Hyperhomocysteinemia is considered a possible risk factor for atherogenesis. The aim of this study was to establish whether lowering of blood homocysteine levels by folic acid treatment in hyperhomocysteinemic subjects is accompanied by positive changes in their coagulation and oxidative status.
Parallel-group observation with the same design was used in the two types of individuals with hyperhomocysteinemia: one group included patients with symptomatic atherosclerosis manifesting itself as peripheral vascular disease (PVD); the other group included elderly subjects still asymptomatic with respect to atherosclerosis. The inclusion criterion in both study groups was a blood homocysteine level >20 μM (normal range, 10–15 μM). The study compared the pre- and posttreatment values of identical parameters in either group. Subjects' characteristics are summarized in Table 1.
In the patient group (n = 33), the inclusion criterion was the presence of PVD. The diagnosis of arterial occlusion was based on the intra-arterial radiographic contrast angiogram. Sixteen patients were treated by bypass surgery and eight by angioplasty, and nine patients were treated conservatively because surgical reconstruction was not feasible. Associated diseases, as diagnosed in these patients, are listed in Table 1. Renal insufficiency was an exclusion criterion. The following concomitant medication was administered: anti-hypertensive agents in 22 patients (diuretic in 10, β-blockers in six, calcium channel blocker in 11, angiotensin-converting enzyme inhibitors in 18, and imidazoline receptor antagonists in four patients); 26 patients were on acetylsalicylic acid, and two were on ticlopidine. Warfarin was given to five patients, vasodilators to 33 patients (pentoxifylline in 16, naftydrofuryl in 17), and hypolipidemic agents to 17 patients (fenofibrates in 12, simvastatin in five). Their therapeutic protocols were not changed during the study period.
Subjects included in the second study group (n = 26) were elderly individuals selected from the general population of the city of Plzeň recruited using a nonmedicinal database. Hyperhomocysteinemic subjects without other abnormalities were identified during an epidemiologic survey aimed at establishing the cardiovascular risk profile of the urban population. Enrollment criteria were absence of a history of a cardiovascular event and absence of symptoms of atherosclerotic disease. Those enrolled did not report lower extremity claudication or drug treatment of cardiovascular or cerebrovascular disease. The only exceptions were a history of an atrial fibrillation episode in one and mild hypertension treated with hydrochlorothiazide (12.5 mg daily) in another individual. Hyperlipoproteinemia was identified during screening in four individuals. In all other subjects, electrocardiographic, blood pressure, and laboratory marker values were within the normal range.
After assigning the enrolled subjects to their respective study groups, and after obtaining informed consent from each participant, an 8-week period with recommended diet started. Instructions were given in verbal and written form by the investigator to reduce the dietary lipid content (consistent with the Recommendations of the Second Joint Task Force of European and Other Societies on Coronary Prevention) (1) and to include fresh fruit and vegetables in their everyday diet (adapted to local conditions). No special precautions were made to monitor compliance to dietary recommendations.
The study design was identical in the two groups. However, the data cannot be compared as those of a test group and a control group because the enrolled probands did not fulfill the criteria of their matched pairs. All the subjects received oral folic acid at a dose of 5 or 10 mg once daily for 3 months; the dose was assigned without knowing the subject's pretreatment homocysteine level. Among the symptomatic patients, 17 received the 5-mg dose and 16 had the 10-mg dose, whereas among the asymptomatic subjects, 19 received the lower dose and seven had the higher dose of folic acid. Blood samples were obtained after an overnight fast.
Total homocysteine was determined by high-performance liquid chromatography (HPLC) according to Fiskerstrand et al. (2) and te Poele-Pothoff et al. (3) with a minor modification. The detection limit was 0.5 μM. The coefficient of variation was <5%. HPLC analysis was performed using a Spectra Physics system (Mountain View, CA, U.S.A.) equipped with a SUPELCOSIL LC-18-DB, 58355-U 25 × 4.6 × 5 column (Bellefonte, PA, U.S.A.) and a fluorescence detector. The eluent was monitored using excitation at 385 nm, and emission was detected at 515 nm; the column flow rate was 1 ml/min. Homocysteine retention time was 4.3 min. The HPLC method was validated as usual. Intra- and interassay variations did not exceed 5%.
Folic acid in erythrocytes (Ery) was estimated using a Folate kit (Chicago, IL, U.S.A.) (ion capture technique, normal range 150–450 μg/l Ery).
Fibrinogen, plasminogen, and anti-thrombin were estimated as coagulation parameters using commercial kits manufactured by Chromogenix and Instrumental Laboratory (Milan, Italy). Normal values, as specified by the manufacturers, were 2–4 g/l for fibrinogen, 70–125% of plasminogen functional activity, and 80–120% for anti-thrombin.
Heparinized blood was used to determine oxidative stress markers. To estimate superoxide dismutase activity in erythrocytes (normal value, 1,100–1,500 U/g hemoglobin) and glutathione peroxidase in whole blood (normal range, 45–75 U/g Hb), kits made by Randox (Crumluin, U.K.) were used. Reduced glutathione in erythrocytes (normal range, 1.7–2.4 m M Ery) was measured using kits made by Oxis International S.A. (Bonnenie, Marie, France). The methods were adapted to a Hitachi 717 analyzer (Roche Analytical Systems, Mannheim, Germany).
The genetic polymorphism of methylenetetrahydrofolate reductase (MTHFR) was estimated by the polymerase chain reaction method. DNA isolation was made using Qiagen kits (Venlo, The Netherlands) after amplification followed by cleavage and electrophoretic detection of a specific DNA fragment (4). All subjects were tested for MTHFR genotypes: C/C (homozygote; having both alleles—i.e., cytidine as nucleotide) normal or C/T (heterozygote) or T/T (homozygote having both alleles—i.e., thymidine as nucleotide) mutated.
The measured parameters were expressed as median values or as mean values with corresponding standard deviation obtained in the corresponding groups. Statistical evaluation of differences between pre- and posttreatment values was made using the Wilcoxon nonparametric paired test. The differences in the parameters investigated according to the MTHFR genotype were tested by analysis of variance (ANOVA).
Patients with symptomatic atherosclerosis
In patients with symptomatic atherosclerosis (Table 2), the median value of baseline plasma homocysteine levels was 26.7 μM in the group of 33 patients before treatment, decreasing to 20.0 μM after 3-month folic acid administration (p < 0.0001). A decrease was observed in 26 patients and an increase in seven patients.
The median value of plasma folic acid levels was 210.0 μg/l Ery before treatment, increasing to 549.0 μg/l Ery after treatment.
Fibrinogen, plasminogen, and anti-thrombin were assessed as coagulation markers. All three indicators changed highly significantly (p < 0.0001); fibrinogen decreased approximately by 0.6 g/l whereas plasminogen and anti-thrombin increased (Fig. 1).
Changes in oxidative status after folic acid treatment took the form of a mild increase in superoxide dismutase enzyme activity and increases in glutathione content as well as in glutathione peroxidase enzyme activity. These changes were statistically significant and could be considered a shift toward diminished oxidative stress.
The average baseline values of total and low-density lipoprotein cholesterol and blood glucose concentrations were above the normal range, indicating disturbances in the lipid and glucose metabolism of patients enrolled in the study. No significant changes were seen after the 3-month folic acid treatment. Blood pressure remained within the normal range.
Elderly subjects asymptomatic for atherosclerosis
The same parameters as those used in the patient group were assessed for elderly subjects asymptomatic for atherosclerosis (Table 3).
The median value of plasma homocysteine was 24.4 μM (n = 26) before treatment, decreasing to 18.6 μM after the 3-month treatment with folic acid (p < 0.0001).
The median of plasma folic acid levels was 209.5 μg/l Ery before treatment; the concentration rose to 783.0 μg/l Ery after treatment. Fibrinogen levels decreased and plasminogen increased significantly (p < 0.0001) whereas changes in anti-thrombin levels did not reach statistical significance (Fig. 2).
The changes in oxidative status markers were less evident in this group than in the patient group. The increases in superoxide dismutase, glutathione, and glutathione peroxidase indicated a shift toward a reduced oxidative status.
The baseline parameters of lipid metabolism, i.e., the slightly increased total and low-density lipoprotein cholesterol, and almost normal triglyceride levels, were not substantially changed by folic acid treatment. No changes in blood glucose were observed; blood pressure also remained within the normal range.
Influence of methylenetetrahydrofolate reductase polymorphism
The observed changes in laboratory parameters were not dependent on the MTHFR genotype (Table 4) when tested in all the 56 subjects using ANOVA, except for fibrinogen, which was highest in subjects with the T/T genotype (p < 0.03).
Based on experimental data, homocysteine is thought to have a direct toxic effect on endothelial cells (5), a prothrombotic effect (6,7), and a smooth muscle cell proliferation–stimulating effect (8). PVD was associated with high homocysteine in the study of Van-den-Berg et al. (9). In our previous study, homocysteine concentrations >15 μM were noted in 94 of 150 PVD patients (10).
Elevated plasma homocysteine proved to be associated with increased cardiovascular risk and death (11,12) in several epidemiologic studies (13). In a study of 586 subjects with definitive coronary heart disease selected from the EuroAspire study, the 90th percentile of homocysteine cumulative distribution was 29.5 μM, whereas in 475 control subjects, homocysteine was 17.6 μM (14). Using 15 μM as the upper normal level, hyperhomocysteinemia was diagnosed in 22% of controls and in 49% of patients with overt coronary heart disease in the PILS II study (12).
The present study showed a decrease in homocysteine after folic acid treatment was accompanied by a decrease in the procoagulatory potential. In both patients and elderly asymptomatic probands, fibrinogen with a procoagulation potential decreased whereas, conversely, plasminogen with an anti-coagulatory potential increased. An increase in anti-thrombin was observed in atherosclerotic patients only. The results are consistent with those of Undas et al. (15), who demonstrated a fall in thrombin/anti-thrombin complexes after vitamin B6, B12, and folic acid supplementation.
Endothelial injury by free oxygen radicals has been considered a potential participating mechanism in diseases leading to atherosclerosis such as diabetes mellitus, hypercholesterolemia, and hypertension (16). Experimental data indicate that homocysteine induces endothelial cell injury facilitating cytotoxic oxygen radical production (17–19).
In our study, oxidative status markers were modified through folic acid treatment in the sense of lower oxidative stress. The effect was more pronounced in atherosclerotic patients than in subjects asymptomatic for atherosclerosis.
Superoxide dismutase and glutathione peroxidase are the key enzymes involved in intracellular anti-oxidative defense. An increase in their activities during folic acid supplementation could promote the capability of cells to metabolize free radicals. Lower consumption of anti-oxidant capacity resulted in an increase of glutathione. Data on oxidative status markers in the current study were also analyzed with regard to smoking status. No significant differences were found, although oxidative status markers were slightly higher in smokers both before and after treatment.
The daily dose of folic acid was 5 or 10 mg. The observed changes in the parameters monitored were independent of the folic acid dose administered. A possible explanation could be that the doses were unnecessarily high. Some studies reported much lower doses (about 1 mg a day) to be sufficient to normalize blood homocysteine concentrations (20,21). However, it is not known whether such a low dose still affects coagulation and oxidative status.
It is evident that concomitant treatment with fibrates elevates homocysteine levels (22). In our series, insignificantly higher initial homocysteine levels were observed in fenofibrate-treated patients. However, folic acid administration lowered homocysteine levels to levels almost the same as those seen in subjects not receiving this treatment.
No dependence was shown for the basal values of the parameters examined including homocysteine and their changes after the treatment with folic acid on MTHFR gene polymorphism, except for fibrinogen. External factors such as nutritional behavior were probably more important in determining blood homocysteine concentration than the individual genotype in our study participants. In conclusion, our study has shown that folic acid influences not only total plasma homocysteine levels but also coagulation and oxidative status.
The authors thank Mrs. B. Novakova and B. Vackova for their invaluable technical assistance.
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