had a blunted arterial response to treatment despite potentially being randomized to the treatment group. Similarly, there is considerable debate regarding the optimal dose for VitD supplementation and repletion in adults. Expert opinion for supplemental dosing ranges from 600–1000 IU (20–25 µg) to 2000 IU (50 µg) daily , , , although some have argued that even the latter dose may be inadequate and higher doses frequently are used clinically . Given the inconsistencies in the literature, we cannot exclude the possibility that supplementation with even higher doses of VitD for a longer duration would show a benefit in terms of CVD risk; however, interventions that reduce CVD risk also tend to improve endothelial function and arterial stiffness soon after initiating treatment, suggesting that 4 months was a reasonable duration of treatment , , . Based on revised estimates using a mixed effects general linear model and restricted maximum likelihood estimation, with 55 subjects per arm we had over 90% power to detect a 1.5% difference in FMD. For PWV, we observed essentially no change PWV and the SDs were very small, however there still is a small chance that we missed a true difference between groups.
In the largest prospective randomized clinical trial of VitD supplementation that used a dose of VitD that normalized VitD levels in most participants, VitD supplementation did not improve endothelial function, arterial stiffness, reduce CRP or improve blood pressure in healthy, post-menopausal women. This study does not support the use of VitD supplementation to reduce CVD risk; however, long-term outcomes studies of this intervention are needed.
CONSORT Checklist. CONSORT 2010 checklist of information to include when reporting a randomized trial.
Trial Protocol. Effect of Vitamin D Status on Endothelial Function.
The authors would like to thank the staff of the UW Osteoporosis Research Center and the UW Atherosclerosis Imaging Research Program, as well as all of the participants in this study.
Conceived and designed the experiments: RR DK NB JS. Performed the experiments: RR DK CK NB JS. Analyzed the data: AG DK CK NB JS. Contributed reagents/materials/analysis tools: CK NB JS. Wrote the paper: AG JS.
- 1. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357: 266–281.
- 2. Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR (2002) Serum 25-hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from NHANES III. Bone 30: 771–777.
- 3. Autier P, Gandini S (2007) Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials. Arch Intern Med 167: 1730–1737.
- 4. Lavie CJ, Lee JH, Milani RV (2011) Vitamin D and cardiovascular disease will it live up to its hype? J Am Coll Cardiol 58: 1547–1556.
- 5. Anderson JL, May HT, Horne BD, Bair TL, Hall NL, et al. (2010) Relation of vitamin D deficiency to cardiovascular risk factors, disease status, and incident events in a general healthcare population. Am J Cardiol 106: 963–968.
- 6. Lee JH, O'Keefe JH, Bell D, Hensrud DD, Holick MF (2008) Vitamin D deficiency an important, common, and easily treatable cardiovascular risk factor? J Am Coll Cardiol 52: 1949–1956.
- 7. Giovannucci E, Liu Y, Hollis BW, Rimm EB (2008) 25-hydroxyvitamin D and risk of myocardial infarction in men: a prospective study. Arch Intern Med 168: 1174–1180.
- 8. Wang TJ, Pencina MJ, Booth SL, Jacques PF, Ingelsson E, et al. (2008) Vitamin D deficiency and risk of cardiovascular disease. Circulation 117: 503–511.
- 9. Reddy VS, Good M, Howard PA, Vacek JL (2010) Role of vitamin D in cardiovascular health. Am J Cardiol 106: 798–805.
- 10. Mheid IA, Patel R, Murrow J, Morris A, Rahman A, et al. (2011) Vitamin d status is associated with arterial stiffness and vascular dysfunction in healthy humans. J Am Coll Cardiol 58: 186–192.
- 11. Lind L, Hanni A, Lithell H, Hvarfner A, Sorensen OH, et al. (1995) Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men. Am J Hypertens 8: 894–901.
- 12. Scragg R, Jackson R, Holdaway IM, Lim T, Beaglehole R (1990) Myocardial infarction is inversely associated with plasma 25-hydroxyvitamin D3 levels: a community-based study. Int J Epidemiol 19: 559–563.
- 13. Poole KE, Loveridge N, Barker PJ, Halsall DJ, Rose C, et al. (2006) Reduced vitamin D in acute stroke. Stroke 37: 243–245.
- 14. Vita JA, Keaney JF (2002) Endothelial function: a barometer for cardiovascular risk? Circulation 106: 640–642.
- 15. Geleijnse JM (2010) Vitamin D and the Prevention of Hypertension and Cardiovascular Diseases: A Review of the Current Evidence. Am J Hypertens 24: 253–262.
- 16. Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, et al. (2006) Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J 27: 2588–2605.
- 17. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, et al. (2002) Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 39: 257–265.
- 18. Hsia J, Heiss G, Ren H, Allison M, Dolan NC, et al. (2007) Calcium/vitamin D supplementation and cardiovascular events. Circulation 115: 846-854 (Erratum in: Circulation. 2007 May 15: 115(19): e466).
- 19. Sugden JA, Davies JI, Witham MD, Morris AD, Struthers AD (2008) Vitamin D improves endothelial function in patients with Type 2 diabetes mellitus and low vitamin D levels. Diabet Med 25: 320–325.
- 20. Margolis KL, Ray RM, Van Horn L, Manson JE, Allison MA, et al. (2008) Effect of calcium and vitamin D supplementation on blood pressure: the Women's Health Initiative Randomized Trial. Hypertension 52: 847–855.
- 21. Heaney RP, Davies KM, Chen TC, Holick MF, Barger-Lux MJ (2003) Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol. Am J Clin Nutr 77: 204–210.
- 22. Torriani FJ, Komarow L, Parker RA, Cotter BR, Currier JS, et al. (2008) Endothelial function in human immunodeficiency virus-infected antiretroviral-naive subjects before and after starting potent antiretroviral therapy: The ACTG (AIDS Clinical Trials Group) Study 5152s. J Am Coll Cardiol 52: 569–576.
- 23. Johnson HM, Gossett LK, Piper ME, Aeschlimann SE, Korcarz CE, et al. (2010) Effects of smoking and smoking cessation on endothelial function: 1-year outcomes from a randomized clinical trial. J Am Coll Cardiol 55: 1988–1995.
- 24. Korcarz CE, Gepner AD, Peppard PE, Young TB, Stein JH (2010) The effects of sleep-disordered breathing on arterial stiffness are modulated by age. Sleep 33: 1081–1085.
- 25. Binkley N, Drezner MK, Hollis BW (2006) Laboratory reporting of 25-hydroxyvitamin D results: potential for clinical misinterpretation. Clin Chem 52: 2124–2125.
- 26. Lensmeyer GL, Wiebe DA, Binkley N, Drezner MK (2006) HPLC method for 25-hydroxyvitamin D measurement: comparison with contemporary assays. Clin Chem 52: 1120–1126.
- 27. Jablonski KL, Chonchol M, Pierce GL, Walker AE, Seals DR (2011) 25-hydroxyvitamin d deficiency is associated with inflammation-linked vascular endothelial dysfunction in middle-aged and older adults. Hypertension 57: 63–69.
- 28. Witham MD, Dove FJ, Dryburgh M, Sugden JA, Morris AD, et al. (2010) The effect of different doses of vitamin D(3) on markers of vascular health in patients with type 2 diabetes: a randomised controlled trial. Diabetologia 53: 2112–2119.
- 29. Dong Y, Stallmann-Jorgensen IS, Pollock NK, Harris RA, Keeton D, et al. (2010) A 16-week randomized clinical trial of 2000 international units daily vitamin D3 supplementation in black youth: 25-hydroxyvitamin D, adiposity, and arterial stiffness. J Clin Endocrinol Metab 95: 4584–4591.
- 30. Andrade J, Er L, Ignaszewski A, Levin A (2008) Exploration of association of 1,25-OH2D3 with augmentation index, a composite measure of arterial stiffness. Clin J Am Soc Nephrol 3: 1800–1806.
- 31. Sepulveda JL, Mehta JL (2005) C-reactive protein and cardiovascular disease: a critical appraisal. Curr Opin Cardiol 20: 407–416.
- 32. Amer M, Qayyum R (2012) Relation between serum 25-hydroxyvitamin D and C-reactive protein in asymptomatic adults (from the continuous National Health and Nutrition Examination Survey 2001 to 2006). Am J Cardiol 109: 226–230.
- 33. Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, et al. (2001) 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. J Immunol 167: 4974–4980.
- 34. O'Connell TD, Berry JE, Jarvis AK, Somerman MJ, Simpson RU (1997) 1,25-Dihydroxyvitamin D3 regulation of cardiac myocyte proliferation and hypertrophy. Am J Physiol 272: H1751–H1758.
- 35. Inoue M, Wakasugi M, Wakao R, Gan N, Tawata M, et al. (1992) A synthetic analogue of vitamin D3, 22-oxa-1,25-dihydroxy-vitamin D3, stimulates the production of prostacyclin by vascular tissues. Life Sci 51: 1105–1112.
- 36. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, et al. (2002) 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 110: 229–238.
- 37. Shapses SA, Manson JE (2011) Vitamin D and prevention of cardiovascular disease and diabetes: why the evidence falls short. JAMA 305: 2565–2566.
- 38. Martins D, Wolf M, Pan D, Zadshir A, Tareen N, et al. (2007) Prevalence of cardiovascular risk factors and the serum levels of 25-hydroxyvitamin D in the United States: data from the Third National Health and Nutrition Examination Survey. Arch Intern Med 167: 1159–1165.
- 39. Witham MD, Crighton LJ, Gillespie ND, Struthers AD, McMurdo ME (2010) The effects of vitamin D supplementation on physical function and quality of life in older patients with heart failure: a randomized controlled trial. Circ Heart Fail 3: 195–201.
- 40. Schleithoff SS, Zittermann A, Tenderich G, Berthold HK, Stehle P, et al. (2006) Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr 83: 754–759.
- 41. Oliver JJ, Webb DJ (2003) Noninvasive assessment of arterial stiffness and risk of atherosclerotic events. Arterioscler Thromb Vasc Biol 23: 554–566.
- 42. Vieth R (2006) Critique of the considerations for establishing the tolerable upper intake level for vitamin D: critical need for revision upwards. J Nutr 136: 1117–1122.
- 43. Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, et al. (2010) The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine: What Clinicians Need to Know. J Clin Endocrinol Metab 96: 53–58.
- 44. Cannell JJ, Hollis BW, Zasloff M, Heaney RP (2008) Diagnosis and treatment of vitamin D deficiency. Expert Opin Pharmacother 9: 107–118.
- 45. Vieth R (2007) Vitamin D toxicity, policy, and science. J Bone Miner Res 22: Suppl 2V64–V68.
- 46. O'Keefe JH, Lavie CJ, Holick MF (2011) Vitamin D supplementation for cardiovascular disease prevention. JAMA 306: 1546-1547; author reply 1547-1548: