Oral Folic Acid Improves Endothelial Dysfunction in Cigarette Smokers

Journal of Surgical Research 106, 342–345 (2002) doi:10.1006/jsre.2002.6467

Oral Folic Acid Improves Endothelial Dysfunction in Cigarette Smokers H. L. O’Grady, AFRCSI, A. Leahy, FRCSI, P. H. McCormick, AFRCSI, P. Fitzgerald, M.B., C. Kelly, FRCSI, and D. J. Bouchier-Hayes, FRCSI Department of Surgery, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin 9, Ireland Submitted for publication January 15, 2002; published online July 24, 2002

INTRODUCTION Introduction. Endothelial dysfunction is an early manifestation of the atheromatous process and is evident without overt clinical signs or symptoms of the disease. Cigarette smoking has been shown to be associated with endothelial dysfunction in otherwise healthy adults. Although cessation of smoking is the ideal objective, it is not always attainable, and therefore any strategy to prevent early endothelial dysfunction is desirable. Folic acid is currently under review as a rational therapeutic agent in hyperhomocysteinemia. However, folic acid may modify endothelial function independent of its effect on homocysteine. We therefore investigated the effect of folic acid on endothelial function in young otherwise healthy cigarette smokers. Methods. Volunteer cigarette smokers (n ⴝ 10) and control lifelong nonsmokers were enrolled in the study. Baseline folate, vitamin B12, homocysteine, and cholesterol levels were analyzed. Flow-mediated dilatation, an endothelial-dependent phenomenon, was assessed using ultrasonography. This scan was performed at baseline and following 4 weeks of folic acid supplementation (5 mg/day). Results. There were no significant differences in the baseline hematological investigations between the groups. Homocysteine levels were within normal limits in both groups and did not change following folic acid supplementation. Cigarette smokers demonstrated significant endothelial dysfunction compared to controls (P < 0.005). This difference was significantly attenuated by folic acid supplementation (P < 0.005). Conclusion. Folic acid significantly improves endothelial function in otherwise healthy cigarette smokers. This provides a potential therapeutic tool in attenuating the atheromatous process in this group. © 2002 Elsevier Science (USA) Presented at the Annual Meeting of the Association for Academic Surgery, Milwaukee, Wisconsin, November 15–17, 2001.

0022-4804/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

Cigarette smoking is a major public health issue. It is recognized as a risk factor for the development of atherosclerosis with a continuous graded association between smoke exposure and atherosclerosis [1]. There are thousands of chemical compounds in cigarette smoke [2] and the exact mechanism of accelerated atherosclerosis remains unclear; however, one of the first sites to demonstrate an abnormality is the endothelium. The endothelium plays a pivotal role in vascular homeostasis. It has a role in the maintenance of vascular tone and leukocyte and platelet interactions with the endothelium through the production of mediators such as nitric oxide (NO) and endothelin-1. Endothelial dysfunction is defined as the functional impairment of the endothelium as characterized by vasospasm, vasoconstriction, abnormal leukocyte– endothelial interaction, and altered coagulation [3] and is an early manifestation of the atherosclerotic disease process. The dysfunction is not limited to the diseased vessel as evidenced by the alterations in brachial artery endothelial function seen in patients with coronary artery disease [4]. This dysfunction has previously been demonstrated among young smokers [5] and indeed passive smokers [6] prior to clinical manifestations of the atheromatous process. Low serum folate levels have been associated with an increased risk of atherosclerosis [7, 8]. Interest has centered on its association with the plasma amino acid homocysteine (Hcy). Elevations in plasma Hcy have been associated with the development of atherosclerosis [9 –11]. Folic acid, essential in the metabolism of Hcy, has been shown to reduce plasma Hcy levels [12]. Evidence is now emerging that folates have a role to play in cardiovascular disease prevention, independent of their effects on plasma Hcy. Although total cessation in cigarette smoking is the ideal objective, it is not always attainable, and there-

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TABLE 1 Clinical Characteristics and Laboratory Data Baseline

Age (years) Cholesterol (mmol/L) Triglycerides (mmol/L) LDL (mmol/L) HDL (mmol/L) Vitamin B12 (ng/L) Folate (␮g/L) Hcy (␮g/L) Vessel diameter

Controls

Smokers

Post folic acid Smokers

P

25.3 (⫾2.6) 4.1 (⫾0.6) 0.17 (⫾0.45) 1.99 (⫾0.37) 1.57 (⫾0.3) 410 (⫾117) 9.75 (⫾3.2) 9.5 (⫾3.6) 0.45 (⫾.04)

27.1 (⫾1.9) 5.5 (⫾2.1) 3.9 (⫾0.4) 2.9 (⫾1.1) 1.4 (⫾0.4) 340 (⫾70.8) 11.2 (⫾2.4) 8.6 (⫾2.7) 0.45 (⫾.05)

5.6 (⫾2.1) 3.1 (⫾0.3) 3.5 (⫾2.0) 1.4 (⫾0.3) 291 (⫾65.9) 14.5 (⫾1.0) 9.02 (⫾3.9) 0.43 (⫾.05)

0.274 0.642 0.132 0.368 0.083 0.004* 0.910 0.921

Note. LDL, low-density lipoprotein; HDL, high-density lipoprotein; Hcy, homocysteine. * A significant increase in serum folate was seen between the smoking group and smokers post folic acid supplementation. Significance was taken as a P value ⬍ 0.05.

fore any strategy to prevent early endothelial dysfunction in cigarette smoking is desirable. We investigated the effect of folic acid supplementation (5 mg/day) on cigarette-smoking-induced endothelial dysfunction.

were calculated using ANOVA and Scheffe post hoc using a computer software package, SPSS (Microsoft).

METHODS

There was no difference in age, sex ratio, lipid profile, or vitamin B12 status between groups. Serum folate levels were within normal limits in both the control group and the smokers at baseline (9.75 ␮g/L ⫾ 3.2 and 11.2 ␮g/L ⫾ 2.4, respectively). Following folic acid supplementation, serum folate increased to 14.5 ␮g/L ⫾ 1.0 in the smoking group; this was significant (P ⫽ 0.004 ANOVA, Scheffe post hoc). Homocysteine levels were within normal limits in all groups and, interestingly, no significant difference in Hcy levels were noted post folic acid supplementation (Table 1). The effect of reactive hyperemia on brachial artery diameter was assessed. There was no difference in baseline vessel diameter in the control (0.446 cm ⫾ 0.04) versus smoking groups (0.448 cm ⫾ 0.05) or in the smokers post folic acid supplementation (0.433 cm ⫾ 0.05). Flow-mediated dilatation of 9.5% (⫾2.89) was observed in the control group; this was significantly reduced in the cigarette smokers with a mean dilatation of 2.58% (⫾1.58) (P ⬍ 0.005 ANOVA). Following supplementation with folic acid (5 mg/day) for 4 weeks, cigarette smokers demonstrated a significant improvement in endothelial function (P ⬍ 0.005 ANOVA, Scheffe post hoc). Flow-mediated dilatation improved to 6.9% (⫾2.0); however, the flow-dependent response was not restored to that of normal control values (Fig. 1).

Volunteer cigarette smokers (n ⫽ 10) were recruited. All subjects were normotensive, without a family history of premature vascular disease, and nondiabetic. A control group of volunteer, lifelong nonsmokers was also recruited (n ⫽ 10). A lipid profile, serum folate, vitamin B12, and plasma Hcy were analyzed in both groups prior to commencement of the study. Subjects in the smoking group had a mean smoking history of 6.3 pack years (1 pack year is defined as 20 cigarettes per day for 1 year). Supplementation with folic acid (5 mg/day) (Clonmel Healthcare, Ireland) was commenced in the smoking group for 4 weeks. Assessment of endothelial-dependent dilatation. A noninvasive method of assessing endothelial function described by Celemajer et al. [13] was used. This method has been shown to be both accurate and reproducible [14]. Participants were positioned supine in a temperature-controlled room (20°C) and a three-lead cardiac monitor was applied. The subject’s right arm was immobilized in the extended position to allow constant access to the brachial artery for imaging. The brachial artery diameter was recorded using B-mode ultrasonography using a 7.0-MHz linear array transducer ultrasound system and a standard Acuson 128XP/10 (Acuson, Mountain View, CA). All measurements were coordinated with the “p” wave of the cardiac cycle. Following occlusion of the blood supply to the wrist using a sphygmomanometer cuff inflated to 240 mm Hg for 4.5 min, the cuff was deflated and the brachial artery diameter assessed for dilatation in response to hyperemia. Participants in the smoking group were then given a 4-week supply of folic acid (5 mg) (Clonmel Healthcare) and advised to take one tablet per day. After a 4-week period their brachial reactivity was reassessed and repeat blood investigations were performed. Data analysis. All scans were recorded on super VHS videotapes for offline analysis. The same observer assessed vessel diameter, and three cardiac cycles were analyzed for each measurement and the results were averaged. Reactive hyperemic measurements were assessed 45– 60 s after cuff deflation. Flow-mediated dilatation (FMD) was calculated as the percentage of change in brachial artery diameter in response to reactive hyperemia, compared to baseline values. The data are depicted as means (⫹ standard deviation). All statistics

RESULTS

DISCUSSION

Flow-mediated vasodilatation is a nitric-oxidemediated response after a transient brachial occlusion [15]. Loss of FMD has been demonstrated by ultrasonic

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FIG. 1. There was a significant improvement in flow-mediated dilatation in the smoking group post folic acid supplementation.

insonation in the brachial arteries of young otherwise healthy cigarette smokers [5], as well as in patients with dyslipidemia and proven atherosclerosis. In FMD, constitutive nitric oxide synthase (cNOS) expressed in endothelial cells is activated by the mechanical forces of increased shear force and stretch with the resultant release of NO which induces relaxation of smooth muscle cells in the vessel wall. Flow-mediated dilatation can be easily assessed using high-resolution ultrasonography of the brachial artery in response to hyperemia. This method has been found to be both sensitive and specific for predicting the presence of atherosclerotic disease [16]. Using this method, we have confirmed the findings of endothelial dysfunction in cigarette smokers. This dysfunction has been attributed to impaired activity of endothelial-derived nitric oxide. Nitric oxide activity is critical in the many potentially proatherogenic processes, namely monocyte adhesion, platelet aggregation, vascular smooth muscle proliferation, and oxidative modification of low-density lipoprotein (LDL). It is also the regulator of vasomotor tone, impairment of which is one of the earliest measurable features of the atheromatous process predating structural changes and increases in serum lipid levels. Decreased release of NO from endothelial cells might result from deficiencies of substrate or cofactors, increased breakdown of NO, or reduced expression of cNOS. Current perspectives on decreased NO availability in the pathogenesis point to a reduction in NO production rather than increased inactivation. Recently, cNOS has been shown to produce superoxide radicals as well as NO [17, 18]. Under physiological conditions, cNOS produces NO predominantly under control of the regulatory coenzyme calmodulin, the substrate L-arginine, and the cofactor tetrahydrobiopterin (BH4). Under pathophysiological conditions, such as dyslipidemia, production shifts from NO to superoxide production [19]. Evidence has accumulated show-

ing that increased degradation of NO by superoxide, rather than impaired formation of NO, is the predominant cause of impaired NO bioavailability in early atherosclerosis [20 –22]. Folic acid supplementation has been shown previously to improve endothelial function in patients with hyperhomocysteinemia [23]. However, the significance of elevated homocysteine levels remains questionable. In a nested case– control study by Ridker et al., homocysteine was not seen as a risk factor for atherosclerosis [24]. Homocysteine levels were within normal limits in all participants of this study, and indeed, there was no significant change in homocysteine levels over the 4-week treatment period. The beneficial effect of folic acid supplementation seen in the smoking group was therefore independent of any homocysteine lowering effect. The means by which this effect was achieved remain uncertain. Folates have been suggested to be involved in the regeneration of BH4, an essential cofactor for NO synthase; however, increased levels of this cofactor have not been shown following folic acid supplementation. 5-Methyltetrahydrofolate, the active form of folic acid, in addition, has been shown to improve the activity of cNOS with improved production of NO and reduced superoxide production [25]. Despite the uncertainty by which folic acid exerts its protective effect, it has been shown to be a cheap and safe therapeutic tool in early atherosclerosis. REFERENCES 1.

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