Introduction

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Elevated total plasma homocysteine (tHcy) is an independent risk factor for peripheral vascular, cerebrovascular, and coronary artery disease (CAD) (Boushey et al., 1995). Even moderate hyperhomocysteinemia (>9 µM) is prospectively associated with increased risk of mortality in CAD patients (Nygard et al., 1997; Anderson et al., 2000).

Homocysteine concentrations are determined by genetic and nutritional factors (Ueland and Refsum, 1989). Deficiencies of vitamins B₆, B₁₂, and folic acid in particular are associated with hyperhomocysteinemia (Ubbink et al., 1993). Consequently, vitamin supplementation, especially with folic acid, has been demonstrated to effectively lower elevated homocysteine levels (Clarke et al., 1998).

Homocysteine is a highly reactive amino acid derived from methionine metabolism and is known to produce endothelial cell injury in experimental animals (Harker et al., 1983), in cell culture (Wall et al., 1980), and in humans (Tawakol et al., 1997). The vascular endothelium plays a critical role in the control of vascular tone and regulation of blood flow. Vascular dysfunction is an established early step in the development of vascular disease and contributes to the pathogenesis of atherosclerosis (Celermajer et al., 1992) and CAD (Zeiher et al., 1991). Furthermore, systemic endothelial dysfunction as assessed in the brachial artery is closely related to the extent of CAD (Neunteufl et al., 1997).

In healthy humans, impaired endothelial function of conduit vessels is found in hyperhomocysteinemia (Woo et al., 1997) and is even induced through physiological increments of plasma homocysteine (Chambers et al., 1999b). Vitamin C prevents these effects, suggesting a role for oxidant stress in homocysteine-induced impairment of vascular endothelial function (Chambers et al., 1999a). Furthermore, folic acid was recently demonstrated to improve endothelial function in healthy adults with hyperhomocysteinemia (Woo et al., 1999).

All of these studies used flow-mediated vasodilation (FMD) of the brachial artery in healthy subjects to examine endothelial function of conduit vessels. Blood flow to target organs, in the absence of hemodynamically significant stenoses in conduit vessels, is regulated at the level of local resistance vessels (Chillian et al., 1986). Impaired reactivity of coronary resistance vessels has been shown to be associated with exercise-induced ischemia in subjects free of macrovascular coronary artery disease (Zeiher et al., 1995). Recently, endothelial dysfunction in patients with moderate CAD was found to be accompanied by myocardial perfusion defects (Hasdai et al., 1997). Both reports demonstrate the importance of resistance vessel reactivity in the regulation of myocardial blood flow.

At present, the role for homocysteine on resistance vessel function is less clear. In particular, the effect of folic acid supplementation on resistance vessel function in patients with atherosclerotic disease has not been investigated before. The extent of homocysteine lowering required for a beneficial effect on vascular dysfunction is unknown.

This study was designed to investigate the intrinsic effects of folate supplementation and homocysteine lowering on resistance vessel reactivity in patients with angiographically documented multivessel CAD.

	Materials and Methods

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Study Protocol and Subjects. Twenty-seven male patients with angiographically documented CAD, mean age 50.3 ± 6.2 (range 46.0-56.6) years, were enrolled in this two-period crossover study. CAD was defined as 50% stenosis of the lumen diameter in at least one of the major epicardial vessels. All subjects gave written informed consent, and the study was approved by the Ethics Board of the Karl-Franzens University. The investigation conforms with the principles outlined in the Declaration of Helsinki.

Laboratory Assays. Fasting tHcy, plasma folate, cholesterol, triglycerides, vitamins B₆ and B₁₂, lipoprotein (a), and total antioxidant status (TAS) were measured for each subject on each day of investigation.

Measurement of Forearm Blood Flow. For measurement of resistance vessel endothelial function, forearm blood flow was determined by venous strain-gauge plethysmography (EC5R; Hokanson Inc., Bellevue, WA) with electrically calibrated mercury-in-Silastic strain gauges (Wascher et al., 1998). In brief, the experiments were started at 8:00 AM after an overnight fast in a temperature-controlled room (22-23°C). Subjects were in a supine position for 30 min before the measurements of resting blood flow to assure stable baseline conditions. Measurements were performed on the right arm; the strain gauge was positioned at the widest part of the forearm. For occlusion of venous outflow, the upper arm cuff pressure was set at 50 mm Hg; for induction of ischemia a pressure of 200 mm Hg was chosen. During measurements of forearm perfusion, the hand was occluded from the circulation with a second cuff positioned at the wrist, inflated immediately before the measurements. Resting blood flow was determined by calculation of the mean of three measurements within 1 min. RH was induced by 5 min of ischemia. After release of the upper arm cuff, forearm blood flow was measured every 10 s for up to 60 s. Peak blood flow values, termed peak postischemic RH, are observed thereby 10 s after the onset of reperfusion.

Statistical Analysis. The MacLab package (AD Instruments Pty Ltd., Castle Hill, Australia) on an Apple Macintosh computer was used to acquire and analyze data from the measurements of resistance vessel endothelial function. The mean slope of the volume curve from the second to the fifth pulsewave at each measurement was taken as the respective blood flow. Blood flow data are given as milliliters of perfusion per minute in 100-ml forearm tissue.

	Results

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All 27 male subjects (mean age 50.3 years) completed the crossover trial according to protocol. No side effects of the vitamin medication were observed or reported by the participants. The baseline clinical and biochemical measurements are summarized in Table 1. In linear regression analysis, folate and homocyst(e)ine levels were not significantly related at baseline (P = 0.1, r² = 0.11).

At baseline, subjects with above median tHcy reduction had higher homocysteine levels (14.4 ± 1.2 versus 9.7 ± 0.7 µM; P = 0.002) and lower folate levels (5.1 ± 0.6 versus 8.5 ± 1.4 ng/ml; P = 0.03) than subjects responding with below median tHcy reduction. After 6 weeks without folic acid supplementation, plasma folate and tHcy reversed but not quite to baseline values, indicating a carryover effect (Table 2).

Biochemical Measurements. Oral folic acid (5 mg/day) induced a 3.49-fold increase of plasma folate (6.7 ± 0.8 to 23.4 ± 2.5 ng/ml; P < 0.0001) and lowered homocysteine levels 21.3% (12.2 ± 0.8 to 9.6 ± 0.5 µM; P < 0.001). TAS increased significantly after 6 weeks of folic acid supplementation and returned to baseline at day 12. Throughout the observation period, vitamin supplementation was not seen to have any effects on vitamin B₆ and B₁₂ levels, plasma cholesterol, triglycerides, and lipoprotein(a).

Resistance Vessel Reactivity. Resting blood flow remained almost unchanged in both groups over the entire 12 weeks (Table 2). Homocysteine lowering above median (>2 µM) significantly increased peak reactive hyperemic blood flow after 6 weeks (18.97 ± 1.12 versus 23.60 ± 1.41 ml/min¹ per 100 ml; P = 0.01), but subjects with below median reduction showed no improvement (20.22 ± 2.96 versus 22.99 ± 1.81 ml/min¹ per 100 ml; P = 0.28). At the 6-week follow-up visit, the change of total blood flow (1 min) was borderline significant in above median subjects (11.09 ± 0.98 versus 13.03 ± 1.09 ml/min¹ per 100 ml; P = 0.058) and insignificant in below median subjects (10.13 ± 1.32 versus 13.12 ± 1.37 ml/min¹ per 100 ml; P = 0.09). After 6 weeks of washout, resting flow, peak flow (10 s), and total flow (1 min) in responders were not significantly different from baseline.

	Discussion

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The major findings of this study are that lowering plasma homocysteine through supplementation with folic acid improves resistance vessel function in patients with documented CAD. Our results further suggest that the lower homocysteine concentration is responsible for this effect rather than the increase of folate itself.

There is experimental (Wall et al., 1980) and clinical (Bellamy et al., 1998) evidence suggesting that hyperhomocysteinemia may cause endothelial dysfunction as an early key event in homocysteine-related vascular disease, but the mechanisms are not fully understood. Damage to the vascular endothelium is considered to be a crucial step in the development and progression of atherosclerosis and precedes overt manifestation of disease (Ross,1993). Endothelial dysfunction affects conduit and resistance vessels and is a general phenomenon closely associated with CAD (Anderson et al., 1995; Neunteufl et al., 1997). The effect of homocysteine on resistance vessel function and the potential effect of lowering plasma homocysteine concentrations had been unclear.

Most studies investigated endothelial function using FMD in the forearm and experimental hyperhomocysteinemia in healthy subjects. The results reflect endothelium-dependent vasodilatation and relate mainly to release of endothelial nitric oxide (NO) in response to shear stress (Joannides et al., 1995; Upchurch et al., 1997). Hyperhomocysteinemia induced through methionine loading using invasive administration of acetylcholine in the brachial artery was recently demonstrated to impair resistance vessel endothelial function in young healthy volunteers (Kanani et al., 1999). Noninvasive testing of resistance vessel function by measurement of postischemic reactive hyperemia in the human forearm represents an alternative to intra-arterial drug infusion and reflects the vasodilatatory capacity of a vascular bed upon a predefined stimulus (Iwatsubo et al., 1997; Wascher et al., 1998).

Impairment of FMD through methionine loading was prevented by oral administration of antioxidant vitamin C, suggesting a role for oxidative stress in homocysteine-induced vascular (endothelial) dysfunction (Chambers et al., 1999a).

In our study, plasma folate increased 349% in all subjects, but TAS increased only in subjects with no change in tHcy levels. Infusion of 5-methyltetrahydrofolate, the biologically active form of folic acid, improved endothelial function in patients with familial hypercholesterinemia but normal homocysteine concentrations (Verhaar et al., 1998). The response was immediate and did not affect homocysteine levels, suggesting a scavenging capacity of folate with an effect on endothelial function. This is supported by the finding that pretreatment with oral folic acid prevents the lipid-induced decrease in FMD as well as the lipid-induced increase in urinary radical-damage end products in healthy volunteers (Wilmink et al., 2000). Folic acid may therefore increase TAS by adding to antioxidative capacity, especially when homocysteine concentrations are unchanged. Furthermore, unchanged TAS in subjects with above median lowering of homocysteine may reflect a decrease in SH-(thiol) groups with simultaneous decrease of antioxidative capacity.

Homocysteine has been demonstrated to disturb NO bioavailability (Upchurch et al., 1997) and increase oxidative stress through autoxidation of homocysteine yielding hydrogen peroxide (Loscalzo, 1996; Kanani et al., 1999). Under these conditions, folic acid may improve NO bioavailability indirectly through decrease of homocysteine concentration leading to decreased oxidative stress and less impairment of NO-induced vasodilatation, with possibly additional antioxidative effect, however, consumed during homocysteine lowering. This is supported by our observation that in CAD patients nonresponders showed no improvement in endothelial function, although there was an increase in folate (and TAS). Because folate increased in both groups, but peak flow reactivity improvement was limited to subjects responding with a homocysteine-lowering effect of at least 2 µM, this effect is therefore most likely to be due to lower homocysteine concentrations and not to folate itself.

It must be noted that this effect has been found in CAD patients with presumably impaired NO metabolism. The findings are important because they demonstrate a vasoprotective effect through lowering homocysteine in atherosclerotic patients.

Potential Study Limitations. Potential study limitations comprise intake of medication that may influence endothelial function measurements. All participants refrained from intake of lipid-lowering drugs but received the same standard medication for CAD (only acetylsalicylic acid and -blockers), as discontinuation would have been considered unethical.

Implications. In our study, supplementation with 5 mg of folic acid was associated with 21% lowering of homocysteine concentrations, which confirms previous reports that found similar lowering effects using dosages between 0.4 and 10 mg (Clarke et al., 1998). Importantly, Chambers et al. (1999b) have recently shown that a physiologic homocysteine increment of only 2 to 3 µM may induce endothelial dysfunction in healthy humans.