Genetic polymorphisms involved in folate metabolism and concentrations of methylmalonic acid and folate on plasma homocysteine and risk of coronary artery disease

Objectives Alterations in the enzymes involved in homocysteine (Hcy) metabolism or vitamin deficiency could play a role in coronary artery disease (CAD) development. This study investigated the influence of MTHFR and MTR gene polymorphisms, plasma folate and MMA on Hcy concentrations and CAD development. MMA and folate concentrations were also investigated according to the polymorphisms. Methods Two hundred and eighty-three unrelated Caucasian individuals undergoing coronary angiography (175 with CAD and 108 non-CAD) were assessed in a case–control study. Plasma Hcy and MMA were measured by liquid chromatography/tandem mass spectrometry. Plasma folate was measured by competitive immunoassay. Dietary intake was evaluated using a nutritional questionnaire. Polymorphisms MTHFR and MTR were investigated by polymerase chain reaction (PCR) followed by enzyme digestion or allele-specific PCR. Results Hcy mean concentrations were higher in CAD patients compared to controls, but below statistical significance (P = 0.246). Increased MMA mean concentrations were frequently observed in the CAD group (P = 0.048). Individuals with MMA concentrations >0.5 μmol/l (vitamin B₁₂ deficiency) were found only in the CAD group (P = 0.004). A positive correlation between MMA and Hcy mean concentrations was observed in both groups, CAD (P = 0.001) and non-CAD (P = 0.020). MMA mean concentrations were significantly higher in patients with hyperhomocysteinemia in both groups, CAD and non-CAD (P = 0.0063 and P = 0.013, respectively). Folate mean concentration was significantly lower in carriers of the wild-type MTHFR 1298AA genotype (P = 0.010). Conclusion Our results suggest a correlation between the MTHFR A1298C polymorphism and plasma folate concentration. Vitamin B₁₂ deficiency, reflected by increased MMA concentration, is an important risk factor for the development both of hyperhomocysteinemia and CAD.     

Elevated levels of homocysteine in plasma as a risk factor for coronary artery disease

This study was performed to assess the significance of association between coronary artery disease (CAD) and circulating homocysteine concentrations. 100 consecutive CAD patients (78 men and 22 women, aged 31 to 79 years) qualified for PTCA were investigated. At the time of PTCA, the risk factors for CAD and plasma for homocysteine and vitamins were obtained. The controls were without clinical evidence of coronary artery disease and hypertension (90 men and 30 women aged 32 to 81 years). Homocysteine was assayed using ELISA test. Red cell folate and plasma vitamin B12 were assayed by immunofluoroscency (Delphia test). Homocysteine concentrations were higher in patients than in controls (13.61 +/- 4.5 vs 10.99 +/- 4.49 mumol/L, p < 0.001, adjusted for age). Male patients had nonsignificantly higher homocysteine levels than females (13.94 +/- 5.21 vs 11.46 +/- 5.16 mumol/L, p = 0.05, adjusted for age). Elevated homocysteine level--defined as one in the top fifth of the control distribution > or = 12.83 mumol/L--was seen in 46% of the patients compared with 20% of the control group (p = 0.001). The odds ratio (OR) for CAD in persons with elevated homocysteine level was 3.1 (95% Cl 1.6-5.8, p < 0.001, adjusted for age). The OR for CAD of 5 mumol/L increment in homocysteine level was 2.1 (95% Cl 1.4-3.1 p < 0.001, adjusted for age). After adjustment for conventional risk factors (age, smoking, hypertension, family history of CAD, hyperlipidemia), elevated homocysteine level remained independent risk factor for CAD (OR 2.88, 95% Cl 1.1-7.8, p < 0.05). We observed inverse correlation between homocysteine and folate level (r = -0.32, p = 0.005) and between homocysteine and vitamin B12 concentrations (r = -0.24, p = 0.03), especially in men. Patients with elevated homocysteine level had lower levels of folate (629.6 +/- 241.2 nmol/L vs 735.1 +/- 252.4 nmol/L, p < 0.05), and vitamin B12 (213.6 +/- 64.4 pmol/L vs 246.6 +/- 62.3 pmol/L, p < 0.05) than patients with normal level of homocysteine. Elevated plasma homocysteine level is a strong risk factor for coronary artery disease. A 5 mumol/L increment in total homocysteine level may be associated with twofold increase of risk for the disease.     

Relationship between total plasma homocysteine, polymorphisms of homocysteine metabolism related enzymes, risk factors and coronary artery disease in the Australian hospital-based population.

Modest elevations of circulating homocysteine are common in patients with vascular disease. We explored interrelations between total plasma homocysteine levels and mutations in genes for three key enzymes in methionine-homocysteine metabolism. Methyltetrahydrofolate reductase (MTHFR) 677C-->T, cystathionine beta synthase (CBS) 68-bp insertion at exon 8, and methionine synthase (MS) 2756A-->G were typed in 685 Australian caucasian patients aged < or =65 years with and without angiographically documented coronary artery disease (CAD). We also assessed associations between homocysteine levels and extracellular superoxide dismutase (EC-SOD) and other CAD risk factors. There were significant correlations between plasma total homocysteine, and EC-SOD (r = 0.170, p = 0.001 for men; r = 0.241, p = 0.003 for women) and LDL (r = 0.153, p = 0.001 for men; r = 0.132, p = 0.081 for women). Levels were also significantly higher among patients with unstable angina (15.30+/-0.44 micromol/l for men, 14.44+/-0.74 micromol/l for women) than those without angina (13.98+/-0.38 micromol/l for men, 13.41+/-0.98 micromol/l for women) or with stable angina (14.00+/-0.37 micromol/l for men, 12.88+/-0.71 micromol/l for women). There were no significant associations between the levels and the presence or severity of CAD. The mutant MTHFR homozygotes tended to have higher levels and those with the MS and CBS mutations tended to have lower levels. We conclude that there is a significant correlation between plasma homocysteine levels and EC-SOD suggesting that elevated homocysteine may exert oxidative stress and that levels are associated with unstable angina, but not the occurrence or extent of coronary stenosis. The contributions to total plasma homocysteine levels of the common mutations of genes coding for the enzymes controlling homocysteine metabolism are modest.     

Genetic determinants of plasma total homocysteine

Hyperhomocysteinemia (Hhcy) is an established risk factor for various pathologies including arterial vascular disease and venous thrombosis, congenital malformations and other pregnancy complications, and dementia. Homocysteine remethylation, transsulfuration, and export to the blood/extracellular compartment determine homocysteine concentrations. Any disturbance in these routes may lead to Hhcy and potentially increase risk of disease. In this report, we aim to review all known polymorphisms involved in homocysteine and B-vitamin metabolism that have been assessed for their effect on tHcy. In the last section, we summarize the polymorphisms, for which the obtained data provides evidence for their involvement in Hhcy at the population level, and discuss how to continue our search for genetic determinants of tHcy.     

Plasma total homocysteine levels in postmenopausal women with unstable coronary artery disease

An elevated plasma total homocysteine (tHcy) level is considered a risk factor for coronary artery disease (CAD), but the relationship between plasma tHcy and well-defined CAD in women is still unclear. Plasma tHcy concentrations and the covariates serum folate, vitamin B12, and creatinine were analysed in 157 angiographically examined postmenopausal women with unstable CAD and in 101 healthy controls. At coronary angiography, 16% had normal vessels and 84% had coronary atherosclerosis. Mean plasma tHcy concentration (micromol/l, 95% confidence interval) did not differ in patients compared to controls (13.1 (12.3-13.8) vs. 12.5 (11.6-13.5)) or in patients with or without coronary atherosclerosis (13.3 (12.4-14.1) vs. 12.0 (10.8-13.2)). A trend to an increasing plasma tHcy with increasing degree of coronary atherosclerosis was attenuated after adjustment for age and the previous mentioned covariates. Odds ratio for the risk of coronary artery disease and coronary atherosclerosis in hyperhomocysteinemic patients (> or =90th percentile in controls) was approximately 3. However, the confidence interval included unity in half of the groups and the significance was therefore difficult to judge. Receiver operating characteristics showed age to be the only variable with a significant discriminatory ability regarding the presence of coronary atherosclerosis (area 0.77). Mild hyperhomocysteinemia seems not to be related to the risk of unstable CAD in postmenopausal women. The trend towards higher plasma tHcy with increasing degree of coronary atherosclerosis may be a marker of the disease. In future studies adjustment for age and the other three covariates should be considered.     

Fibrinogen and homocysteine levels in coronary artery disease

Conventional risk factors like high serum cholesterol, smoking and hypertension do not explain all the mortality and morbidity due to coronary artery disease in Indian population. Novel factors like plasma fibrinogen and homocysteine have been currently recognised as independent risk factors for coronary artery disease. A case-control study was carried out to examine the role of plasma fibrinogen, homocysteine, lipid profile and anthropometric parameters in angiographically established coronary artery disease patients. The relationship between the biochemical and anthropometric parameters was also examined. Fifty-eight male patients in the age range of 35-60 years with angiographically established coronary artery disease and equal number of matched-controls were the subjects of this study. Cases with coronary artery disease had significantly higher waist-to-hip ratio, waist-to-thigh ratio, plasma fibrinogen and total cholesterol. Mean plasma total homocysteine levels were not significantly different between cases and controls. In Indian population, elevated plasma fibrinogen and abdominal obesity appear to be significantly associated with coronary artery disease.     

Plasma total homocysteine and cysteine levels as cardiovascular risk factors in coronary heart disease

OBJECTIVES: As an important risk factor for coronary atherosclerosis, elevated plasma total homocysteine (t-hcy) concentration has recently received greater attention than have conventional risk factors. Though less reactive than homocysteine, cysteine (cys) is the most abundant plasma thiol and may function as an extracellular regulating factor of thiol/disulfide exchange in order to maintain an adequate redox status. An increase in the total amount of this compound may be noxious depending on environmental conditions. In the present study, the aim was to investigate changes of plasma total cysteine, homocysteine and other determinants in different types of coronary heart disease. DESIGN AND METHODS: Plasma total homocysteine (t-hcy), cysteine (t-cys), cysteinylglycine (t-cysgly), folic acid, vitamin B(12), lipid parameters, total protein, albumin and creatinine levels were studied in plasma from 68 patients with coronary heart disease and 42 healthy controls. After reduction of disulfide bonds with tri-n-buthylphosphine, plasma total thiols were assayed using high performance liquid chromatography (HPLC) and fluorescence detection following derivatization of sulfhydryl groups with 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F). Other parameters were determined by using commercial kits. RESULTS: Plasma t-hcy and t-cys levels were higher in patients (P<0.0001) than in controls, but t-cysgly was unchanged. Hcy and cys levels were correlated with age in the whole study population (r=0.49, r=0.46, P<0.01). Plasma t-hcy positively correlated with plasma t-cys (r=0.53, P<0.01) and t-cysgly (r=0.49, P<0.01) in patients, and with plasma t-cys (r=0.57, P<0.01) in controls. Postmenopausal women had higher t-cys and t-hcy levels than premenopausal women among the controls (P<0.01). Folate and vitamin B(12) levels were similar in both patients and controls. Patients with vitamin B(12) levels below normal had higher plasma t-cys and t-cysgly levels (P<0.05). Interestingly, control subjects with lower vitamin B(12) levels had lower plasma t-hcy levels (P<0.05). Plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, total protein, albumin and creatinine levels in patients and controls were within the normal range, but only HDL-cholesterol levels in patients were lower than in controls (P<0.0001). Triglyceride and VLDL levels of patients were also higher than those of controls (P<0.0001). CONCLUSIONS: Higher plasma total cysteine levels are as important as higher plasma total homocysteine levels. Both parameters are intercorrelated and may act synergistically. To discern their respective roles in atherosclerotic disease, these aminothiol levels have to be considered together.     

氧化型低密度脂蛋白和同型半胱氨酸与冠状动脉病变的相关性研究

目的 通过研究冠状动脉粥样硬化性心脏病（冠心病）患者血浆氧化型低密度脂蛋白（oxidized low-densitylipoprotein cholesterol,OX-LDL-C）和同型半胱氨酸（homoeysteine,HCY）浓度与Gensini评分的相关性,探讨其对冠状动脉病变严重程度的预测价值.方法 选择疑似冠心病患者86例,据冠状动脉造影结果分为冠心病组（67例）及对照组（19例）,冠心病组根据Gensini评分分为轻度亚组（23例）、中度亚组（22例）和重度亚组（22例）.分析各组血浆OX-LDL-C和HCY浓度与Gensini评分的相关性.结果 （1）冠心病组与对照组血浆OX-LDL-C、HCY浓度比较,冠心病组较高,差异有统计学意义（P＜0.05）.（2） Gensini评分三个亚组间血浆OX-LDL-C和HCY浓度比较,差异有统计学意义（P＜0.05）;随着冠状动脉狭窄程度加重,血浆OX-LDL-C、HCY浓度有增高趋势,差异有统计学意义（P＜0.05）.（3） Gensini评分与血浆OX-LDL-C（r=0.805,P＜0.01）、HCY （r=0.700,P＜0.01）浓度呈正相关,且OX-LDL-C与Gensini评分相关性较HCY高;血浆OX-LDL-C与HCY浓度呈正相关（r=0.698,P＜0.01）.结论 血浆OX-LDL-C和HCY浓度与Gensini评分有关,血浆OX-LDL-C与HCY具有相关一致性,联合检测血浆OX-LDL-C和HCY浓度可更好地了解病情、指导治疗及判断预后.     

Increased plasma homocysteine and allantoin levels in coronary artery disease: possible link between homocysteine and uric acid oxidation

OBJECTIVE: Homocysteine increases the damage to the cardiovascular system in different ways, one of them is the formation of reactive oxygen species resulting from the auto-oxidation of homocysteine.At the same time, uric acid is one of the major antioxidants in the plasma and protects the cells towards increased ROS activity. In humans, allantoin is only formed from non-enzymatic oxidation of uric acid by free radicals. We aimed to determine the levels of homocysteine, uric acid and allontoin in patients with coronary artery diseases, and to evaluate the possible correlation between homocysteine and allantoin. METHODS AND RESULTS: Plasma total homocysteine, uric acid and allantoin levels of 50 patients with coronary artery diseases and 23 healthy controls were determined by HPLC methods. Commercial diagnostic kits were used for the determination of other biochemical parameters.We obtained higher homocysteine, uric acid and allantoin levels in patients than in controls (p < 0.0001). Homocysteine levels were positively correlated with uric acid (r = 0.435, p < 0.0001) and allantoin (r = 0.583, p < 0.0001) levels in the whole study population.This correlation was persistent between allantoin and homocysteine after adjustment of these parameters for age, sex and creatinine. We accepted 15.0 micromol/l as a cut-off value between normal and mildly elevated homocysteine levels for patients and controls. Twenty-five patients showed moderate hyperhomocysteinaemia. The mean allantoin and uric acid values of the moderate hyperhomocysteinaemic group were significantly higher than that of the group having lower homocysteine levels than this cut-off value (p < 0.0001 for allantoin, p < 0.02 for uric acid). CONCLUSION: Results imply that there is increased allantoin production resulting from uric acid oxidation by free radicals in hyperhomocysteinaemic patients with coronary artery disease. The possible significance of the relationship between homocysteine and allantoin warrants further study.     

The association of homocysteine and coronary artery disease

Hyperhomocysteinemia has been associated with increased risk of atherosclerosis and myocardial infarction by a number of prospective case-control studies. A variety of genetic mutations, nutritional deficiencies, disease states, and drugs can elevate homocysteine concentrations. Treatment with folic acid with or without B-complex vitamins effectively lowers homocysteine levels. Whether therapy corresponds with decreased risk of coronary events is unknown, but may be promising. This article reviews the biochemistry of homocysteine metabolism, pathogeneisis, and etiology of hyperhomocysteinemia, along with its association with coronary artery disease, screening, and treatment.     

冠心病患者血浆同型半胱氨酸水平及其意义

目的：观察冠心病(CHD)患者血浆同型半胱氨酸(Hcy)水平的变化。方法：选择 CHD 患者78例,测定其血浆 Hcy,并与冠状动脉造影结果进行对照分析。此外选非冠心病患者29例测定血浆 Hcy 作为对照。结果：CHD 患者的血浆 Hcy 水平较非冠心病者的血浆 Hcy 水平明显升高[(14.56±6.15)μmol/L：(9.89±3.98)μmol/L,P＜ 0.01]。急性心肌梗塞(AMI)、不稳定型心绞痛(UAP)患者的血浆 Hcy 水平皆较稳定型心绞痛(SAP)患者的明显升高[(16.69±7.20)μmol/L、(14.70±5.21)μmol/L vs,(11.59±4.51)μmol/L,P 分别＜0.01、0.05],AMI 患者与 UAP 患者的血浆 Hcy 水平无差异(P＞0.05)。3支血管病变较1支、2支血管病变患者血浆 Hcy 明显升高 [(18.14±7.14)μmol/L vs．(11.14±4.99)μmol/L、(13.43±3.51)μmol/L,P 皆＜0.01]。2支血管病变与1支血管病变患血浆 Hcy 无明显差别(P＞0.05)。血浆 Hcy 水平与冠状功脉病变积分呈轻度正相关(r=0.375,P＜ 0.05)。结论：血浆 Hcy 水平越高,提示其冠状动脉病变越重、病变范围越广。     

同型半胱氨酸水平与冠状动脉斑块易损性相关性的研究

目的:探讨血浆同型半胱氨酸水平(HCY)与冠状动脉斑块易损性的相关性。方法:入选2015年1月至2015年12月,我科首次接受冠状动脉内药物洗脱支架置入术治疗的的冠心病患者78例,对病变部位进行血管内超声检查(IVUS),根据其特征分为易损斑块组(49例)及稳定型斑块组(29例)。比较两组患者的一般资料及HCY水平并对冠状动脉病变部位IVUS图像进行定量和定性分析。结果:与稳定斑块组相比,易损斑块组HCY水平显著增加(P0.01);其病变处血管面积(EEMA)、斑块面积(PA)、斑块负荷(PB)、偏心指数(EI)、正性重构及脂质斑块均显著增加(P0.05);负性重构更多见于稳定斑块组(P0.01)。HCY与EEMA、PA、PB、EI、正性重构及脂质斑块呈正相关(P0.05)。结论:高同型半胱氨酸水平增加冠心病患者病变血管斑块的不稳定性,联合IVUS检查有助于对冠状动脉易损斑块的判断。     

Relation between plasma homocysteine, gene polymorphisms of homocysteine metabolism-related enzymes, and angiographically proven coronary artery disease

BackgroundHyperhomocyteinemia (HHcy) is a risk factor for coronary artery disease (CAD), and methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) polymorphisms may contribute to plasma total homocysteine (tHcy) variation. We investigated the association of polymorphisms 1298A→C in the MTHFR gene, 2756A→G in the MTR gene, and 66A→G in the MTRR gene with tHcy levels and with CAD in patients undergoing coronary angiography.MethodsCAD patients (n = 151) and control subjects (n = 79) were compared regarding the prevalence of the polymorphisms, risk factors, and biochemical parameters.ResultsThe mean tHcy concentration was significantly higher in CAD patients than in control subjects (P < 0.001). HHcy (tHcy ≥ 15 μmol/l) conferred an OR of CAD of 4.1 (95% CI 2.2–7.5, P < 0.001). In both cases and controls, smokers had a higher tHcy level than non-smokers and demonstrated a markedly increased risk for CAD (OR = 2.5, 95% CI 1.7–3.3, P < 0.001). The allele frequencies of the MTHFR 1298A→C, MTR 2756A→G, and MTRR 66A→G mutations were 36.7%, 15.7%, and 36.6%, respectively. The 1298C allele frequency was significantly higher in the CAD group than in controls (P < 0.05) and showed a significant association with CAD in heterozygote carriers. There was no statistically significant difference between cases and controls in the frequencies of the A2756G alleles/genotypes in the MTR gene and of the A66G alleles/genotypes in the MTRR gene. The contributions to tHcy levels of the three common mutations were statistically significant. The heterozygosity of the MTHFR 1298AC genotype, MTR 2756G allele, and MTRR 66G allele yielded an OR of 3.4, 2.0, and 2.1, respectively, for having HHcy.ConclusionWe suggest that HHcy confers a risk for CAD, and smokers with tHcy are at a greatly increased risk. Our finding supports an important role of the MTHFR gene in CAD and provides evidence of polygenic regulation of tHcy.     

Cystatin C as a determinant of fasting plasma total homocysteine levels in coronary artery disease patients with normal serum creatinine.

Serum creatinine, a surrogate for both renal function and homocysteine generation, is a determinant of fasting plasma total homocysteine levels in coronary artery disease (CAD) patients. We hypothesized that among stable-CAD patients with normal creatinine levels (ie, 0.2). Consistent with the impact of folic acid fortification of cereal grain flour in the general population, only 1 of the CAD subjects (0.6%) had a plasma folate level <3 ng/mL. We conclude that serum cystatin C levels may reflect subtle decreases in renal function that independently predict fasting total homocysteine levels among stable-CAD patients with normal serum creatinine.     

同型半胱氨酸与冠心病的相关性及其机制探讨

目的　探讨血浆同型半胱氨酸 (HCY)与冠心病 (CHD)的相关性、与CHD传统危险因素的关系及其致CHD的机制。方法　分别测定CHD患者 (10 5例 )及对照组 (32例 )血中HCY、内皮素(ET)、一氧化氮 (NO)、一氧化氮合酶 (NOS)水平及血脂各参数。结果　CHD组高同型半胱氨酸血症(HHCY)的发生率明显高于对照组 (49 5 %比 9 4 % ,P <0 0 1) ,CHD组HCY水平明显高于对照组[(15 2 9± 5 14 ) μmol L比 (10 6 6± 2 78) μmol L ,P <0 0 1]。多元回归分析显示HCY对CHD的相对危险度 (RR)为 1 397(95 %可信区间 :1 137～ 1 6 4 5 ,P <0 0 1) ,表明HCY为CHD的独立危险因素。HCY与年龄、甘油三酯有关 (P <0 0 5 )。HCY与ET呈正相关 (P <0 0 1) ,而与NO、NOS呈负相关 (P <0 0 1)。结论　HHCY是CHD的独立危险因素。HCY可能损伤血管内皮细胞 ,使血管内皮功能失调。     

A case-control study of plasma homocysteine levels in South Indians with and without coronary artery disease

BACKGROUND: An increased level of plasma homocysteine is being recognized as a new risk factor for coronary artery disease. Since there are not enough data about its importance in Indians with coronary artery disease, we aimed to assess the significance of plasma homocysteine as a coronary risk factor in South Indian patients. METHODS AND RESULTS: In a case-control study, fasting plasma homocysteine levels were estimated in 565 subjects, of whom 221 were cases and 344 were controls. Of the 221 clinically defined cases, 112 underwent coronary angiography while 107 of the 344 controls had angiographically proven normal coronary arteries. Ninety healthy volunteers from the community were also included as controls. Fluorescent polarization immunosorbent assay was used to measure plasma homocysteine levels. In 12 patients, this method was compared to high pressure liquid chromatography and was found to give comparable results. The mean plasma homocysteine level was 18.30 +/- 10.08 micromol/L in clinically defined cases and 18.04 +/- 10.69 micromol/L in controls. Similarly, in angiographicallyproven coronary arterydisease patients, the mean plasma homocysteine levelwas 18.49 +/- 10.04 micromol/L and in individuals with angiographically normal coronary arteries, it was 19.16 +/- 11.38 micromol/L. CONCLUSIONS: There is no statistically significant difference in plasma homocysteine levels between controls and cases with coronary artery disease. The mean plasma homocysteine levels in controls as assessed by fluorescent polarization immunosorbent assay in the present study population are higher as compared to other published reports.