Endothelial function in post-menopausal women: effect of folic acid supplementation

Giancarlo Paradisi1,3, Francesco Cucinelli1, Maria Cristina Mele1, Angela Barini1, Antonio Lanzone2 and Alessandro Caruso1

1 Department of Obstetrics and Gynecology, Catholic University of Sacred Heart, Largo A. Gemelli 8, 00168 Rome and 2 Oasi Institute of Research, Troina (Enna), Italy

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Catholic University of Sacred Heart, Via Servilio IV 4, 00178 Rome, Italy. e-mail: giancarlo.paradisi{at}tin.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Higher than normal homocysteine levels are associated with an increased incidence of adverse cardiovascular events in post-menopausal women, perhaps via hyperhomocysteinaemia-induced vascular endothelial damage. Because folic acid supplementation reduces homocysteine levels, we attempted to evaluate whether folic acid supplementation may affect endothelial function in post-menopausal women. METHODS: Brachial artery flow-mediated dilatation (endothelium-dependent) and nitroglycerin-induced dilatation (endothelium-independent) before and after a methionine load were analysed in 15 healthy post-menopausal women. Plasma levels of folate, homocysteine, glucose, insulin and lipids were measured, as was blood pressure. All studies were repeated after 1 month supplementation with 7.5 mg/day of folic acid. RESULTS: After folate, endothelial function rose 37% over pre-folic acid supplementation value (P < 0.001), and flow-mediated dilation before folic acid was reduced by 62% subsequent to methionine loading (P < 0.0001); this reduction was still present after folic acid, but was only 19% (P < 0.001). Nitroglycerin-induced dilatation did not change in response to methionine loading before or after folic acid supplementation. Among the other cardiovascular risk factors studied, only high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol showed significant changes after folic acid supplementation, with a 6% increase (P < 0.03) and a 9% decrease (P < 0.03) respectively. CONCLUSIONS: Although preliminary, these results indicate that folic acid supplementation may improve endothelial function and lipid profile in post-menopausal women, thus contributing to reduce their cardiovascular risk.

Key words: endothelium/folic acid/menopause


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Heart disease is the leading cause of morbidity and mortality among post-menopausal women in industrialized countries (Colditz et al., 1987Go; Falkeborn et al., 2000Go). Recently, elevated blood levels of homocysteine (tHcy), an amino acid formed during the metabolism of methionine, has been added to the list of cardiovascular risk factors. Increased tHcy levels are associated with vascular damage, although the mechanism whereby hyperhomocysteinaemia exerts its deleterious effects remains unclear (Mayer et al., 1996Go; Welch et al., 1998Go; Aronow et al., 2000Go). The major focus of current studies is on the endothelium as the site of initiation of vascular damage. In vitro studies have shown that homocysteine is directly cytotoxic to endothelial cells at higher than normal levels (Stamler et al., 1993Go). Oxidation of homocysteine may also lead to the formation of hydrogen peroxide, which may in turn play a role in endothelial cell damage (Starkebaum et al., 1986Go). Additionally, clinical reports in humans have suggested that homocysteine may inhibit endothelium-dependent flow-mediated dilation, consequent upon inhibition of nitric oxide synthase (Woo et al., 1997Go). Given that the post-menopausal status has been shown to be associated with increased homocysteine levels, it is conceivable that such an increase could have a role in the augmented cardiovascular morbidity of post-menopausal women (Verhoef, 2000Go). Folic acid is required for homocysteine metabolism, and folate deficiency is associated with elevated tHcy concentrations. Supplementation with folic acid reduces tHcy levels in normal subjects and in patients at risk for vascular disease (Rasmussen et al., 2000Go); folic acid therapy may thus preserve or restore endothelial function of subjects at risk for cardiovascular disease. To examine this hypothesis, we evaluated endothelial function, and carbohydrate and lipid metabolism, in post-menopausal women. The investigation was performed under basal conditions and after methionine-induced hyperhomocysteinaemia, and was repeated after 1 month of folic acid supplementation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
During the study, ~100 women were seen at our outpatient meno pause care centre. In all cases, >6 months had elapsed since the last menstrual bleed. Those who smoked were excluded, as were those who drank >60 g of alcohol per day, those who were clinically diagnosed with liver disease, ischaemic heart disease, hypertension, diabetes, renal disease, or who were taking HRT or medications known to affect endothelial function or glycaemic and lipid metabolism. Thirty-two women who met the above conditions were approached to participate in the study. Fifteen agreed to be enrolled. Vitamin B12 status was not formally ascertained although none of the women had any direct or indirect sign of vitamin B12 deficiency.

Study design
The studies were carried out over 2 days. On day 1, sex hormones, glucose, insulin, folate and lipid evaluations were performed. On day 2, bioelectrical impedance, blood pressure, endothelial function and plasma levels of total homocysteine at fasting and 2 h after administration of oral methionine (L-methionine, 0.1 g/kg body weight) were measured. The investigations were performed in the morning after overnight fasting in a supine position at a temperature of ~25°C. All subjects were studied under basal conditions and after 1 month supplementation with 7.5 mg/day of folic acid. The study was conducted at the Department of Obstetrics and Gynecology at Catholic University in Rome and was approved by the Institutional Review Board. Informed consent was obtained from each subject before the study.

Measurement of endothelial function
The ultrasound investigation for measuring endothelium-dependent and endothelium-independent arterial dilatation was performed as described previously (Celermajer et al., 1992Go). Briefly, brachial artery diameter was measured by B-mode ultrasound image, by the use of a 7.5 MHz linear-array transducer and a standard ESAOTE AU 570 A system (Ansaldo, Italy). In all studies, scans were obtained with the subject at rest, during reactive hyperaemia, again with the subject at rest, and after sublingual administration of nitroglycerin. The velocity of arterial flow was measured with a pulsed Doppler signal. Increased flow was induced by the inflation of a pneumatic tourniquet placed around the forearm (distal to the scanned part of the artery) to a pressure of 250 mmHg for 4.5 min, followed by release. A scan was performed continuously for 30 s before and 90 s after deflation of the cuff, including a repeat recording of flow velocity for the first 15 s after the cuff was released. Thereafter, 10–15 min was allowed for recovery of the vessel, after which an additional resting scan was performed. Sublingual nitroglycerin spray (400 µm) was then administered, and 3–4 min later the last scan was performed. For the reactive hyperaemia scan, measurements of diameter were taken 50–60 s after deflation of the cuff. The vessel diameter in scans obtained after reactive hyperaemia (flow-mediated dilatation, FMD) and the administration of nitroglycerin (nitrate-induced dilatation, NID) was expressed as a percentage of the average diameter of the artery in the two resting (or control) scans (considered as 100%). Reactive hyperaemia was calculated as the maximal flow recorded in the first 15 s after cuff deflation, divided by the flow during the first resting (baseline) scan. Each subject was studied in the morning after abstaining from alcohol, caffeine, and food for 8 h. Flow-mediated and nitrate-induced dilatation was assessed while fasting and 2 h after oral methionine administration.

Analytical methods
Plasma glucose levels were measured by the glucose oxidase method (Beckman, USA), and all hormone levels by commercial radioimmunoassay kits (Radim, Italy). Free testosterone index was calculated as previously reported (Vermeulen et al., 1999Go).

Total cholesterol and triglyceride concentrations were determined by an enzymatic assay (Bristol, France). High-density lipoprotein cholesterol (HDL-C) concentrations were determined after precipitation of chylomicrons, very-low-density lipoprotein cholesterol (VLDL-C), and low-density lipoprotein cholesterol (LDL-C) (Boehringer, Germany). A magnesium chloride/phosphotungstic acid technique was used to precipitate LDL-C from the bottom fraction after ultracentrifugation. Free fatty acids were determined by an acyl-coenzyme A oxidase-based colorimetric method. Total plasma concentrations of homocysteine were estimated by high-performance liquid chromatography. Red blood cell folate concentrations were measured by radioassay (Folate Elecsys 2010; Roche Diagnostics GmbH, Germany).

Body composition
The bioelectrical impedance to estimate the subject’s body composition was performed with a tetrapolar impedance plethysmograph [Soft Tissue Analyzer (STA/BIA); Akern Bioresearch, Italy] according to Lukasky et al. (1986Go). Briefly, at 07:00 h each woman was supine on a bed made of non-conductive materials. Detecting electrodes (Red Dot; 3M Health Care, USA) were placed in the middle of the dorsum of hands and feet proximal to the metacarpal-phalangeal metatarso-phalangeal joints respectively, and also medially between the distal prominences of the radius and the ulna and between the medial and lateral malleoli at the ankle. The current-introducing electrodes were placed at a minimum distance of the diameter of the wrist or ankle beyond the paired detector electrode. An exitation current of 800 mA, AC, at 50 kHz was introduced at the distal electrodes and the voltage drop across the patient was detected by the proximal electrodes. The percentages of body fat, fat free mass and total body water were calculated by using the appropriate software (Bodygram; Akern Bioresearch, Italy).

Statistical analysis
Comparison within groups was performed by Student’s paired t-test. Simple linear regression analysis was performed to assess the relationship between percentage increase in homocysteine concentrations and difference in FMD in response to methionine administration. The sample size for FMD was calculated for power 0.80, assuming a difference in means of ~5 and SD ~3 from our preliminary findings.

Results are shown as the mean ± SEM. P < 0.05 was considered statistically significant.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Demographic and hormonal features of post-menopausal women under basal conditions and after folic acid supplementation were similar (Table I). Among the cardiovascular risk factors studied, only HDL-C and LDL-C showed significant changes after folic acid supplementation, with a 6% increase (P < 0.03) and a 9% decrease (P < 0.03) respectively. No significant difference was seen with regard to other lipids studied, although mean values of total cholesterol, triglycerides and free fatty acids were somewhat lower after folic acid supplementation than at baseline. Insulin and glucose did not show any difference after folate. Similarly, blood pressure was not modified in response to folic acid supplementation. As expected, red blood cell folate was significantly increased after folic acid.


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Table I. Demographic and clinical characteristics of post-menopausal women before (study 1) and after (study 2) folic acid supplementation
 
Vascular characteristics are shown in Table II. Brachial artery diameter (vessel size) was similar at baseline and after methionine loading, before and after folic acid supplementation. Similarly, baseline velocity and the percentage increase in blood velocity after ischaemic stimulus (reactive hyperaemia) was comparable across measurements. FMD under basal condition (i.e. endothelium-dependent dilatation) was reduced by 62% after methionine loading (P < 0.0001). Interestingly, after folic acid, FMD was still reduced subsequent to methionine, but only by 19% (P < 0.001). After folate, basal FMD exhibited a remarkable 37% increase with respect to pre-folic acid supplementation value (P < 0.001). Nitroglycerin-dependent dilatation (endothelium-independent) did not change in response to methionine loading, either before or after folic acid supplementation. Homocysteine values increased significantly 2 h after methionine loading during both pre- and post-folic acid supplementation. There was no difference in fasting homocysteine between pre- and post-folic acid study, while the 2 h post-methionine peak was significantly reduced after folate.


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Table II. Vascular characteristics and homocysteine concentration at basal and post-methionine load, before (study 1) and after (study 2) folic acid supplementation
 
To investigate whether some of the other variables studied were associated with endothelial function, we performed a simple correlation between FMD and hormonal, metabolic and demographic indices. Estradiol was positively related to FMD, before and after folic acid supplementation (r = 0.54, P = 0.037; r = 0.52, P = 0.047 respectively). Inverse correlations were observed between FMD and body mass index (BMI) and between FMD and free testosterone index, before and after folic acid supplementation, but did not reach statistical significance (r = –0.46, P = 0.079; r = –0.50, P = 0.058; r = –0.40, P = 0.145; r = –0.39, P = 0.157 respectively).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The beneficial effect of folic acid on endothelial impairment in hyperhomocysteinaemic subjects is described in the literature (Holven et al., 2001Go; Woo et al., 2002Go). There is now evidence that this favourable effect is present not only in subjects at high risk for macrovascular disease, but also in healthy normo-homocysteinaemic post-menopausal women: we found a 37% improvement of FMD after 1 month of folic acid supplementation (Woo et al., 1999Go; Title et al., 2000Go). As expected, the worsening of endothelial function after acute methionine-induced hyperhomocysteinaemia was much less marked after folic acid supplementation, thus supporting previous findings showing that high levels of red blood cell folate are protective against the deleterious effect of hyperhomocysteinaemia on the vasculature (Usui et al., 1999Go). The improved endothelial function seen during hyperhomocysteinaemia after folate was coupled with a parallel significant reduction of methionine-induced homocysteine peak. Although the magnitude of the decrease in homocysteine peak seen after folic acid supplementation was lower than the corresponding improvement of post-methionine FMD, it is likely that the lower homocysteine achieved in response to methionine load could account, at least in part, for the amelioration of endothelial function. Interestingly, the improved vascular function under basal conditions after folate supplementation was not associated with a significant decrease in fasting homocysteine. This result is in accord with findings of in vivo (Doshi et al., 2002Go) and in vitro (Verhaar et al., 1998Go; Stroes et al., 2000Go; Doshi et al., 2001Go) studies suggesting that the beneficial endothelial effect of folic acid, rather than occurring directly via a reduction in homocysteine, may occur as either a direct or indirect reduction in oxidative stress. On the other hand, we can hypothesize that the chronic lower post-meal homocysteine concentrations obtained during folate supplementation may extend to the favourable effect on endothelium also in fasting conditions. It is important to underline, however, that we did not study the mechanism whereby folate supplementation acts on endothelial function during hyperhomocysteinaemia. In addition, the study was not designed as a double-blind randomized placebo study. Therefore, our findings have to be considered with caution.

In vitro studies have shown that the auto-oxidation of homocysteine is accompanied by the generation of oxygen radicals which, in turn, may lead to oxidative modification of low-density lipoproteins with an increase in LDL values (Weiss et al., 1999Go). Supplementation with folic acid, reducing the hyperhomocysteine-induced generation of oxygen radicals, has been shown to decrease the extent of LDL oxidation and to increase HDL-C levels in subjects at high risk for cardiovascular disease (McGregor et al., 2000Go; Ziakka et al., 2001Go). In accordance with these observations, we found an improvement in lipid pattern after folate supplementation, with a 6% increase of HDL-C and a 9% decrease of LDL-C, thus extending previous findings to normo-homocysteinaemic post-menopausal women. It has been reported that HDL-C is directly associated with endothelial function, via an HDL-mediated increase in endothelial nitric oxide synthase expression (Kuvin et al., 2002Go; Spieker et al., 2002Go). Conversely, high levels of LDL-C have been shown to be associated with endothelial dysfunction (Shechter et al., 2000Go). Although we did not find significant correlations among FMD, HDL-C and LDL-C before or after supplementation (data not shown), it is likely that the improved lipid profile subsequent to folic acid might contribute to the ameliorated endothelial function seen at basal fasting condition.

Estrogen has a protective action on the vessel wall. Epidemiological studies have demonstrated that menopause and consequent estrogen deprivation increase the risk of cardiovascular disease in women (Kannel et al., 1976Go; Colditz et al., 1987Go). Several cardioprotective effects have been ascribed to estrogens, although the mechanism whereby estrogens modulate their effects is still unclear and subject of intense investigation. According to other reports, in this study we found that estradiol showed a direct significant correlation with vascular vasodilatation, irrespective of folate administration, thus indicating that endogenous estrogens may have a role in endothelial function in post-menopausal women. However, given that estradiol values were not changed after folic acid supplementation, it is unlikely that this hormone could account for the improved FMD observed after folate supplementation.

As we and others have previously demonstrated, there is an inverse correlation between endothelial function and BMI as well as with free testosterone (Steinberg et al., 1996Go; Paradisi et al., 2001Go). Adiposity and androgen levels are known factors that negatively affect endothelial function in both pre- and post-menopausal women, probably via their effect on carbohydrate and lipid metabolism. In the present study, no correlation reached statistical significance either before or after folic acid supplementation, perhaps reflecting the small sample size. However, because there were no changes in BMI and free testesterone after folate, it is unlikely that these factors could have a role in the improved endothelial function seen after folic acid supplementation.

In conclusion, in post-menopausal women, 1 month of folic acid supplementation is associated with improved endothelial function, under basal conditions and in response to acute hyperhomocysteinaemia. These changes, along with an improved lipid profile, seem to indicate that folic acid supplementation may contribute to reduce some of cardiovascular risk factors in these subjects.


    Acknowledgements
 
This study was presented in part at the 84th Annual Meeting of the Endocrine Society, San Francisco, USA in June 2002. We are indebted to Federica Mancinelli for technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aronow WS and Ahn C (2000) Increased plasma homocysteine is an independent predictor of new coronary events in older persons. Am J Cardiol 8,346–347.[CrossRef]

Celermajer DS, Sorensen KE and Gooch VM (1992) Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340,1111–1115.[Medline]

Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE and Hennekens CH (1987) Menopause and the risk of coronary heart disease in women. N Engl J Med 316,1105–1110.[Abstract]

Doshi SN, McDowell IF, Moat SJ, Lang D, Newcombe RG, Kredan MB, Lewis MJ and Goodfellow J (2001) Folate improves endothelial function in coronary artery disease: an effect mediated by reduction of intracellular superoxide? Arterioscler Thromb Vasc Biol 21,1196–1202.[Abstract/Free Full Text]

Doshi SN, McDowell IF, Moat SJ, Payne N, Durrant HJ, Lewis MJ and Goodfellow J (2002) Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Circulation 105,22–26.[Abstract/Free Full Text]

Falkeborn M, Schairer C, Naessen T and Persson I (2000) Risk of myocardial infarction after oophorectomy and hysterectomy. J Clin Epidemiol 53,832–837.[CrossRef][Medline]

Holven KB, Holm T, Aukrust P, Christensen B, Kjekshus J, Andreassen AK, Gullestad L, Hagve TA, Svilaas A, Ose L et al (2001) Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects. Am J Med 110,536–542.[CrossRef][Medline]

Kannel WB, Hjortland MC, McNamara, PM and Gordon T (1976) Menopause and risk of cardiovascular disease: the Framingham study. Ann Intern Med 85,447–452.[Medline]

Kuvin JT, Ramet ME, Patel AR, Pandian NG, Mendelsohn ME and Karas RH (2002) A novel mechanism for the beneficial vascular effects of high-density lipoprotein cholesterol: enhanced vasorelaxation and increased endothelial nitric oxide synthase expression. Am Heart J 144,165–172.[CrossRef][Medline]

Lukasky HC, Bolonchuk WW, Hall CB and Siders WA (1986) Validation of tetrapolar bioelectrical impedance method to assess human body composition. J Appl Physiol 60,1327–1332.[Abstract/Free Full Text]

Mayer EL, Jacobsen DW and Robinson K (1996) Homocysteine and coronary atherosclerosis. J Am Coll Cardiol 27,517–527.[CrossRef][Medline]

McGregor D, Shand B and Lynn K (2000) A controlled trial of the effect of folate supplements on homocysteine, lipids and hemorheology in end-stage renal disease. Nephron 85,215–220.[CrossRef][Medline]

Paradisi G, Steinberg HO, Hempfling A, Cronin J, Hook G, Shepard MK and Baron AD (2001) Polycystic ovary syndrome is associated with endothelial dysfunction. Circulation 103,1410–1415.[Abstract/Free Full Text]

Rasmussen LB, Ovesen L, Bulow I, Knudsen N, Laurberg P and Perrild H (2000) Folate intake, lifestyle factors, and homocysteine concentrations in younger and older women. Am J Clin Nutr 72,1156–63.[Abstract/Free Full Text]

Shechter M, Sharir M, Labrador MJ, Forrester J and Merz CN (2000) Improvement in endothelium-dependent brachial artery flow-mediated vasodilation with low-density lipoprotein cholesterol levels <100 mg/dl. Am J Cardiol 86,1256–1259.[CrossRef][Medline]

Spieker LE, Sudano I, Hurlimann D, Lerch PG, Lang MG, Binggeli C, Corti R, Ruschitzka F, Luscher TF and Noll G (2002) High-density lipoprotein restores endothelial function in hypercholesterolemic men. Circulation 26,1399–1402.[CrossRef]

Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D and Loscalzo J (1993) Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 91,308–318.[Medline]

Starkebaum G and Harlan JM (1986) Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77,1370–1376.[Medline]

Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G and Baron AD (1996) Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest 97,2601–2610.[Abstract/Free Full Text]

Stroes ES, van Faassen EE, Yo M, Martasek P, Boer P, Govers R and Rabelink TJ (2000) Folic acid reverts dysfunction of endothelial nitric oxide synthase. Circ Res 86,1129–1134.[Abstract/Free Full Text]

Title LM, Cummings PM, Giddens K, Genest JJ Jr and Nassar BA (2000) Effect of folic acid and antioxidant vitamins on endothelial dysfunction in patients with coronary artery disease. J Am Coll Cardiol 36,758–765.[CrossRef][Medline]

Usui M, Matsuoka H, Miyazaki H, Ueda S, Okuda S and Imaizumi T (1999) Endothelial dysfunction by acute hyperhomocyst(e)inaemia: restoration by folic acid. Clin Sci 96,235–239.[CrossRef][Medline]

Verhaar MC, Wever RM, Kastelein JJ, van Dam T, Koomans HA and Rabelink TJ (1998) 5-methyltetrahydrofolate, the active form of folic acid, restores endothelial function in familial hypercholesterolemia. Circulation 97,237–241.[Abstract/Free Full Text]

Verhoef P (2000) Hyperhomocysteinemia and risk of vascular disease in women. Semin Thromb Hemost 26,325–334.[CrossRef][Medline]

Vermeulen A, Verdonck L and Kaufman JM (1999) A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 84,3666–3672.[Abstract/Free Full Text]

Weiss N, Feussner A, Hailer S, Spengel FA, Keller C and Wolfram G (1999) Influence of folic acid, pyridoxal phosphate and cobalamin on plasma homocyst(e)ine levels and the susceptibility of low-density lipoprotein to ex-vivo oxidation. Eur J Med Res 15,425–432.

Weiss N, Keller C, Hoffmann U and Loscalzo J (2002) Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc Med 7,227–239.[CrossRef][Medline]

Welch GN and Loscalzo J (1998) Homocysteine and atherothrombosis. N Engl J Med 338,1042–1050.[Free Full Text]

Woo KS, Chook P, Chan LL, Cheung AS, Fung WH, Qiao M, Lolin YI, Thomas GN, Sanderson JE and Metreweli C (2002) Long-term improvement in homocysteine levels and arterial endothelial function after 1-year folic acid supplementation. Am J Med 112,535–539.[CrossRef][Medline]

Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, Sanderson JE, Metreweli C and Celermajer DS (1997) Hyperhomocysteinemia is a risk factor for arterial endothelial dysfunction in humans. Circulation 96,2542–2544.[Abstract/Free Full Text]

Woo KS, Chook P, Lolin YI, Sanderson JE, Metreweli C and Celermajer DS (1999) Folic acid improves arterial endothelial function in adults with hyperhomocysteinemia. J Am Coll Cardiol 34,2002–2006.[CrossRef][Medline]

Ziakka S, Rammos G, Kountouris S, Doulgerakis C, Karakasis P, Kourvelou C and Papagalanis N (2001) The effect of vitamin B6 and folate supplements on plasma homocysteine and serum lipids levels in patients on regular hemodialysis. Int Urol Nephrol 33,559–562.[CrossRef][Medline]

Submitted on September 18, 2003; accepted on February 4, 2004.