Influence of folic acid on plasma homocysteine levels & arterial endothelial function in patients with unstable angina

Indian J Med Res 129, March 2009, pp 279-284 Influence of folic acid on plasma homocysteine levels & arterial endothelial function in patients with unstable angina Hangyuan Guo, Jufang Chi, Yangbo Xing & Ping Wang Department of Cardiology, Shaoxing People s Hospital, The First Affiliated Hospital of Shaoxing University, Zhejiang, China Received June 25, 2007 Background & objectives: High plasma homocysteine (Hcy) levels are known to be associated with coronary artery disease, but the precise level associated with an increased risk is yet controversial. Whether the beneficial effects of folic acid on arterial endothelial function persist over longer periods is not known. This study was carried out to assess whether folic acid supplementation could produce improvements in Hcy levels and arterial endothelial function in the patients with unstable angina (UA) and hyperhomocysteinaemia. Methods: The plasma Hcy levels of 52 cases with UA and 30 control subjects were measured by using high-performance liquid chromatography (HPLC) with fluorescence detection, plasma folic acid and vitamin B 12 levels were also measured. The patients with hyperhomocysteinaemia were treated with 5 mg of folic acid for 8 wk, and then rechecked the plasma levels of Hcy, folic acid and vitamin B 12 at the end of 4 th and 8 th wk. Arterial endothelial function was measured as flow-mediated dilation of the brachial artery using high-resolution B-mode ultrasound in 22 cases with UA and hyperhomocysteinaemia before and after folic acid treatment. Results: The plasma Hcy level was significant higher in the patients with UA than in the controls (19.2 ± 4.9 vs 10.7 ± 5.3 µmol/l, P<0.01). The plasma levels of folic acid and vitamin B 12 were significant lower in the patients with UA than in the controls. There were 22(42.3%) patients with hyperhomocysteinaemia in UA group. After 4 and 8 wk of administration of folic acid, the Hcy level reduced by 20.3 and 55.3 per cent in the UA patients with hyperhomocysteinaemia, respectively. Flow-mediated dilation also improved significantly, from 6.4 ± 1.9 to 9.0 ± 1.2 per cent (P<0.05) after 8 wk treatment with folic acid. Interpretation & conclusions: Plasma Hcy level was elevated in patients with UA. Folic acid can reduce the plasma Hcy levels and improve arterial endothelial function in the UA patients with hyperhomocysteinaemia. Key words Acute coronary syndrome - endothelial function - folic acid - homocysteine - unstable angina - vitamin B 12 Homocystinuria was first reported in 1962 1 and the relationship between homocysteine (Hcy) and vascular disease in 1969 2. Many independent risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia, smoking, gender and age, etc., are known to contribute to the development of coronary artery disease. Mild 279

280 INDIAN J MED RES, MARCH 2009 to moderate elevation of plasma Hcy concentration has also been recognized as carrying an increased risk for the development of atherosclerotic vascular disease 3. The link between hyperhomocystinaemia and coronary artery disease (CAD) is the deficiency of the cystathionine beta-synthase enzyme, which is deficient in homocystinuria 4. Alterations in the methylenetetra hydrofolate reductase enzyme, which is necessary for the metabolism of Hcy, may also play a significant role in hyperhomocystinaemia. Hyperhomocysteinaemia is thus an independent risk factor for atherosclerosis, and impaired arterial endothelial function is detectable in healthy adults with hyperhomocysteinaemia 3,5. High plasma Hcy levels are associated with coronary artery disease, but the precise level associated with an increased risk is yet controversial 6. Whether the beneficial effects of folic acid on arterial endothelial function persist over longer periods is not known. Our previous studies 7-9 showed that plasma Hcy level was elevated significantly in patients with 3 risk factors of coronary artery disease, and folic acid supplementation may be useful in reducing plasma Hcy level and improving arterial endothelial function in high risk patients with hyperhomocysteinaemia. We undertook this study to determine the plasma levels of Hcy and the presence of hyperhomocysteinaemia in the patients with unstable angina (UA), and to observe the effects of interventional therapy of folic acid on Hcy levels and arterial endothelial function in these patients. Material & Methods Study patients: Eighty two (52 inpatients & 30 outpatients) patients were randomly enrolled in Department of Cardiology at Shaoxing People s Hospital, Zhejiang, China from August, 2005 to February, 2007, and divided into 2 groups: 52 patients with UA (UA group) were recruited (26 males, 26 females, mean age 51.2 ± 14.1 yr) and 30 control subjects with no coronary artery disease (control group) were chosen (15 males, 15 females, average age 51.0 ± 11.1 yr). Age, gender, the history of present illness, the family history for CAD, smoking, hypertension and diabetes mellitus were carefully asked and recorded. Blood lipid profile and glucose levels were estimated. All patients underwent coronary angiography. The general characteristics of the study participants in the two groups were similar except that the number of subjects with hypertension (P<0.01) and diabetes (P<0.05) was significantly more in study group (Table I). Those taking any medicine before the study which would interfere the metabolism of plasma Hcy such as folic acid, Vitamin B 6 and B 12 were excluded, and patients having obvious renal dysfunction (creatinine>2.0 mg/dl), cerebral accidents (bleeding and infarction), anaemia (haemoglobin<13.5 g/dl in males or <12 g/dl in females), tumour and severe peripheral vascular disease were also excluded. This study was approved by the ethics committee of the institution. All patients in the UA group met the clinical criteria for the diagnosis of unstable angina and had electrocardiographic alterations compatible with that diagnosis. Inclusion criteria included patients with acute chest pain but without persistent ST-segment elevation (they had rather persistent or transient STsegment depression or T-wave inversion, flat T-waves, pseudonormalization of T-waves, or no ECG changes at presentation), and the measurement of troponins was negative. These patients were stratified for the risk of death or/and infarction into low, moderate, and high risk, according to Braunwald's criteria 10 : low risk, 26.9 per cent; moderate risk, 57.7 per cent; and high risk, 15.4 per cent. In the UA group, depression of the ST segment was observed in 10 (19.2%) patients, elevation of the ST segment in 7 (13.5%), inverted T in 27 (51.9%), and no alteration in the electrocardiogram in 8 (15.4%) patients. Among the 52 patients in UA group 47 (90.4%) were on aspirin, 35 (67.3%) on angiotensin converting enzyme (ACE) inhibitors, 28 (53.9%) on beta blockers, 19 (36.5%) on calcium antagonists, 10 (19.2%) on diuretics, and 46 (88.5%) on nitrates. Procedural protocol: The plasma Hcy, folic acid and Vitamin B 12 levels of 52 cases with UA and 30 Table I. General characteristics of the study participants in the two groups Age (yr) Sex (m:f) Hypertension (%) Diabetes (%) Hypercholesterolaemia (%) Smoking (%) Unstable angina (UA) group (n=52) 51.2±14.1 1:1 46.2** 28.8* 23.1 28.8 Control group (n=30) 51.0±11.1 1:1 23.3 16.7 20.0 30.0 Diabetes (positive past history of diabetes and new diabetics, fasting plasma glucose 120 mg/dl or two hours after glucose load 200 mg/dl); 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); hypercholesterolemia (>200mg/dl); smoking ( 400 cigarettes/year). P*<0.005, **<0.001 compared to control group

GUO et al: EFFECTS OF FOLIC ACID ON HOMOCYSTEINE & ET IN UA PATIENTS 281 control subjects were measured. The patients with hyperhomocysteinaemia (UA group: 22; control group: 4) were treated with 5mg of folic acid for 8 wk, and then rechecked the plasma levels of Hcy, folic acid and Vitamin B 12 at the end of 4 th and 8 th wk. Arterial endothelial function was measured as flowmediated dilation of the brachial artery using highresolution B-mode ultrasound in 22 cases with UA and hyperhomocysteinaemia before and after treatment. Measurement of plasma levels of Hcy, folic acid and Vitamin B 12 : Fasting blood (2ml) was drawn to EDTA-Na anti-thrombosis test tubes, Plasma was separated by centrifugation at 500 g for 10 min at 4 C within 30 min after drawing, and stored at -70 C until analysis. Plasma Hcy was measured according to the method of Ubbink et al 11 using high-performance liquid chromatography (HPLC, Waters600, USA) with fluorescence detection (420-AC) at 390 nm, launch wavelength was 515 nm, temperature 45 O C, volume 20 µl. The reagents were supported by Bio-Rad company in USA. Another 3 ml blood was drawn to dry tubes for measuring the plasma levels of folic acid and Vitamin B 12. Homocysteine was measured by immunoassay (DPC dual-count solid-phase no-boil kits, Diagnosyics products corporation, Los Angeles, California, USA) 3. The normal fasting plasma level of Hcy is 5-15 µmol/l, >15 µmol/l is considered as hyperhomocysteinaemia. At plasma Hcy concentration of 7.41 µmol/l the inter- and intra-assay coefficients of variation (CV) were 5.1and 2.3 per cent, respectively. Interventional therapy of folic acid: The patients with hyperhomocysteinaemia in the two groups were given folic acid (5 mg/day) for 8 wk. Then plasma Hcy, vitamin B 12 and folic acid levels were rechecked at the end of 4 th and 8 th wk. Vascular reactivity: Endothelial function of the brachial artery was measured using high-resolution ultrasound 12 in 22 patients with UA and hyperhomocysteinaemia. The diameter of the brachial artery was measured with B-mode ultrasound images (Acuson Computer Sonography 128 P/10C, USA). Endothelial responses of the brachial artery measured by this method correlated with coronary endothelial function. Measurements of vascular reactivity were performed by the same investigator who was blinded to the timing of the vascular examination and the phase of study protocol. Statistical analysis: The changes in various parameters were compared using a Student t-test both paired and unpaired. All statistics were carried out in IBM computer with version 15 SPSS software. P<0.05 was considered as significant. Results The Hcy concentration in the UA group was significantly (P<0.01) higher than in the control group, while the concentrations of folic acid and Vitamin B 12 were significantly lower (P<0.05) (Table II). All coronary risk factors including male sex, old age, cigarette smoking, diabetes mellitus, high blood pressure and high cholesterol level were related to the elevated Hcy levels (Table III). After interventional therapy of 5mg/day folic acid in the 22 UA patients with hyperhomocysteinaemia for 8 wk, the changes of homocysteine and folic acid Table II. Changes of Hcy, folic acid and vitamin B 12 levels in the two groups Control group (n=30) UA group (n=52) Hcy (µmol/l) Folic acid (µg/l) Vitamin B 12 (ng/l) 10.7 ± 5.3 7.0 ± 2.5 586.7 ± 164.5 19.2 ± 4.9** 5.1 ± 2.0* 471.6 ± 135.4* Values are mean±sd Compared with control group: *P<0.05, **P<0.01 Table III. The relationship between homocysteine levels and classical coronary risk factors in UA group Coronary risk factors (n) Homocysteine P (µmol/l) Sex Male (26) 21.3 ± 4.1 <0.05 Female (26) 17.1 ± 5.3 Age (yr) >60 (18) 20.8 ± 6.6 <0.05 <60 (34) 17.6 ± 4.8 Hypertension Positive (24) 26.9 ± 5.0 <0.01 Negative (28) 11.5 ± 4.7 Hypercholesterolaemia Positive (12) 21.6 ± 7.3 <0.05 Negative (40) 16.8 ± 3.8 Diabetes Positive (15) 27.5 ± 6.4 <0.01 Negative (37) 10.9 ± 4.2 Smoking Positive (15) 25.3 ± 5.4 <0.01 Negative (37) 13.1 ± 3.9

282 INDIAN J MED RES, MARCH 2009 Table IV. Changes of Hcy and folic acid levels after therapy of folic acid in patients with UA and hyperhomocysteinaemia (n=22) Hcy (µmol/l) Folic acid (µg/l) Vit B 12 (ng/l) Cholesterol (mmol/l) Before therapy 23.7 ± 9.5 4.1 ± 1.5 430.6 ± 97.8 6.0 ± 0.7 4 th weekend 18.9 ± 7.9* 11.2 ± 5.0** 441.3 ± 109.4 6.1 ± 1.3 8 th weekend 10.6±6.4 ** 16.7±4.8 ** 422.5±117.0 6.3±1.1 Compared with before therapy: *P<0.05;**P<0.01; compared with 4 th weekend: P<0.05; P<0.01 levels are shown in Table 5. All 22 subjects with UA and hyperhomocysteinaemia completed the 8 wk period with folic acid supplementation. No obvious adverse events were encountered. Four subjects (13.6%) had no substantial changes in homocysteine levels despite an increase in their folic levels. There were no significant changes in cholesterol, or vitamin B 12 levels (Table IV). Eight weeks of folic acid supplementation led to a significant improvement in endothelium-dependent dilation, from 6.4 ± 1.9 to 9.0 ± 1.2 per cent (P<0.05). In adjusted analyses, there was no significant correlation between improvement in arterial endothelial function and changes in folic acid or homocysteine levels. Discussion Circulating homocysteine can be increased by genetic deficiency of enzymatic pathways involved in its catabolism as well as by environmental factors including nutritional deficiencies, lifestyle, physiological conditions, drugs and some diseases, which mainly induce deficiency of folic acid, vitamin B 12 and B 6. Therefore, plasma homocysteine can be reduced by interventional therapy with folic acid and vitamin B 12 13. Hyperhomocysteinaemia is an independent risk factor of CAD 1,3. Hyperhomocysteinaemia may cause injury to the endodermis of the vessels, activate the platelets, improve the congregation of the platelets, enhance the production of fibrinogen, and promote migration and proliferation of smooth muscle cells. Hcy can also activate protein kinase C, promote the expression of c-fos and c-myb genes in vascular smooth muscle cells 3,14-17. Long term folic acid treatment improves arterial endothelial function and has potential implications for the prevention of atherosclerosis in adults with hyperhomocysteinaemia 18. The first prospective randomized placebo-controlled intervention study suggested that coronary endothelial function improves after treatment with folic acid and cobalamin 19. Folic acid significantly improves endothelial function in otherwise healthy cigarette smokers and during pregnancy 20-22. Hcy exerts atherogenic effects by enhancing chemokine responses in cells involved in atherogenesis and folic acid supplementation may downregulate these inflammatory responses 23. Folic acid supplementation to hyperhomocysteinaemic subjects resulted in a decrease in total blood homocysteine concentrations; moreover, there was a tendency to reverse the coagulation status and oxidative stress 24. Many studies did not show the relation between plasma Hcy levels, nonfatal myocardial infarction, and coronary artery disease 25-27. Their data do not support the hypothesis that high plasma Hcy levels increase the risk of coronary artery disease, suggesting another possible mechanism in which Hcy may be a predictor in a later stage of the disease. Elevated Hcy levels are shown to be associated with a higher risk of ischaemic myocardial injury in patients presenting with acute coronary syndrome 28. The serum Hcy level on hospital admission is an independent predictor of long-term survival in patients with acute coronary syndromes 29. Elevated total homocysteine levels on admission strongly predict late cardiac events in acute coronary syndromes 30. Our study showed that the plasma levels of Hcy in patients with UA were significant higher than controls. Folic acid supplementation reduced the level of plasma Hcy in the patients with hyperhomocysteinaemia, and was more effective at the end of 8 th wk than the 4 th weekend. Four patients showed no change in hyperhomocysteinaemia, despite supplementation with folic acid. This may be due to the lack of supplementation with vitamin B 12. It is also possible that folic acid itself might have direct antioxidant effects on the endothelium and therefore could directly improve endothelial function. Long-term folic acid treatment improves arterial endothelial function and has potential implications for the prevention of atherosclerosis in adults with hyperhomocysteinaemia 31. Folic acid significantly improves endothelial function in otherwise healthy cigarette smokers 32. Folic acid supplementation in hyperhomocysteinaemia was associated with decreasing levels of both endothalin-1 and intraplatelet

GUO et al: EFFECTS OF FOLIC ACID ON HOMOCYSTEINE & ET IN UA PATIENTS 283 cgmp, and the absence of an increase in the levels of the inflammatory mediator neopterin 33. However, available data indicate that hyperhomocysteinaemia could be an independent risk factor for the recurrence of cardiovascular events in patients with coronary artery disease and in high risk patients. Finally, the possibility that a reduction in plasma Hcy induced by interventional therapy can diminish the risk of cardiovascular events is under evaluation in several controlled longitudinal studies focusing mainly on primary prevention 3,17. A double-blind randomized controlled clinical trial would have been better. Another limitation was the small number of cases and also a very short follow up period of 8 wk. It will be interesting to see the longterm effect of folic acid supplementation on specially the arterial function in this group of patients. 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