Folic acid (pteroylglutaminacid), a water-soluble vitamin, and in particular its active metabolite 5-methyltetrahydrofolate (5-MTHF), is likely to play an important protective role in the cardiovascular system. Indeed, oral intake of folate improves endothelial function in hypercholesterolemia1 independent of changes of homocysteine levels.2 Folic acid in combination with the vitamins B6 and B12 decreases the restenosis rate after coronary stenting.3 Twenty minutes after oral administration of folic acid, its plasma levels are detectable. In the body, the major function of folic acid relates to nucleotide synthesis. On the other hand, folic acid deficiency leads to megaloblastic anemia and neural tube defects in embryogenesis.
In hypertension baroreceptor modulation of heart rate is impaired, resulting in a decreased vagal cardiac control.4 The role of baroreceptor-mediated modulation of vascular tone via peripheral sympathetic muscle-nerve activity is still controversial because there are reports of impaired5 as well as preserved6 sympathetic baroreceptor function in hypertensive patients.
Paracrine factors are able to modify baroreceptor sensitivity. Indeed, locally produced prostacyclin increases baroreceptor sensitivity,7 whereas nitric oxide8 and reactive oxygen species9 decrease baroreceptor sensitivity. Folic acid has antioxidative properties,10 which are able to reduce the levels of reactive oxygen species. Therefore, the aim of this study was to test the hypothesis that folic acid improves baroreceptor function in hypertension.
Twenty-one male patients with mild to moderate hypertension participated in the study. Their baseline characteristics are listed in Table 1. Exclusion criteria were smoking, hypercholesterolemia, secondary hypertension, stroke or transient ischemic attacks within 1 year, documented myocardial infarction, angina, or revascularization within 1 year. Neither chronic treatment with nonsteroidal antiinflammatory drugs nor substitution of vitamins was allowed. All participants gave written informed consent, and the study protocol was approved by the local research ethics committee of the University Hospital, Zürich, Switzerland.
At visit 1 the patients underwent physical examination, and an ECG was performed. After inclusion all antihypertensive medication were stopped for 2 weeks, while concomitant therapy with acetylsalicylic acid was allowed. Then at visit 2 (+2 weeks), baroreceptor function was assessed by two different methods, ie, cardiac interval with the stimulus-independent α-coefficient method11 and a stimulus-dependent test in which we infused a bolus of phenylephrine or sodium nitroprusside as described previously.12 For quantification of the baroreceptor vascular sympathetic interval, we used microneurography to obtain muscle sympathetic activity (MSA) in a standardized fashion as described.13 We used the N. peroneus of the right leg to obtain MSA. Signals were amplified, filtered (700-2000 Hz), integrated (time constant 0.1 seconds), and digitized using an analog-digital board (MIO-16L, National Instruments, Austin, TX) and a modified commercial software (LabView, National Instruments). Data were recorded with a computer (Apple Macintosh Power PC 7100) and analyzed offline (MatLab, MathWorks, Natick, MA). Heart rate, blood pressure [conventional (Riva Rocci) by Dynamap® and continuously by Finapress®, an oscillometric device] were measured.
The patients were randomized either to a single dose of folic acid (5 mg PO, Hänseler AG, Switzerland) or matching placebo group in a 2:1 ratio. Baseline recordings were done over a 20-minute period; then 200 μg phenylephrine (PE) (Inj. Lös 0.01% KA, Vial, Switzerland) was applied intravenously. Ten minutes later, 1.2 μg/kg nitroprusside sodium (SNP) (Nipruss®, Schwarz Pharma, Germany) was applied. Then placebo or 5 mg folic acid was given orally. After a period of 30 minutes to allow drug resorption, the baroreceptor testing maneuvers with PE and SNP were repeated in the same manner.
Oxidative Stress (8-Isoprostane)
The measurement of 8-isoprostane plasma levels was done with an ELISA (Cayman Chemical, MI). 8-Isoprostane is an eicosanoid and a marker of oxidative stress.14
Data Analysis and Statistics
Off-line calculation of baroreceptor sensitivity with the α-coefficient method was performed twice, at baseline (after a resting time of 15 minutes) and 30 minutes after folic acid/placebo. The analysis was based on the spectral analysis technique; in brief, subdivisions of the continuous recorded heart rate and blood pressure ranging from 300 heartbeats were made, and the segments simultaneously analyzed by fast Fourier transformation (FFT) providing R-R interval and systolic blood pressure spectral powers. The square root of the ratio of these parameters was then calculated. This method is well established and has been validated against other methods, ie, sequence method to gain baroreceptor sensitivity.15,16 The PE and SNP baroreceptor cardiac intervals were calculated from changes of R-R interval and systolic blood pressure after the intravenous bolus injection of these vasoactive drugs. The slope of the regression line fitting the systolic blood pressure and R-R interval changes serves as a measure of cardiac BRS.12 Both results are expressed as milliseconds/mm Hg. The baroreceptor vascular sympathetic interval was calculated as percent change of the integrated MSA activity (%ia) and changes of diastolic blood pressure and expressed as %ia/mm Hg. The changes of MSA were calculated over a period of 1 minute, 30 seconds after the bolus of PE or SNP, respectively, and compared with the penultimate minute preceding the bolus application. All stored experiments on the computer were blinded to the analyzing investigator. Results are presented as means ± SEM unless stated otherwise. Single comparisons were made using with paired and unpaired Student t test and two-way ANOVA with Bonferonni/Dunn post-hoc test for repeated measures (StatView 4.5, Abacus Concepts, Berkeley, CA). Statistical significance was accepted at P < 0.05.
There were no significant differences in the baseline characteristics between the two groups (Table 1), and no changes in heart rate, blood pressure, or MSA were noted before or after placebo or folic acid.
In the folic acid group the cardiac α-coefficient baroreceptor sensitivity improved significantly by 1.3 ± 0.3 milliseconds/mm Hg as compared with −0.1 ± 0.2 milliseconds/mm Hg in the placebo group (P = 0.005; Fig. 1). The phenylephrine baroreceptor cardiac interval improved in the folic acid group by 1.6 ± 0.5 milliseconds/mm Hg as compared with placebo (0.3 ± 0.2 milliseconds/mm Hg, P = 0.031). The sodium nitroprusside baroreceptor cardiac interval improved by −2.0 ± 0.5 milliseconds/mm Hg as compared with placebo (0.1 ± 0.4 milliseconds/mm Hg, P = 0.013). In the folic acid group the phenylephrine baroreceptor vascular sympathetic interval improved by −1.3 ± 0.3 %ia/mm Hg as compared with placebo (0.3 ± 0.2 %ia/mm Hg, P = 0.038), and the sodium nitroprusside baroreceptor vascular sympathetic interval improved by 1.0 ± 0.3 as compared with −0.3 ± 0.5 %ia/mm Hg in the placebo group (P = 0.017; Fig. 2).
This study demonstrates that in patients with mild to moderate hypertension, a single dose of folic acid improves cardiac and vascular sympathetic baroreceptor sensitivity, suggesting an improved vagal control of the heart and an enhanced baroreceptor modulation of peripheral sympathetic vasomotor tone with this treatment.
Hypertension is associated with impaired baroreceptor function.17 This alteration is reflected by the following changes: (1) baroreceptor reflex control of sympathetic nerve activity and heart rate is shifted to higher blood pressure levels, and (2) baroreceptor sensitivity is reduced, leading to a reduced cardiovagal control. In hypertension the role of baroreceptor-mediated modulation of vasomotor tone via sympathetic muscle nerve activity is still unclear, as there are reports of altered5 as well as preserved6 vascular sympathetic baroreceptor function.
Folic acid is a derivative of pteridine and an important cofactor in the transfer and utilization of 1-carbon moieties and improves endothelial function in patients with coronary artery disease2 and hypercholesterolemia,1 independent of homocysteine plasma levels. Three different potential effects might be involved in improving endothelial function. (1) Folate lowers homocysteine levels because folate is the substrate in the remethylation of homocysteine to methionine.18 However, there is no evidence of an involvement of this mechanism in the modulation of baroreceptor sensitivity. (2) An interaction of folate and endothelial NO synthase (eNOS) might be involved. eNOS catalyzes the formation of NO via the transformation of L-arginine to citrulline using NADPH and tetrahydrobiopterin (BH4) as cofactors. Under certain circumstances an uncoupling of eNOS may occur, which leads to the production of superoxide ions instead of NO. Folate stabilizes BH4 and thus eNOS is sheltered from uncoupling. This in turn stabilizes the production of NO.19 (3) The antioxidant potential of folic acid might reduce reactive oxygen species by inhibiting superoxide production by eNOS and xanthine oxidase.10 Taken together as potential explanations (further investigation is needed) of our findings, the positive effects of folate on baroreceptor sensitivity seem to be mediated by a reduction of oxidative stress because oxidative stress directly interferes with nerve endings of baroneurons in the arterial wall20 and reduces baroreceptor sensitivity independently of an increased bioavailability of NO. This explanation is in line with the fact that in animal models infusion of scavengers such as catalase and superoxide dismutase improved baroreceptor sensitivity.9
Besides eNOS, which is expressed in endothelial cells, there are other isoforms of the enzyme such as neuronal NOS (nNOS), expressed in central and peripheral neuronal cells. Baroneurons contain nNOS, and NO serves as a neurotransmitter.9 The effect of NO within the nervous system has been little explored; nevertheless, in animal experiments NO exerts a central sympathoinhibitory effect in the hypothalamus and medulla.21 This supports the concept that folate might exert its effects on baroreceptor function not only by reducing oxidative stress but also via neuronal effects in the central nervous system. Indeed, folate as a molecule can easily cross the blood-brain barrier.22
Hypertension and coronary artery disease commonly occur together; thus, an improvement of baroreceptor sensitivity in such patients might reduce arrhythmic events through better cardiovagal control of the heart. Indeed, the ATRAMI study revealed less frequent arrhythmic events among such coronary artery disease patients who had preserved baroreceptor sensitivity.23
At this stage it remains uncertain whether the effects of folate on baroreceptor function are maintained under chronic conditions. However, the antioxidative effects of folic acid are not receptor or enzyme mediated, which makes adaptive mechanisms such as down- or up-regulation of enzymes or receptors an unlikely possibility. Thus, most probably the chronic effects of folic acid on baroreceptors are sustained. Nevertheless, this question must be addressed by appropriate long-term studies.
This study reports a positive effect of orally applied folic acid on cardiac and vascular sympathetic baroreceptor function in hypertensive patients. In hypertensive patients, an improved baroreceptor sensitivity would lead to a better cardiovagal control and peripheral regulation of sympathetic vasomotor tone.4 This finding, one could speculate, may represent a novel treatment in prevention of orthostatic dysregulation and/or arrhythmic complications of baroreceptor dysfunction.
This research was supported by the Swiss National Research Foundation (32-52690-97) and the Swiss Heart Foundation.
1. Verhaar MC, Wever RM, Kastelein JJ, et al. Effects of oral folic acid
supplementation on endothelial function in familial hypercholesterolemia. A randomized placebo-controlled trial. Circulation.
2. Doshi SN, McDowell IF, Moat SJ, et al. Folic acid
improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Circulation.
3. Schnyder G, Roffi M, Pin R, et al. Decreased rate of coronary restenosis after lowering of plasma homocysteine levels. N Engl J Med.
4. Lucini D, Mela GS, Malliani A, et al. Impairment in cardiac autonomic regulation preceding arterial hypertension
in humans: insights from spectral analysis of beat-by-beat cardiovascular variability. Circulation.
5. Matsukawa T, Gotoh E, Miyajima E, et al. Angiotensin II inhibits baroreflex control of muscle sympathetic nerve activity and the heart rate in patients with essential hypertension
. J Hypertens Suppl.
6. Grassi G, Cattaneo BM, Seravalle G, et al. Baroreflex control of sympathetic nerve activity in essential and secondary hypertension
7. Xie PL, Chapleau MW, McDowell TS, et al. Mechanism of decreased baroreceptor activity in chronic hypertensive rabbits. Role of endogenous prostanoids. J Clin Invest.
8. Matsuda T, Bates JN, Lewis SJ, et al. Modulation of baroreceptor activity by nitric oxide and S-nitrosocysteine. Circ Res.
9. Li Z, Mao HZ, Abboud FM, et al. Oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerotic rabbits. Circ Res.
10. Verhaar MC, Wever RM, Kastelein JJ, et al. 5′-Methyltetrahydrofolate, the active form of folic acid
, restores endothelial function in familial hypercholesterolemia. Circulation.
11. Pagani P, Somers V, Furlan R, et al. Changes in autonomic regulation induced by physical training in mild hypertension
12. Smyth HS, Sleight P, Pickering GW. Reflex regulation of arterial pressure during sleep in man. A quantitative method of assessing baroreflex sensitivity. Circ Res.
13. Delius W, Hagbarth KE, Hongell A, et al. General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand.
14. Morrow JD, Frei B, Longmire AW, et al. Increase in circulating products of lipid peroxidation (F2
-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med.
15. Parati G, Di Rienzo M, Mancia G. How to measure baroreflex sensitivity: from the cardiovascular laboratory to daily life. J Hypertens.
16. Parati G, Fratolla A, Di Rienzo M, et al. Effects of aging on 24 hour dynamic baroreceptor control of heart rate in ambulant subjects. Am J Physiol.
17. Grassi G, Cattaneo BM, Seravalle G, et al. Baroreflex impairment by low sodium diet in mild or moderate essential hypertension
18. Brouwer IA, van Dusseldorp M, Duran M, et al. Low-dose folic acid
supplementation does not influence plasma methionine concentrations in young non-pregnant women. Br J Nutr.
19. Stroes ES, van Faassen EE, van Londen GJ, et al. Oxygen radical stress in vascular disease: the role of endothelial nitric oxide synthase. J Cardiovasc Pharmacol.
20. Chapleau MW, Cunningham JT, Sullivan MJ, et al. Structural versus functional modulation of the arterial baroreflex. Hypertension.
21. Zhang K, Patel KP. Effect of nitric oxide within the paraventricular nucleus on renal sympathetic nerve discharge: role of GABA. Am J Physiol.
22. Wu D, Pardridge WM. Blood-brain barrier transport of reduced folic acid
. Pharm Res.
23. La Rovere MT, Pinna GD, Hohnloser SH, et al. Baroreflex sensitivity and heart rate variability in the identification of patients at risk for life-threatening arrhythmias: implications for clinical trials. Circulation.