Background
Homocysteine is a sulfhydryl containing amino acid produced by demethylation of an essential amino acid (methionine)
Metabolism of methionine/homocysteine [38]
Metabolism of methionine/homocysteine [38].
Except in cases of severe hyperhomocysteinemia (plasma total homocysteine (tHcy) > 100 μmol/lit), usually due to metabolic disorders of methyonine metabolism, mild or moderate cases of hyperhomocysteinemia (tHcy > 15 μmol/lit), prevalent in the general population, can be the result of dietary deficiency of factors such as vitamin B12 and folic acid
Meanwhile, we do not have sufficient data regarding the role of hyperhomocysteinemia in development of premature CAD and this question needs further investigations
In addition to CAD development, high plasma levels of homocysteine have a relation with the incidence of several neurologic disorders such as congenital neural tube defect, Alzheimer's disease and suspicious carcinogenic potentials
This study was performed to assess the role of homocysteine, folic acid and vitamin B12 (as probable causes of hyperhomocysteinemia) in the occurrence of premature CAD while considering other known risk factors of CAD.
Methods
This case-control analytical study was performed in a group of 294 participants (225 male and 69 female) including consecutive patients aged less than 45 years old, who underwent selective coronary angiography into two medical and educational centers in Tehran. From the 294 patients included in this study: 144 were referring angina (56 stable angina and 88 unstable angina); 41 had a previous myocardial infarction; 65 had a positive ergometric test and 44 had a positive scintigraphy test. The sample population was stratified in patients with CAD (127) and normal (167) groups. There were two sources for data collection. Some data were gathered by interview and filling a questionnaire covering all personal data and medical history. This questionnaire was designed based on standard Rose questionnaire for anginal pain, known CAD risk factors and confounding factors.
The second source of data was lab exams [performed for homocysteine, folic acid, vitamin B12, triglyceride, and cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL)] and the results of selective coronary angiography. We excluded patients with myocardial infarction in the last 6 weeks, those who had taken any medication containing vitamin B12 or folic acid and N-Acetyl Cysteine (NAC) within 6 weeks of enrollment, those suffering from hypothyroidism ,anemia (hemoglobin<12 g/dl) and creatinine levels equal or greater than 1.5 mg/dl plus any features of chronic renal failure. By applying these criteria, 69 participants (46 males and 23 females) were excluded from the study.
Laboratory analyses
Blood samples were withdrawn from patients after an overnight fast, and the plasma were put in a cooled container and immediately carried to the "Massoud" laboratory, where the plasma was separated within 1 hour of sampling by centrifugation (20 min, RT, at 2000 RPM) and aliquots were stored at -70°C until analysis. Plasma level of homocysteine was determined by high performance liquid chromatography (HPLC) method, coupled with fluorescence detector and plasma level of folic acid and vitamin B12 by Radio immunoassay (RIA). The serum level of lipids and lipoproteins were determined by calorimetric method. Folic acid levels less than 2.2 ng/ml and vitamin B12 levels less than 175 pg/ml were classified as folate and B12 deficiency, respectively.
Definitions of risk factors
The traditional or classical coronary risk factors include: (1) diabetes mellitus (positive past history of diabetes and new diabetics, fasting plasma glucose ≥ 120 mg/dl or two hours after glucose load ≥ 200 mg/dl); (2) hypertension (positive past history of hypertension and new hypertensives, systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, based on the average of two or more readings on two or more occasions after initial screening); (3) Hyperlipidemia (total cholesterol> 250 mg/dl, LDL > 160 mg/dl or if the patient was on lipid-lowering therapy); (4) smoking (Regularly using tobacco for the last 6 months); and (5) family history of CAD (one or more family member, including parents, brothers and sisters, with documented CAD).
Statistical analysis
After collection of crude necessary data, these data were statistically analyzed by statistical analysis system (SAS) software for windows 9.1. We used chi-square and t-test to compare qualitative variables and quantitative variables, respectively. Logistic regression analysis was used for detecting independent variables. The differences were considered significant if P value was less than 0.05.
Ethical consideration
The committee for medical ethics of the Tehran Heart Center approved the study. Each participant signed an informed consent form before completing the questionnaire.
Results
A total number of 225 participants were enrolled from which 179 (79.6%) were male and 46 (20.4%) were female. All participants were under 45 years old.
Considering results of coronary angiogram, 97 participants (43.1%) had premature coronary artery disease of which 86 patients (88.7%) were male and 11 (11.3%) were female ;128 participants (56.9%) were free of significant CAD of which 93 (72.7%) were male and 35 (27.3%) were female that stratified in control group. In patients with significant CAD; 32 (33%) had single vessel disease, 37 (38.2%) two vessel disease and 28 (28.8%) had three vessel disease.
The mean age of the study group was (40.1 ± 4.2) years in males and (39.4 ± 4.8) in females. No significant difference was noted between the patient and control group in this regard. Major CAD risk factors are compared between controls and patients in Table
Characteristics of study population
Risk factor
Case group
Control group
P value
Age (year ± SD)
41.01 ± 3.13
39.1 ± 4.94
0.05
Male gender, n (%)
86(88.7)
93(72.6)
0.01
Family history, n (%)
31(31.9)
34(26.5)
0.75
Hypertension, n (%)
24(24.7)
21(16.4)
0.18
Diabetes mellitus, n (%)
13(13.4)
3(0.02)
0.02
Smoking, n (%)
57(58.7)
57(44.5)
0.36
Hyperlipidemia, n (%)
39(40.2)
25(19.5)
0.01
Laboratory data
Triglyceride (mg/dl)
194.68 ± 8.5
177.21 ± 12.4
0.25
Cholesterol (mg/dl)
209.8 ± 4.7
189.34 ± 4.0
0.77
LDL (mg/dl)
132.1 ± 4.1
113.33 ± 2.9
0.16
HDL (mg/dl)
37.7 ± 0.83
41.25 ± 0.92
0.02
Homocysteine (μmol/L)
1.7 ± 19.3
0.9 ± 13.9
0.00
SD, standard deviation; LDL, low density lipoprotein; HDL, high density lipoprotein
The mean serum level of homocysteine was (16.6 ± 1.01 μmol/lit) in the study group and was significantly higher in males (17.9 ± 1.2 μmol/lit) compared to females (11.5 ± 0.9 μmol/lit) (P < 0.01). The mean plasma level of homocysteine in patients (19.3 ± 1.7 μmol/lit) was significantly higher than the control group (13.9 ± 0.9 μmol/lit) (P < 0.005). Considering the sex factor, a similar relationship was present in male participants (P < 0.01) but not in females (P = 0.87) (Table
Plasma homocysteine level in study population
Number (%)
homocysteine (μmol/L)
P value
Male
Control
93(52)
14.9 ± 1.2
0.01
Case
86(48)
20.3 ± 1.9
Female
Control
35(76)
11.5 ± 1.1
0.87
Case
11(24)
11.8 ± 1.3
In order to evaluate the risk of hyperhomocysteinemia for premature CAD in the context of other known CAD risk factors such as diabetes mellitus, hypertension, hyperlipidemia, lipoprotein disorders, positive family history and cigarette smoking, logistic regression test was performed. Results showed that the risk of premature CAD in those participants with a homocysteine plasma level of more than 15 μmol/lit was 2.4 times more than others (OR = 2.42, 95% CI: 1.28–4.56: P = 0.007) (Table
Multivariate logistic regression analysis for development of coronary artery disease
Risk factors
Odds ratio (OR)
Confidence interval 95% (CI)
P value
Male gender
3.05
1.28 – 7.27
0.012
Diabetes mellitus
4.91
1.18 – 20.4
0.029
Hyperlipidemia
2.29
1.16 – 4.53
0.017
HDL
2.05
1.09 – 3.84
0.026
Hyperhomocysteinemia
2.42
1.28 – 4.56
0.007
HDL, high density lipoprotein
Logistic regression test in males with and without hyperhomocysteinemia compared to total subjects showed similar results in effect of hyperhomocysteinemia on development of CAD (OR = 2.54, 95% CI: 1.23–5.22: P = 0.01) while in females there was no increased chance for premature CAD in individuals with hyperhomocysteinemia. However, prevalence of premature CAD was higher in females with hyperhomocysteinemia (above 15 μmol/lit) than who did not have hyperhomocysteinemia (42.9% versus 20.5%). The role of other known risk factors of CAD in increasing the risk of premature CAD is also demonstrated in Table
We studied the relation between hyperhomocysteinemia and plasma level of folic acid, vitamin B12, male gender, increasing age, cigarette smoking, diabetes mellitus and hypertension. Mean plasma level of folic acid in the study group was 6.33 ± 0.29 ng/ml (5.6 ± 0.2 ng/ml in males and 8.8 ± 0.7 ng/ml in females). Mean plasma level of vitamin B12 was 282.5 ± 9.1 pg/ml (283.8 ± 10.5 pg/ml in males and 277.5 ± 18.6 pg/ml in females). Therefore, 10.7% of the study group (24 participants) had folic acid deficiency (13.1% of male participants and 2% of females). Also 24.3% (55 participants) had vitamin B12 deficiency (26.6% of males and 16.3% of females).
Although generally speaking there is a reverse relationship between plasma level of homocysteine and folic acid level (Pearson correlation coefficient = -0.148, P = 0.02) or vitamin B12 (Pearson correlation coefficient = -0.22, P = 0.001), the logistic regression test for the study group showed that male sex (OR = 3.87, 95% CI : 1.57–9.50, P = 0.003) and vitamin B12 deficiency (OR = 2.06, 95% CI : 1.06–3.98 : P < 0.0001) were two factors that caused significant increase in homocysteine levels.
Other factors such as cigarette smoking, increasing age, diabetes mellitus and hypertension did not have any statistically significant role in hyperhomocysteinemia (tHcy>15 μmol/lit). Although the mean plasma level of homocysteine in cigarette smokers (17.5 ± 1.4 μmol/lit) was higher than non-smokers (14.9 ± 1.2 μmol/lit), this was not statistically significant (P = 0.16). Mean plasma level of homocysteine in hypertensive individuals was not significantly different from normotensives (16.2 ± 2.6 μmol/lit versus 16.3 ± 0.9 μmol/lit, P = 0.97). The differences in diabetic and non diabetic individuals were also non significant (13.6 ± 1.4 μmol/lit versus 16.4 ± 1.02 μmol/lit, P = 0.44). No significant difference in plasma homocysteine level was observed in either of age groups (above and bellow 35 years) (P = 0.1).
Discussion
In the present study, the prevalence of hyperhomocysteinemia in patients with premature CAD was 47.4%. American Heart Association (AHA) reported a prevalence of 12 to 47% in 1999 for patients with CAD
Pinto et al in Bellvitge hospital (Spain) also found a correlation between high plasma level of homocysteine and premature CAD (P < 0.001; OR = 3.2). In their study hyperhomocysteinemia was presented as a risk factor for premature CAD
Lolin et al (China) showed that in patients with premature CAD, plasma levels of homocysteine was significantly higher than normal participants (6.41 μmol/lit versus 5.35 μmol/lit, P = 0.042)
Although, in none of the above mentioned studies was there any difference between males and females in this regard, Foody et al (Cleveland – USA) showed that despite the presence of a correlation between hyperhomocysteinemia and premature CAD in males (OR = 1.93; P = 0.05) no such correlation was noted in females (OR = 1.06; P = 0.89) which resembles our findings
Based on the results of our study and other previous studies it seems that hyperhomocysteinemia is a risk factor for premature CAD in men, but not proven so in women. A Correlation between plasma levels of homocysteine, severity of CAD and number of involved vessels was studied and no significant correlation was found. Brilakis et al have also studied the correlation between plasma level of homocysteine and angiographic findings in patients with CAD and found no significant correlation
Plasma level of homocysteine in male participants was significantly higher than females (17.9 ± 1.2 μmol/lit versus 11.5 ± 5.9 μmol/lit, P = 0.01). Logistic regression analysis yeilded the same results, meaning male sex was an effective factor in plasma level of homocysteine in our study group (OR = 3.87, 95% CI: 1.57–9.5; P = 0.003). Like wise, Jucques et al(Boston- USA) showed that the mean plasma level of homocysteine in men was 11% more than women (P < 0.001). This difference could be having several explanations
In our study, the prevalence of hyperhomocysteinemia was higher than other populations. It could be due to geographical variations, racial and ethnic differences
Based on results of the present study, the prevalence of folic acid deficiency was 10.7% (13.1% in men and 2% in women) while vitamin B12 deficiency had a rate of 24.4% (26.6% in men and 16.7% in women). This deficiency (especially in vitamin B12) is considerable. Logistic regression tests demonstrate that vitamin B12 deficiency was an effective factor in hyperhomocysteinemia in our study group (OR = 2.06, 95% CI: 1.06–3.98; P < 0.0001) which resembles Jucques et al findings
The present study is one of the few studies in which the control group is chosen precisely and based on angiographic results, while in most of studies, lack of CAD symptoms and signs have been the inclusion criteria for the control group
Conclusion
We may conclude that hyperhomocysteinemia is an independent risk factor for CAD in young patients (bellow 45 years old) (especially in men) and vitamin B12 deficiency is a preventable cause of hyperhomocysteinemia.
Abbreviations
AHA: American Heart Association, CAD: Coronary Artery Disease, CBS: cystathionine-β-synthase, HDL: high density lipoprotein, HPLC: High performance liquid chromatography, LDL: low density lipoprotein, MTHFR: methylene tetrahydrofolate reductase, NAC: N-Acetyl Cysteine, OR: odds ratio, RIA: Radio immunoassay, SAS: statistical analysis system, tHcy: plasma total homocysteine
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
1) SS: principle investigator, has made substantial contributions to conception and design of the study
2) FF: performed angiography
3) MS: performed angiography
4) GD: performed angiography and has been involved in drafting
5) MM: has made substantial contributions to data acquisition and has been involved in drafting and interpretation of data
6) NF: data analysis and substantial contribution
7) SD: has been involved in drafting and interpretation of data
8) AK: has given final approval of the version to be published and revising it critically for important intellectual content
Declaration: All authors read and approved the final manuscript