Vitamin D Deficiency, Coronary Artery Disease, and Endothelial Dysfunction: Observations From a Coronary Angiographic Study in Indian Patients

Abstract: Background and Methods. Vitamin D deficiency has been linked to an increased risk of coronary artery disease (CAD) and cardiovascular (CV) death. Endothelial dysfunction plays an important role in pathogenesis of CAD and vitamin D deficiency is postulated to promote endothelial dysfunction. Despite rising trends of CAD in Asians, only limited data are available on the relationship between vitamin D, CAD, and endothelial dysfunction. Results. In a study of 100 patients undergoing coronary angiography, mean 25(OH)D level was 14.8 ± 9.1 ng/mL; vitamin D deficiency was present in 80% and only 7% had optimal 25(OH)D levels. Nearly one-third (36%) were severely deficient, with 25(OH)D levels <10 ng/mL. Those with vitamin D deficiency had significantly higher prevalence of double- or triple-vessel CAD (53% vs 38%), diffuse CAD (56% vs 34%), and higher number of coronary vessels involved as compared to those with higher 25(OH)D levels. Those with lower 25(OH)D levels had significantly lower brachial artery flow-mediated dilation (FMD; 4.57% vs 10.68%: P<.001) and significantly higher prevalence of impaired FMD (values <4.5%; 50.6% vs 7%; P<.002). A graded relationship between 25(OH)D levels and FMD was observed; impaired FMD was noted in 62.2%, 38.6%, and 13.3% in those with 25(OH)D levels <10 ng/mL, 10-20 ng/mL, and >20 ng/mL, respectively. Conclusion. Indian patients with angiographically documented CAD frequently have vitamin D deficiency. Patients with lower 25(OH)D levels had higher prevalence of double- or triple-vessel CAD and diffuse CAD. Endothelial dysfunction as assessed by brachial artery FMD was also more frequently observed in those with low 25(OH)D levels.

J INVASIVE CARDIOL 2012;24(8):385-389

Key words: vitamin D deficiency, coronary artery disease

_____________________________________________________

Though vitamin D has been traditionally associated with bone health, adequate levels are also important for optimal cardiovascular (CV) function. The quest to identify new predictors of CV disease has focused the attention on vitamin D, given its association with various established risk factors for CAD, including hypertension, diabetes, obesity, metabolic syndrome, congestive heart failure, and prevalent coronary artery disease.^1,2 Vitamin D deficiency has also been linked to increased risk of adverse CV events including higher risk of myocardial infarction, CV death, and overall mortality.^3-5 However, other studies have reported absence of a significant correlation of vitamin D with CVD and the overall epidemiologic evidence is inconclusive, necessitating further studies to corroborate a potential link between the two.^6-9

Endothelial dysfunction is an important antecedent event in the development of CHD and atherosclerosis.^10,11 Vitamin D is known to affect vascular endothelium directly or indirectly through up-regulation of the renin-angiotensin system or via induction of smooth muscle proliferation and a pro-inflammatory state.^12-14 Hence, it is important to study the relationship between vitamin D deficiency and endothelial dysfunction assessed by brachial artery flow mediated vasodilatation, given their role in the development of CVD.

Despite abundant sunshine, vitamin D deficiency is frequent among Indians.¹⁵ This is due to lack of vitamin D supplementation in the diet, malnutrition and high degree of coverage of the body with clothing, thus precluding adequate synthesis of the vitamin. Moreover, a darker skin pigmentation, as noted with Asians, requires greater degree of exposure to the sun to be able to synthesize equivalent amounts of vitamin D compared to people with lighter skin color.¹⁶ Despite the rising prevalence of CAD in Asian Indians, studies of vitamin D levels in patients with angiographically documented CAD in such patient populations are limited and have reported conflicting results.^17,18

We prospectively studied the prevalence of vitamin D deficiency in 100 consecutive patients undergoing coronary angiography at our institution for suspected CAD, and also tested the hypothesis of whether vascular endothelial function, assessed by endothelium-dependent dilation, is related to serum 25(OH)D status. 

Methods

A total of 100 patients undergoing coronary angiography for evaluation of CAD were included in this study, which conformed to the institutional ethical guidelines. 

Hypertension was defined as blood pressure ≥140/90 mm Hg or history of antihypertensive drug use, while diabetes was defined as fasting blood glucose level ≥100 mg/dL or history of oral hypoglycemic drug or insulin use. Coronary angiography was performed in the standard manner in all patients through either radial or femoral artery access. Angiographic CAD was defined as >50% of diameter stenosis in any of the major epicardial coronary arteries, while diffuse CAD was defined as involvement of >20 mm segment in a particular epicardial vessel.

Flow-mediated dilation (FMD) measurement. Endothelium-dependent brachial artery FMD was measured following reactive hyperemia as previously described.¹⁹ Using high-resolution ultrasound (7.0 MHz linear array transducer), measurements of the brachial artery were taken at rest and after cuff deflation completing suprasystolic compression (30 mm Hg above systolic BP for 4.5 minutes) of right upper arm. Scans of brachial artery were taken proximal to the bifurcation of the radial and the ulnar artery by the same ultrasound operator. Diameter measurements were taken from one media-adventitia interface to the other for at least 3 times at baseline and following hyperemia and mean reading was taken. Maximum flow velocity was measured in all subjects at rest and within 15 seconds of cuff deflation. Vasodilatation was then calculated as the percent change in diameter compared to baseline and impaired FMD was defined as values <4.5%.²⁰ Routine biochemistry including hemogram, lipid profile, blood sugar, renal function tests, serum calcium, and phosphorous analysis was performed in all patients. Serum 25(OH)D levels were measured by commercially available assay (DiaSorin Stillwater). The detection limit for 25(OH)D was 5 ng/mL, with a reported inter-assay variation coefficient of approximately 10%. Vitamin D deficiency was defined as 25(OH)D level of <20 ng/mL, vitamin D insufficiency as levels between 21 to 29 ng/mL, while optimal levels were defined as 25(OH)D >30 ng/mL.²¹

Statistical considerations. All data were analyzed using SPSS 16.0 statistical software (SPSS Inc). All data are expressed as mean ± standard deviation. Student t-test was used to compare means between the groups, and the chi-square test was used to compare proportions between the groups.

The Pearson correlation was used to analyze the correlations between impaired FMD and 25(OH)D levels. P<.05 was considered statistically significant.

Results

The basal demographics of the patient population are summarized in Table 1. The mean age was 56.7 ± 8.4 years (range, 37 to 75 years), 81% were males, hypertension was present in 46%, diabetes in 32%, and smoking in 31%. Chronic stable angina was present in 44% and a history of acute coronary syndrome was present in 56%. Coronary angiography revealed normal coronaries or insignificant CAD in 15%, while single-, double- and triple-vessel CAD was present in 34%, 21%, and 30%, respectively.

Vitamin D status and CAD. The mean 25(OH)D level of the patient population was 14.8 ± 9.1 ng/mL (range, 2.0 -49.8 ng/mL); vitamin D deficiency was present in 81%, with insufficiency in 14%, while only 7% had optimal levels. Severe vitamin D deficiency, ie, 25(OH)D levels <10 ng/mL, was observed in 37% of patients. There was a trend toward lower mean 25(OH)D levels with increasing severity of CAD, but this was not statistically significant, with 25(OH)D levels in patients with triple-vessel disease, double-vessel disease, and single-vessel disease of 12.9 ± 9.1 ng/mL, 14.9 ± 9.4 ng/mL, and 15.6 ± 10.6 ng/mL, respectively).

We did not find any significant correlation of 25(OH)D levels with age, gender, lipid levels, presence or absence of hypertension, diabetes, or smoking. However, an association between vitamin D levels and angiographic extent of CAD could be demonstrated. Among patients with triple-vessel disease on coronary angiography, 89% were vitamin D deficient; similarly, among patients with diffuse angiographic CAD, 84% had vitamin D deficiency. The relative risk of having triple-vessel disease on coronary angiography was 2.25 (95% confidence interval [CI], 0.75-6.17 in those with 25(OH)D levels <20 ng/mL).

Patients were assigned into risk groups based on presence or absence of risk factors including diabetes mellitus, hypertension, dyslipidemia, smoking, and age >55 years in males and >65 years in females (each given 1 point per risk factor). We observed that patients with scores >3 had significantly lower 25(OH)D levels as compared to those with scores ≤3.

Analysis of patients with 25(OH)D levels <20 ng/mL vs >20 ng/mL. On categorizing patients into two groups (those with vitamin D deficiency and those without; Table 2), there was no significant difference between the two groups in terms of age (56.1 ± 8.7 years vs 59.0 ± 6.9 years; P=NS), body mass index (26.6 ± 4.2 vs 25.1 ± 3.7; P=NS), prevalence of HT (46 vs 47%; P=NS), DM (36 vs 26%; P=NS), smoking (19% vs 20%; P=NS), mean lipid levels, or mode of clinical presentation of CAD. Patients with lower 25(OH)D levels expectedly had lower total and ionic calcium levels. Coronary angiography revealed that severe CAD (double- or triple-vessel CAD) was more frequent in those with 25(OH)D levels <20 ng/mL as compared to those with levels >20 ng/mL (53% vs 38%; P=.03). Diffuse CAD on coronary angiography was also more frequently present in those with lower 25(OH)D levels (56% vs 38%; P=.03). The mean number of coronary vessels involved was also higher in those with lower 25(OH)D levels (1.78 ± 0.76 vs 1.24 ± 0.43; P=.05).

Association between Vitamin D levels and impaired FMD. The mean brachial artery flow mediated vasodilatation of the overall patient population was 5.85 ± 5.3% and impaired FMD (<4.5%) was present in 43% of patients. Patients with 25(OH)D levels <20 ng/mL had significant endothelial dysfunction as compared to those with higher levels; mean brachial artery FMD values were significantly lower (4.57% vs 10.68%; P<.001) and overall impaired FMD (values <4.5%) was also significantly more common in them (50.6% vs 7.0%; P<.002). Categorizing the patients into three groups (group 1: 25(OH)D levels >20 ng/mL; group 2: 10-20 ng/mL; and group 3: <10 ng/mL) revealed significantly lower mean FMD values with progressively decreasing 25(OH)D levels. Mean FMD value was 10.68%, 5.44%, and 3.53%, respectively, for the three groups (P<.001 for group 1 vs groups 2 and 3; P<.01 for group 2 vs group 3) (Figure 1). The prevalence of impaired FMD was 13.3%, 38.6%, and 62.2%, respectively, in patients with 25(OH)D levels >20 ng/mL, 10-20 ng/mL, and <10 ng/mL; P=.01 for all; Figure 2) 

Impaired FMD (<4.5) was found to be negatively correlated with 25(OH) D levels, (r = -0.302; P=.01) both for absolute values of 25(OH)D as well as for patient classification based on 25(OH)D levels <10/10-20 and >20 ng/mL (r = -0.370; P=.01).

Discussion

In this study of Indian patients undergoing coronary angiography at a tertiary care hospital, the mean 25(OH)D level was 14.8 ± 9.1 ng/mL. Eighty percent of the patient population was categorized as deficient in vitamin D; an additional 13% had vitamin D insufficiency, while only 7% had normal vitamin D levels. The high prevalence of vitamin D deficiency in our patient population is reflective of the generalized high prevalence rates of hypovitaminosis D in India.^15,22,23 The high prevalence rates in our country despite its sunny climate and proximity to the equator are explained by the darker skin complexion of the population, generalized malnutrition, vegetarian food habits, inadequate sun exposure, and lack of vitamin D food fortification program.

Coronary angiographic findings in our study revealed that patients with vitamin D deficiency had higher frequency of double- or triple-vessel CAD (53% vs 38%), diffuse CAD (53% vs 38%), and higher mean number of coronary vessels involved (1.78 vs 1.24). Despite the more extensive pattern of angiographic CAD, the prevalence of risk factors like diabetes, hypertension, smoking, lipid profile, and mode of clinical presentation of CAD was not significantly different in patients with or without vitamin D deficiency.

Though vitamin D deficiency has previously been shown to be associated with established CV risk factors, higher cardiovascular death, and overall mortality, studies have been heterogenous, and only small numbers of longitudinal studies are available. Recent meta-analyses have been conflicting, with both positive and neutral associations reported.^24-26 Moreover, data on the spectrum and severity of vitamin D deficiency and extent of angiographically determined CAD are limited. Among patients undergoing coronary angiography in the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study cohort, although low levels of vitamin D were independently associated with heart failure, all-cause and CV mortality, the relationship between vitamin D levels and angiographic severity of CAD as demonstrated by us was not reported.^5,27 In patients with myocardial infarction undergoing coronary angiography, although Lee et al reported a high prevalence of vitamin D deficiency, the correlation of vitamin D status with angiographic CAD was not commented upon.²⁸

Previous studies of 25(OH)D levels in Indian patients with angiographically documented CAD have reported conflicting results. Though Shanker et al found that low vitamin D levels were associated with increased risk for CAD and patients in the lower vitamin D quartile had significantly higher risk for CAD, they did not find an association with severity of CAD.¹⁶ Rajasree et al reported paradoxically increased odds of ischemic heart disease among patients with 25(OH)D levels >89 ng/mL compared to those with lower levels, attributing it to high intake of regional/local foods rich in vitamin D, which were perhaps deleterious.¹⁷

Though we did not find any significant correlation of vitamin D levels with age, gender, lipid levels, presence or absence of hypertension, diabetes, or smoking, an association between vitamin D levels and angiographic extent of CAD was demonstrable. Patients with triple-vessel disease on coronary angiography had a high prevalence of vitamin D deficiency (89%); similarly, patients with diffuse angiographic CAD also had a high prevalence of vitamin D deficiency (84%). There was a trend toward lower mean 25(OH)D levels with increasing severity of CAD (in patients with single/double or triple-vessel CAD, respectively), but this was statistically not significant. Possibly, studies with larger numbers of patients would be able to demonstrate more robust and statistically significant associations. Despite the fact that association of vitamin D with CAD was independent of presence or absence of traditional cardiovascular risk factors, patients with more risk factors (>3) had significantly lower 25(OH)D levels as compared to those with <3 risk factors.

It is postulated that low levels of vitamin D might increase the CVD risk by promoting endothelial dysfunction. We observed that vitamin D deficiency was associated with significantly depressed vascular endothelial function as measured by brachial artery FMD. Mean FMD values were markedly reduced in patients with vitamin D deficiency (4.57% vs 10.68% in those with 25(OH)D levels <20 ng/mL or >20 ng/mL, respectively); impaired FMD (<4.5%) was also significantly more common in those with vitamin D deficiency (59.4% vs 7.7%). A graded relationship between 25(OH)D levels and FMD was observed; 13.3%, 38.6%, and 62.2% had impaired FMD in patients with 25(OH)D levels >20 ng/mL, 10-20 ng/mL, and <10 ng/mL, respectively. Lower FMD values in vitamin D deficiency have been reported in previous studies, but such studies have included either asymptomatic subjects, or those with diabetes or renal disease, and no data are available in patients with angiographically documented CAD, as reported in our study.^14,29,30 Effect of vitamin D on endothelial function is postulated to be either direct via modulation of calcium influx or by indirect mechanisms, including protection against oxidative stress and lipid peroxidation.^31,32 It has also been shown that vitamin D supplementation in deficient individuals is associated with improvements in parameters of vascular function, thus further strengthening its association with impaired FMD.^33-35 Given the postulated role of vitamin D in pathogenesis of CVD, it is important to study its effect on endothelial function in larger patient numbers, since this would provide a mechanistic insight into how vitamin D deficiency could lead to higher risk of development of CVD.

Study limitations. A limitation of the study is that all patients underwent 25(OH)D level assessment at the time of the coronary angiography procedure, which was from January-March 2011. These are predominantly winter months and may have affected the vitamin D levels due to coverage of the body with clothing, a practice which is common in our country. However, this would not be expected to affect the association between vitamin D deficiency and impaired FMD.

Conclusion

Our results add to existing evidence suggesting that low vitamin D levels may be an independent, potentially modifiable cardiovascular risk factor. We observed a high prevalence of hypovitaminosis D in Indian patients with angiographically documented CAD. Patients with lower levels of vitamin D had higher prevalence of severe (double- and triple-vessel CAD) and diffuse disease on coronary angiography, independent of established CV risk factors. Endothelial dysfunction as assessed by brachial artery FMD was also more frequently observed in those with 25(OH)D levels.

Acknowledgment. We would like to acknowledge the help of Mr PK Awasthi in analyzing the 25(OH)D levels.

References

1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357(3):266-281.
2. Zittermann A. Vitamin D and disease prevention with special reference to cardiovascular disease. Prog Biophys Mol Biol. 2006;92(1):39-48.
3. Wang TJ, Pencina MJ, Booth SL, et al. Vitamin D deficiency and risk of cardiovascular disease. Circulation. 2008;117(4):503-511.
4. Giovannucci E, Liu Y, Hollis BW, Rimm EB. 25-hydroxyvitamin D and risk of myocardial infarction in men: a prospective study. Arch Intern Med. 2008;168(11):1174-1180.
5. Dobnig H, Pilz S, Scharnagl H, et al. Independent association of low serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels with all-cause and cardiovascular mortality. Arch Intern Med. 2008;168(12):1340-1349.
6. Melamed ML, Michos ED, Post W, et al. 25-Hydroxyvitamin D levels and the risk of mortality in the general population. Arch Intern Med. 2008;168(15):1629.B-1637.B.
7. Kim DH, Sabour S, Sagar UN, Adams S, Whellan DJ. Prevalence of hypovitaminosis D in cardiovascular diseases (from the National Health and Nutrition Examination Survey 2001 to 2004). Am J Cardiol. 2008;102(11):1540-1544.
8. Marniemi J, Alanen E, Impivaara O, et al. Dietary and serum vitamins and minerals as predictors of myocardial infarction and stroke in elderly subjects. Nutr Metab Cardiovasc Dis. 2005;15(3):188-197.
9. Grandi NC, Breitling LP, Vossen CY, et al. Serum vitamin D and risk of secondary cardiovascular disease events in patients with stable coronary heart disease. Am Heart J. 2010;159(6):1044-1051.
10. Quyyumi AA. Prognostic value of endothelial function. Am J Cardiol. 2003;91(12A):19H-24H. 
11. Weber T, Auer J, O’Rourke MF, et al. Arterial stiffness, wave reflections, and the risk of coronary artery disease. Circulation. 2004;109(2):184-189.
12. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP. 1,25-dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest. 2002;110(2):229-238.
13. Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE. 1 alpha, 25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo. Circ Res. 2000;87(3):214-220.
14. Al Mheid I, Patel R, Murrow J, et al. Vitamin D status is associated with arterial stiffness and vascular dysfunction in healthy humans. J Am Coll Cardiol. 2011;58(2):186-192. 
15. Goswami R, Gupta N, Goswami D, Marwaha RK, Tandon N, Kochupillai N. Prevalence and significance of low 25-hydroxyvitamin D concentrations in healthy subjects in Delhi. Am J Clin Nutr. 2000;72(2):472-475.
16. Clemens TL, Adams JS, Henderson SL, Holick MF. Increased skin pigment reduces the capacity of skin to synthesise vitamin D3. Lancet. 1982;1(8263):74-76.
17. Shanker J, Maitra A, Arvind P, et al. Role of vitamin D levels and vitamin D receptor polymorphisms in relation to coronary artery disease: the Indian atherosclerosis research study. Coron Artery Dis. 2011;22(5):324-332.
18. Rajasree S, Rajpal K, Kartha CC, et al. Serum 25-hydroxyvitamin D3 levels are elevated in South Indian patients with ischemic heart disease. Eur J Epidemiol. 2001;17(6):567-571.
19. Corretti MC, Anderson TJ, Benjamin EJ, et al; International Brachial Artery Reactivity Task Force. 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. 2002;39(2):257-265. 

20. Schroeder S, Enderle MD, Ossen R, et al. Noninvasive determination of endothelium-mediated vasodilation as a screening test for coronary artery disease: pilot study to assess the predictive value in comparison with angina pectoris, exercise electrocardiography, and myocardial perfusion imaging. Am Heart J. 1999;138(4 Pt 1):731-739.
21. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr. 2006;84(1):18-28.
22. Arya V, Bhambri R, Godbole MM, Mithal A. Vitamin D status and its relationship with bone mineral density in healthy Asian Indians. Osteoporos Int. 2004;15(1):56-61.
23. Harinarayan CV, Ramalakshmi T, Prasad UV, et al. High prevalence of low dietary calcium, high phytate consumption, and vitamin D deficiency in healthy south Indians. Am J Clin Nutr. 2007;85(4):1062-1067. 
24. Elamin MB, Abu Elnour NO, Elamin KB, et al. Vitamin D and cardiovascular outcomes: a systematic review and meta-analysis. J Clin Endocrinol Metab. 2011;96(7):1931-1942. Epub 2011 Jun 15.
25. Autier P, Gandini S. Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials. Arch Intern Med. 2007;167(16):1730-1737. 
26. Grandi NC, Breitling LP, Brenner H. Vitamin D and cardiovascular disease: systematic review and meta-analysis of prospective studies. Prev Med. 2010;51(3-4):228-233.
27. Pilz S, März W, Wellnitz B, et al. Association of vitamin D deficiency with heart failure and sudden cardiac death in a large cross-sectional study of patients referred for coronary angiography. J Clin Endocrinol Metab. 2008;93(10):3927-3935. 
28. Lee JH, Gadi R, Spertus JA, Tang F, O’Keefe JH. Prevalence of vitamin D deficiency in patients with acute myocardial infarction. Am J Cardiol. 2011;107(11):1636-1638. Epub 2011 Mar 23.
29. Jablonski KL, Chonchol M, Pierce GL, Walker AE, Seals DR. 25-hydroxyvitamin D deficiency is associated with inflammation-linked vascular endothelial dysfunction in middle-aged and older adults. Hypertension. 2011;57(1):63-69. 
30. Andrade J, Er L, Ignaszewski A, Levin A. Exploration of association of 1,25-OH2D3 with augmentation index, a composite measure of arterial stiffness. Clin J Am Soc Nephrol. 2008;3:(6)1800-1806.
31. Wiseman H. Vitamin D is a membrane antioxidant. Ability to inhibit iron-dependent lipid peroxidation in liposomes compared to cholesterol, ergosterol and tamoxifen and relevance to anticancer action. FEBS Lett. 1993;326(1-3):285-288
32. Kuzmenko AI, Morozova RP, Nikolenko IA, Korniets GV, Kholodova YuD. Effects of vitamin D3 and ecdysterone on free-radical lipid peroxidation. Biochemistry (Mosc). 1997;62(6):609-612
33. Harris RA, Pedersen-White J, Guo DH, et al. Vitamin D3 supplementation for 16 weeks improves flow-mediated dilation in overweight African-American adults. Am J Hypertens. 2011;24(5):557-562. 
34. Sugden JA, Davies JI, Witham MD, Morris AD, Struthers AD. Vitamin D improves endothelial function in patients with type 2 diabetes mellitus and low vitamin D levels. Diabet Med. 2008;25(3):320-325.
35. Tarcin O, Yavuz DG, Ozben B, et al. Effect of vitamin D deficiency and replacement on endothelial function in asymptomatic subjects. J Clin Endocrinol Metab. 2009;94(10):4023-4030.

_____________________________________________________

From the Departments of ¹Cardiology and ²Endocrinology, Sanjay Gandhi PGIMS, Lucknow, India.
Disclosure: The authors have completed and returned the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors report no conflicts of interest regarding the content herein.
Manuscript submitted February 22, 2012 and accepted March 20, 2012.
Address for correspondence: Dr Aditya Kapoor, Professor of Cardiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Luck now 226014, India. Email: akapoor65@gmail.com