Association between low-dose folic acid supplementation and blood lipids concentrations in male and female subjects with atherosclerosis risk factors

Background

Folic acid (FA), also called folate or vitamin B9, is one of the B complex vitamins [1]. Preventive effects of FA on developmental birth defects, Alzheimer’s disease, and megaloblastic anemia have been confirmed [2,3]. Introduction of the folate food fortification program in North America resulted in coronary plaque regression through lowering of homocysteine (Hcy) [4], which is an atherosclerosis risk factor [5]. It is considered that even a slight FA deficiency promotes atherosclerosis lesions formation in arterial endothelium [6]. Deficiency of FA coexisting with hyperhomocysteinemia can be observed in healthy individuals and individuals with established cardiovascular disease [7]. However, the preventive effect of FA on cardiovascular disease remains unclear. Large-scale clinical trials failed to show a benefit from Hcy-lowering treatment in individuals with established cardiovascular disease or with history of myocardial infarction. However, those studies evaluated high-dose FA supplementation as secondary prevention and were not investigating low-dose FA supplementation as primary prevention of cardiovascular disease [8–10].

High levels of cholesterol (Ch) in the blood, especially the low-density lipoprotein cholesterol (LDL-C), have been linked to arterial damage and cardiovascular disease, because of atherosclerosis promotion [11]. Hcy activates 3-hydroxy-3-methylglutaryl coenzyme A reductase, which plays a key role in Ch biosynthesis [1]. FA, acting through reducing Hcy, may contribute to decreased Ch synthesis [13]. Therefore, folate could have a preventive effect on atherosclerosis development. Apolipoproteins are structural components of lipids and transport them through the lymphatic and circulatory systems. There are 6 classes of apolipoproteins, which differ in regard to biological function. Apolipoprotein AI (apoAI) is the major component of high-density lipoprotein (HDL); apolipoprotein B (apoB) binds with chylomicrons and LDL. Lipoprotein(a) (Lp(a)) is a lipoprotein subclass linked with atherosclerosis and is also responsible for clot generation, because of reducing plasmin generation. Numerous studies have identified Lp(a) as a risk factor for coronary heart disease, cerebrovascular disease, thrombosis, and stroke [14–16].

The aim of this study was to analyze the association of low-dose folic acid supplementation, used as primary prevention, with blood lipids concentrations in subjects with atherosclerosis risk factors.

Material and Methods

LABORATORY METHODS:

Fasting blood for biochemistry was collected. Blood tests comprised the following: measurements of FA and Hcy concentrations, lipid panel (total cholesterol, HDL-C, LDL-C, triglycerides [TG]), apoAI, apoB, and Lp(a). The FA level was determined by an Abbott test kit (Abbott Laboratories, Chicago, IL, USA) using the ion capture method on an IMX immunochemical analyser (Abbott). Total Hcy was determined by high performance liquid chromatography (HPLC) using test kits from Bio-Rad, on a Hewlett-Packard analyser with a fluorescence detector. TG and total cholesterol levels were determined by enzymatic methods. LDL-C and HDL-C were obtained using the precipitation method and cholesterol concentration in each fraction was measured. Apolipoproteins B and AI were measured using a photometric method using an antigen-antibody binding. Measurements were done using reagents from the commercial test kits from Roche, and a Clinilab analyser from bioMérieux. Lp(a) was measured using a photometric method using antigen-antibody binding on a latex carrier, with the use of test kits from Dialab and a Clinilab analyser.

STATISTICAL ANALYSIS:

Statistical analysis was performed by the STATISTICA StatSoft Polska v.9.0 package (StatSoft Inc., Tulsa, OK, USA) and the examined parameters were first evaluated for normal distribution (Shapiro-Wilk test). The paired t test was used for the comparison of mean values of measured parameters after structuring subjects in the study by sex. The results obtained, which suggested the occurrence of significant metabolic differences between males and females, convinced us that this division should be maintained during further analysis. Significance level was set at p≤0.05.

Results

Table 1 presents characteristics of examined subjects. Tables 2 and 3 show that low-dose FA supplementation resulted in statistically significant elevation of FA levels in studied females (6.3 vs. 12.5 ng/dL; p=0.001) and males (6.4 vs. 11.4 ng/dL; p=0.001) and, concomitantly, a decrease in Hcy levels (10.6 vs. 8.3 μmol/L; p=0.001 and 11.5 vs. 9.3; p=0.001, respectively). A significant reduction in mean concentration of total cholesterol in females (203.4 vs. 193.1 mg/dL; p=0.001) and in males (209.5 vs. 201.9; p=0.002) was observed. The LDL-C levels also decreased in females and in males (107.4 vs. 99.9 mg/dL; p=0.001 and 121.5 vs. 115.1; p=0.002, respectively). There were no significant differences in HDL-C and TG concentrations before and after FA supplementation in both sexes. However, the total cholesterol/HDL-C ratio and LDL-C/HDL-C ratio differed significantly in both females (3.6 vs. 3.4; p=0.001 and 1.9 vs. 1.8; p=0.001, respectively) and males (4.4 vs. 4.2; p=0.02 and 2.6 vs. 2.4; p=0.01, respectively). There were no significant differences in apolipoproteins concentrations before and after FA supplementation in either sex. Tables 4 and 5 show that low-dose FA supplementation resulted in statistically significant elevation of FA levels in studied females and males with dyslipidemia, overweight and obese (BMI ≥25 kg/m2), and smokers, and, concomitantly, a decrease in Hcy levels in all those groups. The concentrations of total cholesterol, LDL-C, total cholesterol/HDL-C ratio, and LDL-C/HDL-C ratio decreased among studied subjects, except male smokers. There were no significant differences in HDL-C and TG concentrations before and after FA supplementation in either sex divided into dyslipidemia, BMI ≥25 kg/m2, and smokers subgroups. The apoAI concentrations increased significantly among smoking women and men with BMI ≥25 kg/m2 (154.3 vs. 157.6 mg/dL; p=0.032 and 140.4 vs. 145.0; p=0.024, respectively). There were no significant differences in apoB, apoB/apoAI ratio, or Lp(a) concentrations before and after FA supplementation in either sex divided into dyslipidemia, BMI ≥25 kg/m2, and smokers subgroups.

Discussion

In our study, the low-dose FA supplementation used as primary prevention showed encouraging results in terms of the reduction of Hcy and beneficial change in lipoproteins profile (significant decrease of the concentrations of total cholesterol and LDL-C in the studied group, and increased apoAI in some individuals).

The 3-month low-dose FA supplementation nearly doubled the folate level and reduced Hcy to below 10 μmol/L in 80% of participants, which is considered within normal range. The increased folate and reduced Hcy achieved in our study are in agreement with results of other authors [8]. Hcy, through activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in vascular endothelial cells, increases synthesis of Ch [13]. Moreover application of a hypolipidemic drug – simvastatin – to endothelial cells reduced cellular cholesterol and inhibited the suppression of nitric oxide (NO) production by Hcy. These findings explain the proatherogenic effect of Hcy. FA, acting through reducing Hcy, may play an important role in atherogenesis prevention. A significant decrease in LDL-C and Hcy concentrations after 8 weeks of FA, fiber, and B vitamins was reported by Sprecher et al. [20]. No adverse changes of TG or HDL-C levels were noted. Results obtained in this study are consistent with our observations. However, it should be noted that, in general, the degree of the reduction in LDL-C was higher with larger initial LDL-C values.

Apart from decreasing Hcy, total cholesterol, and LDL-C levels, we found that the low-dose FA supplementation increases apoAI, the primary HDL-C apolipoprotein. We observed this effect only in smoking women and men with BMI ≥25 kg/m2. Experimental studies in mice conducted by Mikael et al. showed that an increased Hcy level decreases apoAI synthesis in the livers of mice [21]. The authors also showed a negative correlation between Hcy levels and apoAI levels in humans (males) with coronary artery disease. These results explain the inverse correlation between Hcy and HDL-C. They also suggest that the inhibition of apoA-I synthesis, induced by Hcy, is a subsequent mechanism through which Hcy is linked with lipid metabolism and atherosclerosis development. HDLs have antiatherogenic properties, associated not only with a reverse transport of Ch from cells in peripheral tissues, but also with their antioxidative, antithrombotic, and anti-inflammatory effects [22].

The beneficial results observed in our study are not consistent with the results of the 2004 VISP study, HOPE 2, and NORVIT studies published in 2006 [8,22,23]. These large-scale and long-follow-up studies investigated the effect of preventive FA use on cardiovascular incidents. However, these trials used FA as secondary prevention in individuals with established cardiovascular disease. In our study, FA was used as primary prevention in healthy individuals with atherosclerosis risk factors but without already existing cardiovascular disease. It should also be mentioned that there is a significant difference between those studies and ours in the dose of FA. In the HOPE2 and VISP studies (high-dose formulation group) the FA dose was 6 times higher, and in NORVIT twice as high as in our study. The lack of beneficial effects in those trials could be attributed to the fact that high doses of FA, which is a methyl group donor, not only may cause methylation of Hcy to methionine, but can also be a substrate for other methylation reactions (e.g., methylation of arginine may lead to the asymmetric dimethylarginine (ADMA) formation, which inhibits the endothelial nitric oxide synthase) [24]. Moreover, in those studies, the use of FA (optionally with other group B vitamins) was not associated with a lower risk of new cardiovascular incidents (recurrent myocardial infarction, stroke, or resultant death), and in the NORVIT study the number of incidents was even increased (in subjects with recent myocardial infarction or with stents). Among examined cardiovascular incidents, only in the HOPE2 study there was a reduction in stroke episodes in comparison to the placebo group. Another difference between those trials and our study was that the VISP and NORVIT studies investigated episodes of stroke and myocardial infarction occurrence and did not investigate the effects of preventive FA doses on the blood lipids, as our study did. Only in HOPE2 were the lipid panel values in subjects receiving the FA supplementation compared to the placebo group, but the differences were not statistically significant. However, VISP, HOPE2, and NORVIT were conducted on high-risk groups (individuals with diabetes, cardiovascular disease, and those after myocardial infarction or a stroke).

The use of low-dose FA supplementation demands further research, as it may have more beneficial effects than the use of high doses. Indeed, the most recent studies have shown that in patients with coronary artery disease, low-dose FA (400 μg/d) administered for 7 weeks improves vascular function through increases in nitric oxide-mediated endothelium-dependent vasomotor responses. On the other hand, high-dose FA (5 mg/d) did not provide any additional cardiovascular system benefits [18].

Conclusions

In view of our results, it seems that low-dose FA supplementation, used as primary prevention, has a beneficial effect on blood lipids through decreasing concentrations of total cholesterol and LDL and increasing concentrations of apoAI. To determine the value of FA in primary cardiovascular disease prevention, further studies are needed.