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Vitamin D deficiency may be involved in the development of atherosclerosis and coronary heart disease (CHD) in humans. We assessed prospectively whether plasma 25(OH)vitamin D (25(OH)D) concentrations are associated with risk of CHD.
A nested case-control study was conducted among 18,225 male participants of the Health Professionals Follow-up Study aged 40 to 75 years who were free of diagnosed cardiovascular disease at the time of blood draw (1993–1995). During 10 years of follow-up through January 31, 2004, 454 men developed MI (nonfatal myocardial infarction) or fatal CHD. Using risk set sampling, controls were selected in a 2:1 ratio matched for age, date of blood draw, and smoking status (n=900).
After adjustment for matched variables, men deficient (≤15 ng/mL) in 25(OH)D were at increased risk of MI compared to those considered to be sufficient (≥30 ng/mL) in 25(OH)D (relative risk (RR)=2.42; 95% confidence interval (CI), 1.53–3.84; P for trend <.001). After additional adjustment for family history of MI, body mass index, alcohol consumption, physical activity, history of diabetes and hypertension, ethnicity, region, marine omega-3 intake, low- and high-density lipoprotein cholesterol and triglyceride levels, this relationship remained significant (RR=2.09; 95% CI, 1.24–3.54; P for trend=0.02). Even men with intermediate values for 25(OH)D were at elevated risk relative to those with sufficient 25(OH)D (22.6–29.9 ng/mL: RR=1.60; 95% CI, 1.10–2.32; 15.0–22.5 ng/mL: RR=1.43; 95% CI, 0.96–2.13).
This study provides evidence that optimal levels of 25(OH)D should be at least 30 ng/mL to lower risk of MI.
A number of observations are not easily explained by known cardiovascular disease (CVD) risk factors. In most populations studied, the rate of CVD death is elevated at higher latitudes, increases during the winter months, and is lower at high altitudes. As has been noted previously by others, this pattern is consistent with an adverse effect of hypovitaminosis D, which is more prevalent in higher latitudes, during the winter, and at lower altitudes.1 Alternative explanations for these observations are possible, but a number of plausible biologic mechanisms support a role of vitamin D. The vitamin D axis influences vascular smooth cell proliferation, inflammation, vascular calcification, and the renin-angiotensin system (RAS) and blood pressure, 1all which influence risk of CVD and of myocardial infarction (MI).
Despite these suggestive ecologic data and plausible mechanisms, data directly linking vitamin D levels to risk of MI are sparse. A Danish study2 examined 25(OH)D levels measured in 128 patients admitted with ischemic heart disease (75 with angina pectoris and 53 with acute MI) and 409 controls and found that 25(OH)D levels were significantly lower in those with angina (23.5 ng/ml) or MI (24.0 ng/ml) than in the controls (28.8 ng/ml). In a New Zealand case-control study of 179 MI cases,3 cases had a lower mean 25(OH)D level (P=0.017), which was more pronounced in the winter-spring (P=0.029) than in the summer-autumn (P=0.21). The relative risk (RR) of MI decreased across increasing quartiles of 25(OH)D: <10 ng/mL: RR =1 (reference); 10–13 ng/mL: RR=0.56 (95% CI, 0.32–1.03); 13.1–16.8 ng/mL: RR=0.33 (95% CI, 0.17–0.64); >16.8 ng/mL: RR=0.30 (95% CI, 0.15–0.61). Multivariate analyses of major CVD risk factors did not appreciably alter the results. A small nested-case control study of MI based in the Tromso Heart Study (northern Norway) based on only 30 cases and 60 matched controls found a slightly non-significant lower 25(OH)D level in cases (23.6 ng/mL) compared to controls (25.4 ng/mL).4
Because hypovitaminosis D is highly prevalent and easily correctable,5 establishing the relationship between vitamin D and risk of MI is important. Thus, we examined prospectively 25(OH)D concentrations in relation to risk of MI in the Health Professionals Follow-Up Study (HPFS), a large cohort of U.S. men.
The HPFS is a prospective cohort investigation among 51 529 US male health care professionals aged 40 to 75 years at baseline in 1986. This cohort was designed primarily to evaluate associations between diet and chronic disease incidence.6 We assessed information about health and disease every 2 years by a self-administered questionnaire, and diet every 4 years by a self-administered food frequency questionnaire.7 Between 1993 and 1995, a blood sample was requested from all surviving cohort participants, of which 18 225 provided samples. The respondents were somewhat younger but were otherwise similar to non-participants. Based on the cohort who provided samples, and after exclusion of participants with a history of CVD prior to 1994, we identified 454 participants with incident nonfatal MI or fatal coronary heart disease (CHD) between date of blood draw and January 31, 2004. We were able to assess disease status through January 31, 2004 for 97.3% of the men. Controls were randomly selected from participants with a blood sample and who were alive and who did not have history of CVD at the time of case ascertainment. For the controls, we used a 2:1 ratio and matched for age, month and year of blood draw, and smoking status (risk set sampling).8 Our analysis includes 8 participants who were selected as a control and subsequently had an event later during follow-up, leaving 900 controls available for analysis. No control was selected twice during the random selection process. All participants gave written informed consent, and the Harvard School of Public Health Human Subjects Committee Review Board approved the study protocol.
Study physicians reviewed the medical records of all participants for whom nonfatal MI or fatal CHD was reported during follow-up. The reviewers were blinded to participants' exposure status. Each questionnaire that is mailed biennially to HPFS participants contains a question on whether the man has had “professionally diagnosed … myocardial infarction (heart attack)” in the preceding 2 years. Myocardial infarction (MI) was confirmed if it met the World Health Organization's criteria, which include symptoms plus either diagnostic electrocardiographic changes or elevated levels of cardiac enzymes.9 For about 70% of the men who self-reported MI, we confirmed the diagnosis using these methods. Reasons for nonconfirmation of MI were either that no further information was available, typically because the participant did not consent or the hospital did not send the hospital records, or that a reported case was rejected based on the medical record information received. We excluded nonconfirmed participants from the control selection process. Deaths were identified from state vital statistics records and the National Death Index or reported by next of kin or by the postal system. Fatal CHD was considered to have occurred if there was fatal MI confirmed by hospital records or on autopsy or if CHD was listed as the cause of death on the death certificate, if it was the underlying and most plausible cause, and if evidence of previous CHD was available. In our analysis, 352 participants had nonfatal MI and 102 had fatal CHD as the qualifying event.
For the analysis, anthropometric data, lifestyle factors, and diet were based on the 1994 questionnaire. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Nutrient intake was computed based on a validated semiquantitative food frequency questionnaire, which inquires about average food intake during the past year, using composition values from the US Department of Agriculture sources,10 supplemented with other data. Physical activity was expressed as metabolic equivalent task (MET)–hours based on self-reported types and durations of activities over the previous year.11 One MET-hour is equivalent to energy expenditure while sitting quietly for 1 hour. We derived medical history information from the questionnaires completed between 1986 and 1994. We have previously reported on the validity and reproducibility of the collected data and measurements.7, 12–16
We collected blood samples in three 10-mL liquid EDTA blood tubes, placed on ice packs, stored in Styrofoam containers, and returned to our laboratory via overnight courier. More than 95% arrived within 24 hours of blood draw. The blood samples were then centrifuged and aliquoted for storage in the vapor phase of liquid nitrogen freezers (−130°C or colder). Fewer than 15% of the samples were slightly hemolyzed and very few were moderately hemolyzed (<3%), lipemic (<1%), or not cooled on arrival (<0.5%).
Plasma 25(OH)D was determined by radioimmunoassay (RIA) in the laboratory of Dr. Bruce Hollis as previously described.17 The coefficient of variation for 25(OH)D was 11.5%. In a sub-cohort of the 144 men who provided baseline blood samples in 1993–94 and again in 1997 (mean of 3.03 0.46 years apart), the Pearson correlation coefficient for 25(OH)D was 0.70 (P<0.0001).18 LDL cholesterol was measured by a homogeneous direct method from Genzyme Corp, Cambridge, MA,19 HDL cholesterol using a direct enzymatic colorimetric assay,20 and triglycerides enzymatically with correction for endogenous glycerol.21 The assays used for lipoprotein and lipid analysis are approved by the US Food and Drug Administration for clinical use, and coefficients of variation were less than 6%.
Plasma 25(OH)D levels were categorized into four categories based on common definitions of “deficient” (≤15 ng/mL), “insufficient” (15.1–29.9 ng/mL) and “sufficient” 25(OH)D (≥30 ng/mL) levels.22 We further dichotomized the “insufficient” range at its midpoint into 15.1–22.5 ng/mL and 22.6 to 29.9 ng/mL. For our main analysis to investigate the association between 25(OH)D concentrations and incidence of nonfatal MI or fatal CHD (events), we used conditional logistic regression. For tests for trend, we modeled the median value of the four categories as continuous variables in the regression model. In our multivariable model, we further adjusted for family history of MI before age 60 years (yes/no), alcohol intake (nondrinker; 0.1–4.9, 5.0–14.9, 15.0–29.9, or 30.0 g/d; or missing), body mass index (continuous), physical activity (quintiles), and history of diabetes (yes/no) and hypertension (yes/no), ethnicity (Caucasian/other), region (Northeast/mid-Atlantic, Midwest, South) marine omega-3 intake (quintiles), low- and high-density lipoprotein cholesterol and triglyceride levels (quintiles) at baseline. Because of the design of our study, the odds ratio derived from logistic regression directly estimates the incidence rate (hazard) ratio and, therefore, the relative risk.8, 23 We assessed the goodness of fit of the models using the method described by Hosmer and Lemeshow,24 which did not show any significant lack of fit.
For stratified analysis, we used unconditional logistic regression adjusted for matched variables (age <50, 50–54, 55–59, 60–64, or 65 years; smoking status never, past, or current; and month of blood draw in 5 categories). For the main analysis, unconditional logistic regression with adjustment for matched variables yielded similar results as conditional logistic regression. Tests for interaction were based on the Wald test for the interaction term (variable times 25(OH)D) with both the variable and vitamin D in the model as continuous variables. All P values presented are 2-tailed; P<.05 was considered statistically significant. All analyses were performed using SAS software, version 9.1 (SAS Institute Inc, Cary, NC).
Selected characteristics and biomarker levels of cases and controls are presented in Table 1. As expected, cases had a higher BMI, lower level of physical activity and less alcohol intake, and were more likely to have a family history of MI before age 60 years and history of diabetes and hypertension. In addition, cases had higher levels of total and LDL cholesterol and of triglycerides, but lower levels of HDL cholesterol. Plasma 25(OH)D levels were significantly lower (P=0.002) in cases (23.0 ng/mL) compared to controls (24.5 ng/mL).
We next measured levels or percentages of selected risk factors across 25(OH)D concentrations in controls, shown in Table 2. Men with lower 25(OH)D levels were more likely to be current smokers, less physically active and heavier, and less likely to be white and to have a parental history of MI. They were also less likely to take a multivitamin supplement, drank less alcohol, and were more likely to live in northern states. Men with lower 25(OH)D levels also had a suggestive but not statistically significant higher prevalence of diabetes and hypertension, and significantly higher levels of triglycerides and lower levels of HDL cholesterol.
Table 3 presents RRs of MI (incident nonfatal MI plus fatal CHD) over 10 years of follow-up across the categories of plasma 25(OH)D. After adjustment for matching factors, men with deficient levels of 25(OH)D had a significantly elevated risk of MI (RR=2.42; 95% CI, 1.53–3.84; P for trend=0.0006). In the multivariable model without plasma lipids, the RR was somewhat attenuated but remain statistically significant (RR=2.01; 95% CI, 1.22–3.30; P for trend=0.02). After further adjustment for plasma lipids, the RR=2.09 (95% CI, 1.24–3.54; P for trend=0.02). The results did not change appreciably after excluding the first two years of follow-up (RR=1.97; 95% CI, 1.10–3.51; P for trend=0.05 across levels of 25(OH)D).
In alternative analyses, we examined season-specific quintiles of 25(OH)D in relation to MI risk. For those in the bottom versus top quintiles, the multivariable RR=1.94 (95% CI, 1.23–3.06). Compared with men with adequate 25(OH)D, those with insufficient values of 25(OH)D were associated with approximately a 50% increase in risk. In the full multivariable model, the β coefficient for a 1 ng/mL increment in 25(OH)D was −0.0214 (SE=0.00987), indicating the risk of MI decreased by 2.1% per a 1 ng/mL increment in 25(OH)D. We did not have adequate power to test for differences between non-fatal (n=352) and fatal (n=102) cases, but the β coefficient for MI risk per 1 ng/mL increase of 25(OH)D suggested a stronger association for fatal (4.3% decrease in risk) compared to non-fatal MI (1.8% decrease in risk). Further, the multivariable RR for deficient vs. sufficient 25(OH)D did not change appreciably when we included C-reactive protein (RR=2.04; 95% CI, 1.20–3.46) or included estimated glomerular filtration rate based on measured plasma creatinine, age, sex, and race (RR=2.09; 95% CI, 1.23–3.54) in the full multivariable model.
We tested for interaction across various factors to examine whether the association between 25(OH)D level and MI risk varied. We did not find statistical evidence of interaction by age (P for interaction=0.85), high blood pressure (P=0.84), BMI (P=0.87), aspirin use (P=0.92), physical activity (P=0.21), alcohol (P=0.53), LDL cholesterol (P=0.33), or triglycerides (P=0.73). Suggestive evidence was observed for an interaction by HDL cholesterol level, with a suggestive stronger inverse association with 25(OH)D among men with a higher HDL cholesterol level (P=0.06). Excluding men taking cholesterol-lowering drugs at baseline, the multivariable RR for deficient versus sufficient 25(OH)D was 2.30; 95% CI, 1.33–3.97. Because our subset included only a limited number of participants with diabetes at baseline, we were not able to calculate effect estimates within this subgroup; however, exclusion of these participants did not substantively alter the results as presented in Table 3. Similarly, exclusion of participants with a parental history of MI before age 60 years or current smokers did not affect the results.
In this cohort study, men with circulating 25(OH)D levels of at least 30 ng/mL had about half the risk of MI, independent of other CVD factors. The association was suggestively stronger for fatal CHD, but the number of cases was too small to make definitive conclusions. Although traditional CVD risk factors (e.g., lipids, hypertension, diabetes, smoking) remained strong risk factors in this population, vitamin D deficiency appeared to be an independent risk factor. Only 23% of the men in the HPFS had levels of 25(OH)D of at least 30 ng/mL. This percentage is typical of many populations, and the prevalence of deficiency is even higher in sub-populations, such as dark-skinned individuals and the elderly. Of note, individuals in sun-rich environments, where clothing or cultural practices do not appreciably limit vitamin D production, 25(OH)D levels of 54–90 ng/mL are attained,25 but from our study we cannot evaluate whether levels above 35 ng/mL would be associated with an even greater MI risk reduction.
The prospective design, high follow-up rate, and use of a plasma marker largely precluded major sources of bias, such as recall bias or selection bias. We excluded men with diagnosed CVD at baseline, and furthermore, results were similar after excluding the first two years of follow-up, arguing against reverse causation bias, which might occur if men predisposed to a MI stayed indoors and thereby avoided sun exposure. We controlled for and stratified by major covariates that could influence MI risk and influence 25(OH)D concentrations, including exercise, which increases sun exposure, BMI, region, race, multivitamin use and marine n-3s, as fatty fish is the only significant natural source of dietary vitamin D. We controlled for the major lipid risk factors of MI. In addition, we considered estimated glomerular filtration rate because circulating 25(OH)D levels could be lower in those with chronic kidney disease, which is a risk factor for CVD.26 Controlling for estimated glomerular filtration rate did not change our results. Given the strength of the association that we observed between 25(OH)D level and MI risk, and the fact that controlling for these factors did not appreciably influence the magnitude of the association, substantial residual confounding by these factors is not likely, but cannot be ruled out.
Because 25(OH)D levels are largely influenced by sun exposure, it is plausible that some other consequence of sun exposure other than vitamin D production is responsible for the observed association with MI. Nonetheless, a large body of evidence supports mechanisms whereby vitamin D could influence CVD risk. Among the potentially relevant mechanisms, vitamin D influences vascular smooth cell proliferation, inflammation, vascular calcification and blood pressure through the renin-angiotensin system.1
The renin-angiotensin system (RAS) helps regulate blood pressure, electrolyte and volume homeostasis, and excessive RAS stimulation is associated with hypertension. Animal studies show that vitamin D is an important regulator of the RAS system, and that 1,25(OH)2D, the activated form of vitamin D, suppresses renin gene expression.27 Disruption of the vitamin D receptor gene leads to elevated renin production, cardiac hypertrophy, and elevated blood pressure in mice.28 In an RCT of either UV-B or UV-A administered through tanning booths, UV-B, which increased 25(OH)D by 162%, was effective in reducing 24-h ambulatory blood pressure (by −6/−6 mmHg; P < 0.001) whereas UV-A radiation did not influence 25(OH)D levels or blood pressure.29 In another RCT of subjects with low vitamin D status (<20 ng/mL; mean approximately 10 ng/mL),30 supplementation with 800 IU vitamin D, resulted in an increase in serum 25(OH)D of 72% (P < 0.01), a decrease in systolic blood pressure (SBP) of 9.3% (P=0.02) and a suggestive decrease of diastolic blood pressure of 8.5% (P=0.10). In the HPFS and Nurses’ Health Study, during 4 years of follow-up, men and women who had plasma 25(OH)D levels <15 ng/mL were three times as likely to have a new diagnosis of hypertension within the next 4 years compared to those with 25(OH)D levels >30 ng/mL.31 An inverse association between 25(OH)D levels and blood pressure was also found using NHANES III data.32
Calcification is a common feature of atherosclerosis, and nearly all angiographically significant lesions are calcified.33 Calcification of coronary arteries has been associated with increased risk of MI34 and poorer 5-year survival.35 Atherosclerotic calcification is a process regulated in ways similar to skeletal osteogenesis.36 A significant association exists between osteoporosis and vascular calcification, suggesting that osteoregulatory mechanisms related to bone development may also influence calcification in the vasculature. 1,25(OH)2D levels have been shown to be inversely associated with vascular calcification,36 suggesting that vitamin D may influence MI risk through its effects on vascular calcification.
Other mechanisms could account for or contribute to the association between 25(OH)D and MI risk. Vitamin D deficiency, possibly in combination with low calcium intake, has been associated with impaired fasting glucose and possibly risk of type 2 diabetes,37–40 risk factors for CVD. Vitamin D deficiency has also been associated with a cytokine profile that favors greater inflammation (e.g. higher CRP, IL-6 and lower IL-10),41–46 which could predispose to heightened MI risk. Finally, seasonal respiratory infections, particularly influenza, have been proposed to account for the winter rise in mortality due to CVD,47 and hypovitaminosis D could contribute to these infections.48, 49
Two case-control studies2, 3 and a small prospective study4 found that individuals with low 25(OH)D levels were at higher risk of ischemic heart disease. The strongest test of the hypothesis that vitamin D lowers MI risk would be from an RCT. Two RCTs reported on CVD. In a UK study of 2,686 men and women, the subjects were randomized to 830 IU vitamin D per day (administered as 100,000 IU oral vitamin D3 every 4 months) or placebo for 5 years. The in-study 25(OH)D levels were 29.7 ng/mL in the vitamin D group and 21.4 ng/mL in the placebo group. There was a non-significant decrease in CVD incidence (RR=0.90, 95%CI 0.77–1.06) and CVD mortality (RR=0.84, 95% CI 0.65–1.10) in the intervention group. Based on our study, a difference of 8.3 ng/mL of 25(OH)D would be associated with a RR of 0.92, which is compatible with their results. A recent meta-analysis of total mortality as a secondary endpoint of RCTs with varying levels of vitamin D versus placebo controls found a statistically significant 8% reduction in risk of total mortality in subjects that had received vitamin D.50 Although the authors could not evaluate cause-specific mortality, the relatively immediate effect of a large enough magnitude to influence total mortality would suggest a benefit on CVD risk.
The largest RCT of vitamin D (and calcium) supplementation and CVD risk was from the Women’s Health Initiative, in which 36, 282 postmenopausal women received either calcium (1000 mg daily) and vitamin D3 (400 IU daily) or placebo.51 No reduction was observed in MI or CHD death (hazard ratio, 1.04; 95% CI, 0.92–1.18]. These results appear to be in contrast to our findings, suggesting two possible explanations. First, despite our efforts to exclude confounding, it is possible that uncontrolled or residual confounding explained our results. Alternatively, the range of vitamin D studied was much wider in the HPFS, which allowed us to detect an association. The difference between the medians of the top and bottom category, for which we observed a RR of about 2, was 23.5 ng/mL (35.5 – 12.0 ng/mL), and the calculated reduction in MI risk per increment of 1 ng/mL of 25(OH)D was 2%. In the WHI study, the influence of the treatment on the 25(OH)D levels was not reported, but based on the dose and compliance, is estimated to be only 2 ng/mL.52 Based on our data, such an increment would be expected to have only a 4% reduction in risk. Of note, to increase 25(OH)D levels from 12 to 35.5 ng/mL would require approximately 3000 IU daily of vitamin D.53 Although such intakes may seem high by current standards, an increasing body of evidence demonstrates no toxicity at intakes below 10,000 IU/day.54 Because current sources of vitamin D provide much less (e.g. a glass of milk has about 100 IU), those who achieve such high levels naturally do so largely through sun exposure.
Vitamin D deficiency has been related to an increasing number of conditions5 and to total mortality.50 These results provide further support of an important role of vitamin D on MI risk. If this association is causal, which remains to be established, the amount of vitamin D required for optimal benefit may be much higher than would be provided by current recommendations (200–600 IU per day), especially in those with minimal sun exposure. Thus, our findings add further support that the current dietary requirements of vitamin D need to be increased to have an effect on circulating 25(OH)D substantially large enough for potential health benefits.25
Funding/Support: The project described was supported by Grants P01CA055075 from the National Cancer Institute and R01 HL035464 from the National Heart, Lung and Blood Institute.
Role of the Sponsors: The sponsors had no role in the design and conduct of the study; collection, management, analyses, and interpretation of the data; or preparation, review, or approval of the manuscript. The content is solely the responsibility of the authors and should not be constituted to represent the official views of the National Cancer Institute or the National Institutes of Health.
Author Contributions: Dr. Giovannucci had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Giovannucci, Rimm. Acquisition of data: Giovannucci, Liu, Rimm. Analysis and interpretation of data: Giovannucci, Liu, Hollis, Rimm. Drafting of the manuscript: Giovannucci. Critical revision of the manuscript for important intellectual content: Giovannucci, Liu, Hollis, Rimm. Statistical analysis: Giovannucci, Liu. Obtained funding: Giovannucci, Rimm. Administrative, technical, and material support: Giovannucci. Study supervision: Giovannucci.
Financial Disclosures: Dr. Hollis is a consultant for Diasorin Corp. No other disclosures are reported.