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Department of Obstetrics and Gynecology (P.V., C.P., R.S., F.C., A.L.), and Institute of Biological Chemistry (M.R., C.M.), Catholic University of Sacred Heart, 00168 Rome, Italy; and National Research Center, Institute for System Analysis and Informatics Biomatlab (S.P.), 00168 Rome, Italy
Address all correspondence and requests for reprints to: Dr. Antonio Lanzone, Department of Obstetrics and Gynecology, Catholic University of Sacred Heart, L. go Gemelli 8, 00168 Rome, Italy. E-mail: alanzone{at}rm.unicatt.it.
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Abstract |
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Objective: The aim of this study was to examine the effect of L-folic acid supplementation on homocysteine (Hcy) and related thiols, such as cysteine (Cys) and Cys-glycine (Cys-Glyc) pathways and their relationship to glucose, insulin, and lipidic metabolism in normoinsulinemic postmenopausal women.
Design: This study was a randomized placebo, not double-blind, trial.
Setting: The study was performed in an academic research center.
Patients or Other Participants: Twenty healthy postmenopausal women were selected. No patient was taking drugs known to affect lipid or glucose metabolism.
Intervention(s): Patients underwent two hospitalizations before and after 8 wk of L-acid folic (7.5 mg/d) or placebo administration. The glycemic metabolism was studied by an oral glucose tolerance test and a hyperinsulinemic euglycemic clamp. Hcy metabolism was studied by a standardized oral methionine-loading test.
Main Outcome Measure(s): Hcy, Cys, and Cys-Glyc, basally and after a methionine loading test, were measured. Basal insulin, glucose, and peptide C levels as well as area under the curve for insulin, area under the curve for peptide, hepatic insulin extraction, and metabolic index were assayed. The total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels and the cholesterol/HDL and LDL/HDL ratios were also measured.
Results: The total basal Hcy concentration and the plasma postmethionine loading Hcy values were significantly decreased (P < 0.01) in L-folic acid-treated patients, whereas postmethionine loading Cys-Glyc levels were markedly increased (P < 0.02). Furthermore, L-folic acid intake induced a significant improvement in carbohydrate metabolism through an increase in fractional hepatic insulin extraction (P < 0.05) and peripheral insulin sensitivity (P < 0.02) in normoinsulinemic women. HDL levels considerably increased, inducing an improvement in other atherosclerotic indexes, such as cholesterol/HDL and LDL/HDL ratios (P < 0.03).
Conclusions: These results show that folic acid supplementation lowers plasma Hcy levels and improves insulin and lipid metabolism, reducing the risk of cardiovascular disease.
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Introduction |
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). Several enzymatic defects in the metabolism of methionine and nutritional deficiencies of vitamin B6, B12, and folic acid can lead to elevated plasma total Hcy (tHcy) concentrations (1, 2), whereas supplementation of folic acid lowers Hcy levels (3).
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Experimental studies suggest that homocysteine promotes atherogenesis by producing endothelial injury, reduction of vascular nitric oxide (NO) production, and bioavailability as well as oxidative modification of low-density lipoprotein (LDL) (5).
Cysteine (Cys), the major aminothiol in human plasma and a substrate for the synthesis of glutathione (GSH), although less reactive than Hcy, promotes detachment of human arterial endothelial cells in culture (6) and exhibits autooxidation properties in the presence of metal ions, thus producing free radicals and in vitro hydrogen peroxide (7).
Cys-glycine (Cys-Glyc) is formed by the breakdown of GSH by 
-glutamyl transpeptidase, an enzyme mostly localized on the external surface of the cell membrane, but also found in plasma (8).
Hcy, Cys, and Cys-Glyc in plasma interact via redox and disulfide reactions; these aminothiol species (reduced, free oxidized, and protein-bound forms) comprise a dynamic system, referred to as redox thiol status, which is linked to the extracellular antioxidant defense system (9).
The methionine-loading test, which stresses the homocysteine recycling/disposal allowing the characterization of a sort of methionine intolerance, is useful in clinical studies because it can screen a sizable percentage of people (from 40–55%) who may have disturbances in methionine metabolism that fasting total plasma Hcy determination alone may fail to identify (3).
Many factors may influence Hcy levels, such as age, gender, nutrition, smoking, stress, chronic inflammation, and sex steroids (10, 11), and insulin has also recently been considered as a modulating factor of Hcy (12). Therefore, many clinical situations with increased levels of insulin, such as noninsulin-dependent diabetes, hypertension with insulin resistance, and polycystic ovary syndrome, have been associated with elevated plasma Hcy levels (13, 14, 15).
After menopause, basal Hcy levels increase progressively, and hormone replacement therapy reduces Hcy concentrations (16, 17). It has been found that low-dose folic acid supplementation reduces plasma Hcy levels in postmenopausal women (18). Menopause is followed by a progressive decrease in insulin sensitivity and a deterioration in carbohydrate and lipid metabolism, leading to a significant rise in the incidence of cardiovascular events (19).
At present there is conflicting evidence about the relationship between Hcy and glucose, insulin, and lipidic metabolism. The aim of our randomized, controlled trial was to examine the effects of folic acid supplementation on Hcy levels and metabolism through a standardized methionine-loading test, evaluating both remethylation and transsulfuration pathways. We particularly studied the complex relationship between Hcy metabolism and carbohydrate and lipid metabolism in healthy normoinsulinemic postmenopausal women.
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Subjects and Methods |
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Twenty healthy postmenopausal women (age, 48–61 yr) who were attending the Gynecological Department of our university were selected. They were 6.4 ± 1.2 yr (mean ± SE) postmenopausal; two of them had undergone hysterectomy with bilateral oophorectomy. Before the beginning of the study, assessment of plasma FSH (>50 IU/liter) and 17ß-estradiol (<73 pmol/liter) concentrations, mammography, cervical cytology, and transvaginal ultrasound examination of ovaries and endometrial thickness were performed. These parameters were found to be normal and compatible with menopausal status. No patient was taking drugs known to affect lipid or glucose metabolism. None smoked more than 10 cigarettes/d or drank more than 300 g alcohol/wk. Diabetes or impaired glucose tolerance, breast cancer, liver or kidney parameter alterations, history of major thromboembolism, thyroid disease, and uncontrolled or treated hypertension (systolic blood pressure > 160 mm Hg or diastolic > 90 mm Hg) were exclusion criteria.
The study was approved by the ethical committee of our department, and informed consent was obtained from each patient.
In a randomized placebo, not double-blind, study, eligible women who agreed to participate were allocated to one of two treatment groups: group A was treated with 7.5 mg/d folic acid (L-isomer; Levofolene, Schering, Rome, Italy), and group B was treated with commercial capsules of calcium as placebo. The active drug and the placebo were administered for 60 d.
Study design and measurements
Patients underwent two hospitalizations before and after 8 wk of L-acid folic or placebo supplementation. During each hospitalization, basal hormones, carbohydrate and lipid parameters, as well as homocysteine pathways were assayed. Glycemic metabolism was studied by an oral glucose tolerance test (OGTT) and a hyperinsulinemic euglycemic clamp. Before and after treatments, after 3 d of a standard carbohydrate diet (300 g/d) and fasting overnight for 10–12 h, patients underwent metabolic evaluation. The OGTT was performed as follows. At 0800 h an indwelling catheter was inserted into the antecubital vein of one arm. Blood samples were collected 0, 30, 60, 90, 120, 180, and 240 min after ingestion of 75 g glucose. Glucose, insulin, and C peptide were assayed for each of the samples. To evaluate the relation between insulin response to glucose load and Hcy plasma levels, we drew blood samples during the OGTT test basally and at 60, 90, and 120 min to assay plasma Hcy levels.
On a different day, a hyperinsulinemic-euglycemic clamp was performed after a 10-h overnight fast to estimate peripheral insulin sensitivity. A retrograde iv catheter was inserted into the antecubital vein for the infusion of glucose and insulin. Another catheter was placed in the dorsal vein of the contralateral hand to permit blood withdrawal and was warmed to 65 C with a warming box to arterialize the venous blood samples. A primed constant infusion of insulin (Actrapid HM, Novo Nordisk, Copenhagen, Denmark) was administered at a rate of 40 mIU/m2·min. After reaching the steady-state velocity for the insulin infusion within 10 min to achieve steady-state insulin levels of approximately 717 pmol/liter during the clamp (range, 574–897 pmol/liter), a variable infusion of 20% glucose was initiated via a separate infusion pump. Plasma glucose samples were taken every 5 min from the arterialized line, and blood glucose infusion was adjusted to maintain plasma glucose between 4.4 and 4.99 mmol/liter.
Samples for plasma glucose concentration as well as for biochemical parameters were assayed immediately; for all other determinations, samples were promptly centrifuged, and plasma was stored at –20 C until assayed. The plasma glucose level was determined using a glucose oxidase technique with a glucose analyzer (Beckman Instruments, Palo Alto, CA). A normal glycemic response to OGTT was defined according to the criteria of the National Diabetes Data Group (20).
All hormones were measured using a commercial RIA (Radim, Pomezia, Italy). The intra- and interassay coefficients of variation were less than 8% and 15%, respectively, for all hormones.
Insulin and C peptide plasma levels were expressed as fasting values and areas under the curve (AUCs) after the glucose load, which was calculated by the trapezoidal rule. The patients were classified as normoinsulinemic according to their insulin response to OGTT, assuming an AUC cutoff value of 107,625 pmol/liter·240 min, as established by standard procedures of our laboratory (21).
Hepatic insulin extraction was estimated according to the following: difference between molar secretory areas of C peptide and insulin divided by molar secretory area of C peptide (22). Insulin sensitivity was calculated as the metabolic index (M) of total body glucose utilization, set between 60 and 240 min of the glucose clamp, expressed as milligrams per kilogram of body weight per minute (23).
Total cholesterol and triglyceride concentrations were determined by an enzymatic assay (Bristol, Paris, France). High-density lipoprotein (HDL) concentrations were determined after precipitation of chylomicrons, very-low-density lipoprotein (VLDL), and LDL (Roche, Mannheim, Germany), VLDL was separated (as the supernatant) from LDL and HDL by lipoprotein ultracentrifugation. A magnesium chloride/phosphotungstic acid technique was used to precipitate LDL from the bottom fraction after ultracentrifugation. Nonesterified fatty acids were determined by an acyl-coenzyme A oxidase-based colorimetric method. All lipid assays were performed according to our standard laboratory procedures.
Hcy metabolism was studied by a standardized oral methionine-loading test, which was performed at baseline and after 2 months of treatment (24). We have assumed a normal plasma Hcy level of less than 15 µmol/liter and a plasma vitamin B12 normal range of 200–480 pg/liter. After consuming their usual diet for 3 wk before hospitalization, the participants were asked to fast for 12 h before the methionine load. For each participant, a fasting blood sample was drawn from the antecubital vein without stasis into a vacuum refrigerated lithium heparin tube (Venoject, Terumo Europe, Leuven, Belgium) to measure the plasma Hcy, serum and red cell folate, and serum vitamin B12 levels. Plasma for thiol determination was immediately separated to avoid time- and temperature-dependent release of Hcy from blood cells (25).
The subjects then underwent a methionine-loading test in which L-methionine (Sigma-Aldrich Corp., St. Louis, MO; 0.1 g/kg body weight) was given orally in 100–120 ml orange juice, and specimens were collected basally and after 6 h. During the methionine-loading test the participants were not allowed to consume any solid food or beverage containing proteins, but only water or tea.
These samples were centrifuged at 3000 x g for 15 min at 4 C within 10 min from blood collection, and plasma was immediately frozen at –80 C until analysis. Plasma tHcy, Cys, and Cys-Glyc were measured by HPLC and fluorescence detection according to the method described by Mansoor et al. (26) using an Alltech Alltima C18, 3 µm Rocket column (Alltech Associates, Inc., Lokeren, Belgium) equipped with a guard column to shorten the elution times from 56 min of the original method to 20 min for the entire chromatographic run. Plasma and erythrocyte folates and plasma vitamin B12 were measured with chemiluminescent methods on an Elexys 2010 (Roche Diagnostic, Milan, Italy). Plasma methionine was measured with by HPLC.
The body mass index was evaluated according to the ratio of weight (kilograms) to height (meters squared).
The waist to hip ratio was used to define body fat distribution (the waist measurement is circumference obtained from the minimum value between the iliac crest and the lateral costal margin; the hip measurement is circumference of the minimum value over the buttocks).
Statistical analysis
Data were stored and analyzed using SPSS software (statistical package for social science, release 6.0, SPSS, Inc., Chicago, IL) on an IBM-compatible computer. Comparison within groups were performed by Wilcoxon signed-rank test for paired data. For all subjects, absolute deltas (posttreatment minus pretreatment values) were computed on each variable. In the comparison between L-folic- and placebo-treated patients, the Mann-Whitney U test was performed on both variables at baseline to verify that the two samples were homogenous at the beginning of the study period and on the absolute deltas to verify that there were differences in the change over time.
Within the two samples of subjects, the Friedman test was used on the variable Hcy to verify that a significant groups difference in trend arose during the OGTT experiment before and after treatment.
The relationship between variables was analyzed by the Spearman rank correlation. A two-tailed P < 0.05 was considered statistically significant. All results are expressed as the mean ± SE.
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Results |
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Table 1 shows the anthropometric and metabolic features before and after folic acid supplementation in healthy postmenopausal women (group A) and placebo-treated subjects (group B). For all considered variables, no significant difference between the two groups was detected at baseline, and no significant change was observed after treatment inside each group.
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In Table 2 the methionine vitamin B 12, Hcy, Cys-Glyc, Cys, and plasma and red blood cell folate concentrations are reported before and after therapy in the two groups of patients. Both groups have similar basal methionine and Hcy concentrations. Noteworthy is the fact that basal folate plasma concentrations in all postmenopausal women were at the lowest limits of normal range. Basal Hcy levels decreased significantly after L-folic acid supplementation (P < 0.01) in group A patients, whereas group B showed a constant trend. In the comparison of delta values between groups A and B, significant differences were obtained for the basal Hcy levels and, as expected, for both plasma and erythrocyte folate concentrations. No significant difference was obtained for deltas computed on the Cys and Cys-Glyc basal concentrations even if Cys showed a modest trend toward increased basal levels in the L-folic acid-treated group.
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Figure 2 shows the means of Hcy levels at 0, 60, 90, and 120 min in the OGTT performed in group A and B patients before and after therapy. Hcy did not undergo any significant change corresponding to insulin peaks during OGTT in either groups.
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Concerning the lipid profile, plasma concentrations of cholesterol, triglycerides, LDL, VLDL, and nonesterified fatty acids did not show any significant variations; however, the HDL plasma levels significantly increased after folic acid treatment (P < 0.01). Comparison of the HDL changes over time showed a significant difference between the two groups (P < 0.03; Table 3).
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Fasting glucose levels as well as insulin and C peptide plasma concentrations were unchanged compared with the baseline in both groups (Table 4).
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Correlation between variables
We found a significant inverse correlation between the difference in basal Hcy values (posttreatment – pretreatment values) and the difference in M values (r = –0.697; P < 0.02).
A significant correlation was found between decremental AUC-I values (the difference between AUC-I values after and before treatment) and incremental PML-Cys-Glyc values (P < 0.01) and between the incremental percentage of PML- Cys-Glyc and the incremental percentage of PML-Cys in the L-folic acid-treated group (P < 0.01), whereas the correlations in the placebo group were not significant.
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Discussion |
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The results of this study provide information about a wide range of effects linked to folic acid supplementation in normohomocysteinemic, normoinsulinemic, menopausal subjects and for the first time about a possible direct relationship between the complex reactions of Hcy remethylation, and transsulfuration and glycolipid metabolism.
In agreement with other studies (18, 24), we showed that folic acid implementation, confirmed by the significantly higher plasma and intraerythrocyte folic acid concentrations, reduces fasting Hcy levels and determines a reduction in the PML-Hcy increase in healthy postmenopausal women.
At the same time after folic acid treatment, we observed a significant increase in postmethionine levels of Cys-Glyc and a trend toward augmented basal Cys levels. Both these proteins are important steps in the redox system. Although Cys-Glyc is a direct metabolite of GSH, cysteine is an important amino acid for GSH synthesis in humans (29, 30).
The increased PML-Cys-Glyc levels found in our study after folate supplementation could be the result of increased intracellular GSH synthesis and turnover generated by a more efficient intracellular flux of substrates through the methionine-Hcy cycle. Considering our data, we hypothesized a more efficient transsulfuration pathway.
The folic acid supplementation may determine a greater availability of methyl and sulfate groups as cofactors/substrates leading to a new synthesis of methionine, but also to an overall activation of other enzymatic pathways in the liver (31) and kidneys (32). Particularly, Hcy, Cys, and Cys-Glyc in plasma interact via redox and disulfide reactions becoming part of a dynamic system referred to as redox thiol status, which is linked to the antioxidant defense system (9).
At present, although the relationship among Hcy, Cys, Cys-Glyc, and other risk factors in the pathogenesis of atherosclerotic mechanisms is not completely known, it is clear that folate supplementation may act directly to influence lipidic and glycemic metabolisms (33, 34).
In the present study there was a significant increase in HDL levels and an improvement in the atherogenic indexes after folic acid treatment. This effect has been observed in previous experimental studies (35), supporting the evidence of an enhanced LDL oxidation and a clearance of LDL after binding to Hcy (33). Other in vivo studies (36) confirm our observations. Moreover, another recent study (10) indicates that folate supplementation improves hepatic metabolism, perhaps by inducing general changes in the anabolic and catabolic states. A crucial role of the liver is indicated, because only oral postmenopausal hormone replacement therapy, not transdermal therapy, is able to reduce Hcy levels (31). In this connection our results also stress the contribution of plasma thiols such as Cys-Glyc or Cys enhanced by folic acid supplementation in improving the liver-led lipidic metabolism.
Hyperhomocysteinemia has been found in patients with noninsulin-dependent diabetes and insulin-dependent diabetes and in patients with insulin resistance, such as obese or polycystic ovary syndrome subjects (13, 14, 15, 37). Hyperinsulinemia seems to influence Hcy metabolism through effects on glomerular filtration or by influencing the activity of key enzymes in Hcy metabolism (38, 39). Therefore, it may be hypothesized that there is a direct influence of hyperhomocysteinemia on insulin resistance through endothelial dysfunction and other cellular oxidative stress in skeletal muscle, liver, and adipose tissue, giving rise to insulin resistance (40, 41). Hcy can directly damage endothelial cells, impairing the release of protective NO and determines a net increase in damaging superoxide (O–2) (42). The oxidant stress may cause a subnormal activity of NO synthase enzyme, which may mediate or increase the peripheral effect of insulin (43). It has been recently shown that folic acid is able to prevent NO synthase dysfunction under different conditions (44). Thus, high-dose folate may influence insulin sensitivity through the action on NO synthase.
However, the direction of causality in the association between hyperhomocysteinemia and hyperinsulinemia is not clear. Even if it remains difficult to assign cause or effect to insulin resistance or hyperhomocysteinemia, our findings seem to indicate that Hcy or other factors involved in folate metabolism directly influence glycemic metabolism.
The in vivo relevance of our study was the finding of the influence of improved Hcy metabolism, due to folic acid availability, on insulin sensitivity. In fact, we showed a significant improvement of M index, a trend toward a reduction of AUC insulin, and an increased hepatic clearance of insulin.
Therefore, the incremental M and decremental AUC-I directly correlate with Hcy and PML-Cys-Glyc changes. However, even if our results do not allow us to consider that the effect is only due to a change in Hcy and a direct effect of folic acid supplementation through implementation of the transsulfuration pathway, we confirm the growing evidence that Hcy-induced oxidant stress may cause insulin resistance. In fact, in vitro studies have recently found that oxidant stress reduces insulin responsiveness by interrupting insulin signals (45). Moreover, another in vivo study has highlighted the fact that the increase in Hcy levels may not be a general complication of the insulin resistance syndrome, because healthy volunteers with altered insulin-mediated glucose disposal have no increased Hcy levels (46). Our results also show that during the OGTT, in correspondence with the highest insulin levels, Hcy levels do not change before or after folate treatment.
In conclusion, our findings may have implications for clinical prevention. Folic acid supplementation is safe and beneficial for lowering plasma Hcy levels, because it improves insulin sensitivity and lipid metabolism, helping to reduce the risk of cardiovascular diseases in postmenopausal women.
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Footnotes |
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Abbreviations: AUC, Area under the curve; AUC-I, insulin AUC; Cys, cysteine; Glyc, glycine; GSH, glutathione; Hcy, homocysteine; HDL, high-density lipoprotein; LDL, low-density lipoprotein; M, metabolic index; 5-MTFH, 5-methylenetetrahydrofolate; NO, nitric oxide; OGTT, oral glucose tolerance test; PML, postmethionine loading; tHcy, total Hcy; VLDL, very-low-density lipoprotein.
Received October 4, 2004.
Accepted May 11, 2005.
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, before L-folic acid treatment; 
, after L-folic acid treatment. Right, Group B: