High Homocysteine Levels are Closely Associated with the Categories of Coronary Artery Diseases as well as Low Status of Folic Acid and Vitamin B 12 Open Access

which Abstract Background: Homocysteine (Hcy) has been considered as an independent risk factor for coronary artery disease (CAD). Folic acid and vitamin B 12 are two vital regulators in Hcy metabolic process. We evaluated the associations between Hcy and the categories of CAD as well as the levels of folic acid and vitamin B 12 . Methods: Hcy, folic acid and vitamin B 12 from 292 CAD patients, including 73 acute myocardial infarction (AMI), 116 unstable angina pectoris (UAP), 103 stable angina pectoris (SAP), and 100 non-CAD controls were measured, and the data were analyzed by SPSS software. Results: Hcy concentrations of AMI patients were the highest, and UAP patients were second, and SAP patients were the third higher, which were significantly higher than controls (p<0.01). Compared to SAP patients, patients with AMI and UAP had higher Hcy levels with approximately average elevated (4-5) µmol/L, while SAP patients were more higher Hcy with approximately 8 µmol/L than controls. However, the levels of folic acid and vitamin B 12 had opposite results, whose levels in AMI group had the lowest, while in controls had the highest. There were strongly moderate negative correlations between Hcy and folic acid (r=-0.67, p<0.001) and vitamin B 12 (r=-0.56, p<0.001). Conclusions: The present study confirms that high Hcy levels are closely associated with the categories of CAD as well as low status of folic acid and vitamin B 12 .


Introduction
Coronary artery disease (CAD) is seriously to harm people's healthy disease in borth developed and developing countries, which was predominantly caused by atherosclerosis with endothelial dysfunction [1,2]. Despite best efforts, available therapies protect only 30-40% of individuals at risk, and no therapeutic cure is anticipated for those who currently suffer from the disease [3]. The endothelium is a single layer of cells lining all blood vessels. It plays an important role in many physiological functions, including the control of blood cell trafficking, vasomotor tone, vessel permeability, and hemostatic balance. Endothelial cells produce a wide variety of substances in response to various physical and chemical stimuli, including vasodilator substances, and vasoconstrictor substances [4]. Researches have confirmed that endothelial dysfunction plays an important role in all categories of CAD from stable angina pectoris (SAP) to unstable angina pectoris (UAP), and to acute myocardial infarction (AMI) [5]. Early warning and immediate risk stratification of patients with different categories of CAD is frequently a challenging task in the current.
A large number of studies have confirmed that elevated homocysteine (Hcy) has been associated with endothelial dysfunction of atherosclerosis owing to oxidative stress [6], endoplasmic reticulum stress [7], increased level of asymmetric dimethylarginine (ADMA) [8], involved inflammation [9,10]. Genetic or nutritional deficiencies in Hcy metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis [11]. Folic acid and vitamin B 12 play an important role in regulating the metabolic process of Hcy [12]. Current studies have shown that folic acid supplementation can significantly improve endothelial dysfunction in patients with CAD [13]. Vitamin B 12 deficiency and HHcy are related to cardiovascular risk factors in patients with coronary artery disease [14]. In present study, we evaluate the correlations between Hcy and folic acid as well as vitamin B 12 , and explore the associations of Hcy with the categories of CAD as well as the levels of folic acid and vitamin B 12 in patients with atherosclerotic CAD.

Materials and Methods
This study included 292 CAD patients (103 SAP, 116 UAP, 73 AMI) and 100 chest pain non-CAD patients confirmed by coronary angiography from the Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital. Patients with the following diseases were excluded from this study: cancer, liver diseases, renal insufficiency, blood diseases, hyperthyroidism, thyroid dysfunction, systemic lupus erythematosus, malnutrition, pregnant woman, and supplemented folic acid and vitamin B 12 . Participating subjects were explained their participation rights and written informed consent was obtained, and were asked about excessive drinking (yes or no, it was defined as yes at least once a week and drinking over 45 degrees of alcohol more than 200 mL) and smoking habits (yes or no, nonsmokers including never smoking and stop smoking more than one year).
Hypercholesterolemia and hypertriglyceridemia were defined as TC ≥ 6.22 mmol/L and TG ≥ 2.26 mmol/L, respectively. According to 2007 China Adult Dyslipidemia Prevention Guide. Diabetes mellitus was diagnosed when patients' fasting plasma glucose (FPG) ≥ 7.0 mmol/L Hypertension was diagnosed when patients' systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg. Overweight and obesity were defined as BMI (24.0-27.9) kg/m 2 , and ≥ 28 kg/m 2 , respectively, according to 2006 Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults.

Statistical Analysis
The data were analyzed by using the statistical package for social science SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). Normal distribution variables were expressed as mean ± SD, which of more than two samples were compared with the One-Way ANOVA, and the differences between two groups were compared by using the SNK-q test when p < 0.05. Categorical variables were expressed as percentage and compared by χ 2 -test. The correlation study between Hcy and folic acid as well as vitamin B 12 were performed on the measured data by using Pearson correlation a coefficient. A p-value < 0.05 was considered as significant.

Comparison of principal characteristics between high Hcy and normal-low Hcy levels in CAD patients
Compared to normal-low Hcy group, High Hcy group were characterized by smoking, Diabetes mellitus, hypercholesterolemia and hypertriglyceridemia. There were no significant differences in the ratio of elder age, male, excessive drinking, hypertension, BMI>24 kg/m 2 between high Hcy and normal-low Hcy groups. The comparison of principal characteristics between high Hcy and normal-low Hcy levels in CAD patients (Table 1).

Comparison of Hcy, folic acid and vitamin B 12 between CAD and non-CAD groups
AMI patients had the highest level of Hcy, and UAP patients were a little lower than AMI but were the second highest. Moreover, SAP patients had the third higher level of Hcy, whose were significantly higher than controls (p<0.01). However, the levels of folic acid and vitamin B 12 had opposite results, whose in AMI group had the lowest, while in controlled group had the highest. The comparison of Hcy, folic acid and vitamin B 12 between AMI, UAP, SAP groups and controls (

Prevalence of high Hcy, and low folic acid and vitamin B 12 in CAD patients
Approximately four-fifths of CAD patients (79.11%) had a prevalence of high Hcy. However, the levels of folic acid and vitamin B 12 in CAD patients were reduced, the prevalence of low folic acid and vitamin B 12 were 41.78% for folic acid, and 34.93% for vitamin B 12 , respectively. The prevalence of high Hcy, and low folic acid and vitamin B 12 in CAD patients (Figure 1). There was a significant trend toward an increase in the prevalence of high Hcy from SAP to AMI (χ 2 = 19.93, p < 0.01). The prevalence of high Hcy progressively increased from 66.02% in SAP group to 81.90% in UAP group, and to 93.15% in AMI group. Similarly, low folic acid and vitamin B 12 also had significant trend toward rise in the prevalence from SAP to AMI (χ 2 = 13.03 and χ 2 = 13.13, respectively, both p<0.01). The prevalence of low folic acid and vitamin B 12 progressively increased from 32.04% and 24.27% in SAP group to 39.66% and 34.48% in UAP group, and to 58.90% and 50.68% in AMI group, respectively.

Comparison of prevalence of low folic acid and vitamin B 12 between high Hcy group and normal-low Hcy group in CAD patient and the correlations between Hcy and folic acid and vitamin B 12
More than half of the CAD patients (51.08%) with high Hcy had low folic acid levels, 7 times higher than that (6.56%) in CAD patients with normal-low Hcy concentrations (χ 2 =39.33, p<0.001), and 41.99% CAD patients with high Hcy had low vitamin B 12 levels, 5 times higher than that (8.20%) in CAD patients with normal-low Hcy concentrations (χ 2 =24.25, p<0.001). There were strongly moderate negative correlations between Hcy and folic acid (r=-0.67, p<0.001) and vitamin B 12 (r=-0.56, p<0.001).

Discussion
Vascular endothelium has important regulatory functions in the cardiovascular system and a pivotal role in regulating blood flow, mediating vasodilatation, coagulation reactions, platelet activation, leukocyte adhesion, and vascular muscle function [15]. Current knowledge suggests that endothelial dysfunction, as an impairment of endothelium-dependent relaxation of blood vessels, occur as the initial event in the pathogenesis of atherosclerosis, which considered to be the initiating factor and the key point of cardiovascular disease [16,17]. A major mechanism of endothelial dysfunction appears to be related to decreased bioavailability of endothelium-derived nitric oxide (NO). NO as a most important mediator of endothelium-dependent relaxation, plays a key role in normal vascular physiology in preserving the vessel wall in a quiescent state by inhibition of inflammation, thrombosis, and cellular proliferation. This gas is a potent vasodilator to activate soluble guanylyl cyclase in vascular muscle, resulting in accumulation of cGMP and relaxation, which is produced from Larginine by the action of endothelial NO synthase (eNOS) in the presence of several cofactors [18].
During the last two decades, overwhelming clinical and epidemiological studies and animal experiments has been actively exploring the roles and mechanisms of Hcy in atherosclerosis with endothelial dysfunction [19,20]. HHcy may lead to increase of the level of ADMA which has been identified as a potential risk factor for atherosclerosis with endothelial dysfunction. Elevated ADMA can results in decreasing NO concentration and bioavailability [21]. Extensive experimental evidence, both in vitro and in vivo, indicates that Hcy is an independent risk factor for cardiovascular disease and elevated serum Hcy level is associated with CAD events [22][23][24]. Homocysteine Studies Collaboration research revealed that elevated approximately 3 µmol/L Hcy will increase about 10% risk of cardiovascular events [25]. Humphrey et al. [26] analyzed has also demonstrated that increased 5 µmol/L Hcy concentration will increase approximately 20% risk of CAD events. In present study, AMI patients had the highest level of Hcy with (23.44 ± 5.78) µmol/L, and UAP patients were a little lower than AMI but were the second highest with (22.62 ± 6.37) µmol/L, SAP patients had the third higher level of Hcy with (18.63 ± 6.73) µmol/L, which were significantly higher than that of controls with (10.81 ± 4.62) µmol/L (p < 0.01). Compared to SAP patients, patients with AMI and UAP had higher Hcy levels with approximately average elevated (4-5) µmol/L, while SAP patients were higher Hcy with approximately 8 µmol/L than controls. There was a significant trend toward an increase in the prevalence of HHcy from SAP to AMI (p<0.01). The prevalence of HHcy progressively increased from 66.02% in SAP group to 81.90% in UAP group, and to 93.15% in AMI group. The present study has provided the valuable evidence that high Hcy levels are closely associated with the categories of CAD. The more serious patients with CAD suffer, the more higher concentration their Hcy have.
Folic acid and vitamin B 12 as two vital regulators play an important role in regulating the metabolic process of Hcy [27,28]. Folic acid and vitamin B 12 deficiency would result in elevated level of Hcy [29,30]. In biological cells, Hcy is derived from methionine after its utilization as a methyl group donor in biological methylation reactions. However, approximately 50% Hcy is produced to remethylate back to methionine by the transmethylation of methionine, while 50% Hcy metabolize via transsulfuration to cystathionine [31]. In this cycle, methionine is activated by condensation with ATP to give the methyl donor, S-adenosylmethionine (SAM). SAM is transformed into Sadenosylhomocysteine (SAH) by donating its methyl group to the substrates of methylation reactions. Subsequently, SAH gives rise to Hcy in a reversible reaction that favors SAH over Hcy production [32]. Methyl-tetra-hydrofolic acid (MTHF) which derivate of folic acid provide methyl to remethylated of Hcy. Vitamin B 12 is agon of methionine synthetase that catalyzed this reaction and participate transfusion of methyl [19]. Folic acid deficiency will prevent remethylation of Hcy because of raw material deficiency. Moreover, Folic acid deficiency will also influence the production of MTHF through to affect activity of methylene tetrahydrofolate reductase (MTHFR) [33,34]. Vitamin B 12 deficiency will also result in HHcy because methyl of MTHF can't transfer, which influence production of methionine and regeneration of tetrahydro-folic acid (THF) [35].
Guo et al. [36] measured plasma levels of Hcy, folic acid and vitamin B 12 levels in 152 patients with > or = 3 risk factors of CAD and 136 patients with 1-2 risk factors and 48 individuals with no risk factors, found that folic acid supplementation may be useful in reducing plasma Hcy level in high risk patients with hyperhomocysteinaemia. Cheng et al. [37] investigated also demonstrated that supplement folic acid, vitamin B 12 in patients with HHcy could reduce Hcy levels. Our study revealed that the levels of folic acid and vitamin B 12 in AMI and in UAP patients were obviously lower compared to those in SAP and non-CAD patients (p<0.05). The prevalence of low folic acid and vitamin B 12 progressively increased from 32.04% and 24.27% in SAP group to 39.66% and 34.48% in UAP group, and to 58.90% and 50.68% in AMI group, respectively. 51.08% CAD patients with HHcy had low folic acid levels, 7 times higher than that (6.56%) in CAD patients with normal-low Hcy concentrations (p<0.001), and 41.99% CAD patients with HHcy had low vitamin B 12 levels, 5 times higher than that (8.20%) in CAD patients with normal-low Hcy concentrations (p<0.001). There were strongly moderate negative correlations between Hcy and folic acid (r=-0.67, p<0.001) and vitamin B 12 (r=-0.56, p<0.001). Our results confirmed that serum folic acid and vitamin B 12 influence Hcy metabolism as cosubstrate and cofactor, respectively, and low status of folic acid and vitamin B 12 are closely associated with High Hcy levels.

Limitations
Since present study was just an investigation that the association between Hcy levels and the categories of CAD, and the correlation of Hcy with folic acid and vitamin B 12 . The larger sample number of multicenter study and longer prospective investigation are necessary to further observe serum Hcy changes and incidence of adverse cardiovascular events by supplementation of folic acid and vitamin B 12 in CAD patients.

Conclusion
The present study confirmed that HHcy and traditional cardiovascular risk factors may be synergistically prompt the formation and development of atherosclerosis in CAD patients. High Hcy levels are closely associated with the categories of CAD as well as low status of folic acid and vitamin B 12 .