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
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Abstract |
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Key words: endothelium/folic acid/menopause
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Introduction |
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Materials and methods |
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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., 1992). 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, 1015 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 34 min later the last scan was performed. For the reactive hyperaemia scan, measurements of diameter were taken 5060 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., 1999).
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 subjects body composition was performed with a tetrapolar impedance plethysmograph [Soft Tissue Analyzer (STA/BIA); Akern Bioresearch, Italy] according to Lukasky et al. (1986). 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 Students 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.
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Results |
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Discussion |
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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., 1999). 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., 2000
; Ziakka et al., 2001
). 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., 2002
; Spieker et al., 2002
). Conversely, high levels of LDL-C have been shown to be associated with endothelial dysfunction (Shechter et al., 2000
). 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., 1976; Colditz et al., 1987
). 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., 1996; Paradisi et al., 2001
). 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.
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Acknowledgements |
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References |
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Submitted on September 18, 2003; accepted on February 4, 2004.