Role of Homocysteine, Vitamins B6, B12 and Folic Acid in Acute Myocardial Infarction Patients

*Institute of Laboratory Medicine & **Dept. of cardiology, K.G. Hospital and Post Graduate Medical Institute, ***Institute of Laboratory Medicine, Dept. of Clinical Biochemistry, Kovai Medical Center and Hospital (KMCH), [Coimbatore-641 014] ****Dept. of Biochemistry & Biotechnology, Faculty of Science, Annamalai University, [Annamalainagar-608002], Tamil Nadu, India

Abstract

Background: Homocysteine is a risk factor for cardiovascular disease. We evaluated the efficacy of homocysteine-lowering treatment with B vitamins for secondary prevention in patients who had had an acute myocardial infarction.
Methods: We investigated the possible correlation between deficiency of vitamins B6, B12 or folic acid and homocysteine in patients with acute myocardial infarction (AMI). A case control study was carried out involving 50 AMI patients and age matched 50 normal healthy subjects.
Results: Mean serum B12 concentration in AMI patients was found to be significantly lower than the mean for controls. Mean serum folate and PLP level in patients was also found to be lower than controls; however, the differences were not statistically significant. Mean plasma homocysteine level in AMI cases was higher than the mean level in controls. Compared to controls, there was significantly greater deficiency of folate, B12 and PLP in AMI patients.
Conclusion Substantial nutritional deficiencies of these three vitamins along with mild hyperhomocysteinemia, perhaps through interplay with the classical cardiovascular risk factors (highly prevalent in this population), could be further aggravating the risk of CAD in the population.

Folic acid, vitamin B6, vitamin B12, homocysteine, AMI

Introduction
Cardiovascular disease (CVD) is the leading cause of death in both industrialized and developing countries. Recent reports have indicated that Indian people belong to an ethnic group, which has the highest rates of coronary artery disease (CAD) (1). Moreover, the relative risk of CAD in South Asian men is highest at early ages and suggesting that both intrinsic and extrinsic factors contribute to the development of CAD in this population (2), (3).
A number of studies during the past few years have indicated a protective role of vitamins B6, B12 and folate against the development of CAD (4), (5), (6). More recent reports have shown an association between deficiency of these B-complex vitamins and hyperhomocysteinemia, a known risk factor for myocardial infarction (7), (8). This has focused attention on these B-complex vitamins and the important role they might play in protection against the development of CAD (9).

The objective of this study is to investigate whether or not our patients suffering from acute myocardial infarction (AMI) have lower levels of plasma B6, B12 or folate and homocysteine compared to healthy subjects. If deficiency of B-complex vitamins is found to exist in this population, then intervention in the form of supplementations might significantly reduce the incidence of CAD.

Material and Methods

Study Population
The populations of study consider two hundred subjects, divided in two groups. Hundred AMI patients (age 30–70 years) admitted to the Kovai Medical Centre and Hospital (KMCH) & K.G. Hospital and Post Graduate Medical Institute Tamil Nadu from January 2007 to February 2008 were included in this study. They were enrolled within the index admission after confirmation of diagnosis based on WHO criteria of clinical history suggestive of myocardial ischemia, ECG indications of myocardial damage, and elevation of biochemical markers (creatine kinase and creatine kinase-MB). All patients were assessed as having risk factors for CVD, such as diabetes mellitus, hypertension, obesity, hypercholesterolemia, smoking and a parental history of ischemic heart disease (IHD). Criteria for diabetes were set as an abnormal fasting blood glucose level >125 mg/dl at admission, or having taken hypoglycemic agents. All those with systolic blood pressure greater than 140 mmHg and/or diastolic blood pressure of 90 mmHg or those on regular anti-hypertensive medications were classified as hypertensive. Those having serum cholesterol level greater than 200 mg/dl were considered to have hypercholesterolemia. A body mass index (BMI) above 30 was classified as obese, and a parental history of IHD was considered positive if any of the parents had IHD at or below the age of 60 years. Subjects were considered smokers if they had been smoking cigarettes regularly (one or more per day). Those cases (as well as controls) that were pregnant, using anti-epileptics, taking oral contraceptives, having malabsorption syndrome, suffering from tuberculosis, liver disease, uremia, or cancer or using vitamin B-complex supplements during the past 6 months were not included.
Similarly, age and sex matched 100 normal healthy subjects were also investigated. They were also assessed for the above-mentioned risk factors. However, more stringent criteria were used for the selection of normal healthy control subjects. In addition to being matched for age, sex and socioeconomic background, they had no evidence of CAD, diabetes mellitus, hypertension, obesity or hypercholesterolemia. The objective was to have an assessment of the levels of folate, B12, B6 and homocysteine in a “normal healthy population� and compare their levels with those obtained from AMI patients.

Estimation of Biochemical Investigation
Biochemical investigation including blood glucose fasting, total cholesterol, triglycerides, HDL, LDL and CK levels were determined by fully automated clinical chemistry analyzer (Hitachi 912, Boehringer Mannheim, Germany). VLDL level was calculated according to Friedewald et al. (10) CK-MB mass were measured with highly specific monoclonal antibodies in a sensitive chemiluminescence assay, with an Elecsys 2010 instrument (Roche Diagnostics, Mannheim, Germany). Troponin I level was estimated using fully automated immunoassay analyzer (AXSYM-Abbott Laboratories, Abbott Park, USA).

Estimation of B12, folate, B6 and homocysteine in serum/plasma
Fasting venous blood was obtained from cases as well as controls. Serum samples were analyzed for folate and vitamin B12 using radio assays [11,12]. Plasma samples from both cases and controls were screened for pyridoxal phosphate (PLP; coenzymic form of vitamin B6) and homocysteine. Plasma PLP concentration was chosen as the standard for vitamin B6 status because this measure appears to reflect tissue stores Lui (13). For determination of PLP in plasma, a modification of the method by Camp et al. (14) as described previously was used Iqbal et al (15). Determination of plasma homocysteine was carried out using a kit based on fluorescence polarization immunoassay (Abbott Laboratories, Ltd., USA).

Statistical Analysis
All data were expressed as mean ± SD. The statistical significance was evaluated by Student’s t test using Statistical Package for the Social Sciences (SPSS Cary, NC, USA) version 10.0.

Results

Demographic data of control and AMI group are shown in (Table/Fig 1). The mean age limit was 47.5 ± 12.5 years in AMI patients and 43.7 ± 10.3 years in control subjects. The increased body mass index (BMI) in AMI patients (28.8 ± 4.2 kg/ m2) when compared to control subjects (23.2 ± 3.7 kg/ m2) was statistically significant. Blood Pressure systolic blood pressure was significantly high (p<0.05) in patients groups as compared with controls.

The comparison of biochemical changes in control and MI subjects are shown in (Table/Fig 2). As expected, the patients had significantly higher level of total cholesterol, triglyceride, LDL-cholesterol and VLDL-cholesterol when compare to control subjects, but HDL-cholesterol in AMI patients was significantly less than in normal healthy subjects. There was no statistical significant in fasting blood glucose. The significantly increases in the level of CK, CK-MB seen in MI patients when compare to control subjects.

(Table/Fig 3) shows the mean concentrations of serum folate, serum B12, plasma PLP and plasma homocysteine in normal healthy subjects (controls) and AMI patients. Analysis the mean serum B12 concentration in AMI patients was significantly less than in normal healthy subjects. Mean serum folate level in AMI patients was found to be lower than the mean level in normal healthy control subjects. Mean plasma PLP concentration in AMI patients was also found to be lower than the controls, however, the values were not significantly different. Mean homocysteine concentration in plasma of AMI patients was higher compared to controls (11.2±4.02 μmol/l vs 19.5 ±7.85 μmol/l).

Discussion

Significantly high percentages of folate, B6, and B12 deficiencies in AMI patients in our study population suggest a causal relationship between nutritional deficiency of these vitamins and development of CAD. The high prevalence of folate and B6 deficiencies in our control group was quite unexpected. With hardly any data available on deficiency of these two vitamins in the general population, our results indicate that folate and B6 deficiencies could be quite common. High prevalence of folate deficiency in our normal as well as patient populations could be due to our urban dietary habits, which include inadequate use of fresh fruit and vegetables and overcooking. There has been a considerable debate on the value of plasma PLP which should be taken as standard for adequate B6 status (16).

Although a mild hyperhomocysteinemia has been observed in our patients suffering from AMI, it does not appear to be an independent risk factor for development of CAD. The causes of hyperhomocysteinemia are multifactorial, but our preliminary data indicate that folate deficiency and B6 deficiency certainly appear to have an association with it. This could be due to the fact that most of the subjects in our cases and control populations belonged to the lower middle class, a group with a relatively modest socio-economic background and perhaps receiving somewhat poor and unbalanced nutrition (3).

A number of studies have shown increased plasma homocysteine levels in aged individuals (17), (18). Our observation of significantly lower levels of serum folate in smokers compared to nonsmokers in the normal control group, as well as in the AMI patients group, is consistent with the results reported by Mansoor et al. (19) who showed a significant decrease in both serum and erythrocyte folate in smokers. Lower intake of fruit and vegetables, free radicals in cigarette smoke, and increased excretion of folate might have contributed to a decline in body stores of folate (20). With low folate and low B12 concentrations in smokers, mean plasma homocysteine concentrations among smokers (both in controls as well as AMI patients), as expected, were found to be significantly higher than in nonsmokers. Nygard et al. (21) in a study involving nearly 16,000 subjects from a general community in Norway have also reported significantly increased levels of total homocysteine in smokers compared to nonsmokers.

On the basis of these observations, it can be suggested that very significant deficiencies of folate, B6 and B12 along with mild hyperhomocysteinemia could act synergistically in concert with other classical risk factors in the study- population, thereby further aggravating the risk of CAD.

Our study reports the prevalence of folate, B6 and B12 deficiency and its relationship with plasma homocysteine levels. Our data show substantial nutritional deficiency of all these three vitamins. Deficiencies of folate, B6 and B12 were even more pronounced in AMI patients, consistent with their important role in preventing the development of CAD. Any single nutritional intervention, such as supplementation with folate alone, may not be enough to protect against CAD in a population that has the highest rates of this disease compared to any other in the world. However, supplementations with all three vitamins would, perhaps, offer some protection. The association between B-complex vitamin deficiency, hyperhomocysteinemia, other classical risk factors and the risk of developing CAD merits our serious attention.