5-methyltetrahydrofolate restores endothelial function in uraemic patients on convective haemodialysis

Gherardo Buccianti1,, Sara Raselli2, Ivano Baragetti1, Fabrizia Bamonti3, Enzo Corghi1, Cristina Novembrino3, Cristina Patrosso4, Franco M. Maggi2 and Alberico L. Catapano2

1 Department of Internal Medicine, Nephrology and Dialysis Unit, Bassini Hospital, Cinisello Balsamo, Milan, 2 Department of Pharmacological Sciences, Center for the Study of Atherosclerosis, University of Milan, 3 Department of Medical Sciences, University of Milan, Maggiore Hospital IRCCS and 4 Clinical Chemistry and Haematology Laboratory, Hospital Niguarda Cà Granda, Milan, Italy



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hyperhomocysteinaemia is an independent risk factor for the development of atherosclerosis. In patients with chronic renal failure, the administration of folic acid or its metabolites reduces but does not normalize plasma homocysteine concentrations. Furthermore, homocysteine induces endothelial dysfunction by an increased inactivation of nitric oxide.

Methods. We examined the effect of the active metabolite of folic acid, 5-methyltetrahydrofolate (5-MTHF), 45 mg/week i.v. for 10 weeks, combined during the last 2 weeks with vitamin B12, 500 µg s.c. twice weekly, on homocysteinaemia and endothelial function in 15 patients undergoing convective haemodialysis. Endothelial function was evaluated by B-mode ultrasonography on the brachial artery. Flow-mediated dilation (FMD) was recorded during reactive hyperaemia produced by inflation of a pneumatic tourniquet. Nitroglycerine-mediated dilation (NMD) was recorded after administration of isosorbide dinitrate. Finally, the presence of the thermolabile variant of methyltetrahydrofolate reductase (t-MTHFR) was assessed by genotype analysis.

Results. Plasma homocysteine concentrations fell by 47% after treatment with 5-MTHF alone and by a further 13.6% after the addition of vitamin B12. The reduction was more marked in homo- and heterozygous patients than in normal genotypes for t-MTHFR. Flow-mediated endothelial vasodilation, measured by ultrasonography of the brachial artery, improved after administration of 5-MTHF (12.52± 2.47% vs 7.03±1.65%; P<0.05), but there were no further changes following the addition of vitamin B12.

Conclusions. Our study demonstrated that 5-MTHF administration not only reduced plasma homocysteine but also improved endothelial function in uraemic patients undergoing convective haemodialysis.

Keywords: 5-methyltetrahydrofolate; atherosclerosis; convective haemodialysis; endothelial function; plasma homocysteine



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Hyperhomocysteinaemia constitutes an independent risk factor for the development of atherosclerosis in patients with chronic renal failure [1] and is frequently associated with modifications of endothelial function [2]. Oral treatment with folic acid at doses varying from 5 to 60 mg reduces homocysteine plasma concentrations, although not to normal levels [3,4].

Folate metabolism is abnormal in patients with chronic renal failure. Plasma folate activities are reduced by plasma inhibitors [5], which probably alter the transformation of folate polyglutamate forms to monoglutamate [6]. Moreover, the anions retained in uraemia inhibit transmembrane transport of folate [7]. Abnormal folate metabolism probably results in an insufficient intracellular concentration of 5-methyltetrahydrofolate (5-MTHF), a metabolically active compound [6].

Intravenous administration of 50 mg/week folinic acid (5-formyltetrahydrofolate), the immediate precursor of 5-10-MTHF, combined with 250 mg pyridoxine three times weekly for 1 year normalized homocysteine plasma concentrations in 78% of one series [8], whereas high-dose 5-MTHF (105 mg/week), which bypasses the transformation mechanisms of the various metabolites at the intestinal level, reduced homocysteine by 70% in another series, normalizing the values in five patients [9].

The vascular endothelium opposes the atherosclerotic process by various mechanisms including the production of nitric oxide [10]. According to numerous studies, homocysteine plays a key role in inducing endothelial dysfunction and consequent vascular damage by altering the release of, and increasing the inactivation of nitric oxide [11].

In healthy subjects, administration of folic acid prevents the endothelial dysfunction produced by acute [12] or persistent hyperhomocysteinaemia [13]. A similar improvement of endothelium-dependent, flow-mediated vasodilation is observed in patients with familial hypercholesterolaemia [14] but not in uraemic patients studied either before [15] or after the start of dialysis treatment [16].

The aim of the present study was to evaluate the effect of intravenous administration of the metabolically active form of folic acid, 5-MTHF, for 10 weeks, combined with subcutaneous vitamin B12 in the last 2 weeks, on homocysteine plasma concentrations and endothelial function as shown by measuring flow-mediated vasodilation.



   Subjects and methods
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 Subjects and methods
 Results
 Discussion
 References
 
Patients
The study series consisted of 15 clinically stable patients, seven men and eight women (mean age 61±3.2 years), with chronic renal failure (autosomal dominant polycystic disease in three cases, chronic pyelonephritis in four, diabetic nephropathy in two, nephroangiosclerosis in five, IgA nephropathy in one), on dialysis for a mean period of 128±20.8 months. They received 4 h of haemodialysis, three times weekly, with the following characteristics: acetate-free biofiltration; AN 69 dialyzer (Nephral 300, Hospal; 1.4 m2); blood flow rate, 300 ml/min; dialysate flow rate, 500 ml/min; reinfusion rate in post dilution, 2000 ml/h; Na, K and Ca2+ in the dialysate, 150, 2–3 and 4 mEq/l, respectively. All patients gave informed consent to participate in the study.

Study design
According to the study design (Figure 1Go), patients first underwent a 12-month washout from folic acid and vitamin B12 supplementation (from T0 to T1). They then received treatment three times a week with slow intravenous administration of 15 mg 5-MTHF (Prefolic; Abbott Italia) diluted in 100 ml of isotonic saline solution at the end of each dialysis session for 8 weeks (T2). Vitamin B12 (500 µg s.c.) was administered twice weekly at the end of dialysis sessions and was then combined with 5-MTHF for an additional 2 weeks (T3).



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Fig. 1.   Study protocol.

 
Plasma homocysteine, serum and erythrocyte folate, and plasma vitamin B12 concentrations were determined at T0, T1, T2 and T3. Endothelial function during reactive hyperemia and after administration of isosorbide dinitrate was evaluated at T1, T2 and T3. Genotyping for the thermolabile variant of MTHF reductase (t-MTHFR) was performed at the start of the washout.

Laboratory methods
From each patient, two predialysis blood specimens were collected and placed into light-protected tubes, either plain for serum vitamin B12 and folate determinations or containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant for erythrocyte folate determinations. Both serum and EDTA whole blood aliquots were immediately frozen. EDTA–plasma aliquots, separated from whole blood within 30 min, were also frozen immediately. All samples were stored at -20°C until analysis. After thawing, serum vitamin B12 and folate determinations, haemolysate preparations and erythrocyte folate assays were performed using MEIA Fluorometric Enzyme-Linked Assays on an IMx analyzer (Abbott). Plasma homocysteine concentrations were measured with an IMx Homocysteine FPIA kit (Axis Biochemical ASA). Haematological values were measured in blood samples from each patient on a Coulter Counter model STKS. The reference intervals of all the measured variables were based on values determined in healthy volunteers.

Genotyping
DNA extraction was performed in all 15 patients. DNA was obtained from 50 µl of whole blood collected in EDTA tubes and processed by a resin matrix (Instagene Whole Blood Kit; Bio-Rad).

MTHFR mutation (MTHFRA223V) analysis was performed by allelic discrimination. The PCR mixture was: 10 mM Tris–HCl pH 8.3, 50 mM KCl, 5 mM MgCl2, 0.8 mM dNTPS with dUTP, 8% glycero1, 0.9 µM of each primer, 0.05 µM FAM probe, 0.15 µM TET probe, 2.5 U TaqGold (Perkin Elmer Cetus, Norwalk, CT, USA), 0.05 U UNG, 5 µl of DNA (100 mg) in a 25-µl total volume. The primers were: TMTHFR for 5'-CACAAAGCAAGAATGTGTCA-3' and TMTHFR rev-5'-GACCTGAAGCACTTGGAGAA-3'; and the probes were 5' FAM-ATGATGAAATCGACTCCCGCAG and 5' TET-ATGATGAAATCGACTCCCGACA.

PCR conditions were as follows: one cycle at 50°C for 2 min; a hot start at 94°C for 10 min; 45 cycles of denaturation for 15 s each and annealing at 60°C for 1 min.

Fluorescence detection of different genotypes was performed by ABI Prism Sequence Detection System (PE Applied Biosystems).

Endothelial function test
Endothelial function was evaluated non-invasively by B-mode ultrasonography (Biosound Au4 idea) with a 10 MHz linear array transducer on a brachial artery. During each test, vessel images were taken at rest, during reactive hyperemia (flow-mediated dilation, FMD) and after sublingual administration of isosorbide dinitrate (nitroglycerin-mediated dilation, NMD).

Vessels were imaged longitudinally, 2–10 cm above the antecubital crease, ensuring optimal visualization of anterior and posterior wall–lumen interfaces and a constant artery diameter. Patients were required to lay at rest for 10 min before the test (temperature 25±2.3°C).

Tests were performed on the same artery with the arm and the hand immobilized in a fixed position to ensure scans in the same vessel portion and projection. During follow-up, each patient was studied at the same hour of the day and on the same day of the week during the interdialytic period. FMD tests were performed by selecting, at rest, three images of the brachial artery at end diastole (B0, B1, B2, respectively). Four images were recorded during reactive hyperaemia, produced by inflation of a pneumatic tourniquet to a pressure of 200 mm Hg for 4.5 min. Measurements were made 30, 90, 150 and 210 s after cuff deflation (T30, T90, T150 and T210, respectively).

The NMD test was performed after at least a 10 min rest. The brachial artery was identified under basal conditions in the same arm position as the FMD test (three images: B3, B4 and B5). Sublingual isosorbide dinitrate was then administered and three vessel images were taken 4–6 min later (ISDN1, ISDN2, ISDN3).

Images were saved on floppy disks, converted from AU4 files to bitmap files, and printed using a high resolution HP laser printer. Each printed image was measured by a blinded, independent operator using a manual calliper at one fixed point of the vessel.

FMD was calculated by:

(001)

NMD was calculated by:

(002)

The reproducibility of FMD evaluation was tested in eight subjects examined on two occasions, with a mean interval of 1 week. The mean coefficient of variation was 9%.

Statistical analysis
Values are expressed as means±SE. Data were analysed using SPSS for Windows. The normal distribution of our data required the use of parametric tests. Analysis of variance (ANOVA) was utilized to compare mean values of plasma homocysteine, serum and erythrocyte folates and plasma vitamin B12 at T0, T1, T2 and T3, differences in homocysteine concentration according to t-MTHFR genotypes, and endothelium-dependent and -independent vasodilation findings at T1, T2 and T3.



   Results
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 Subjects and methods
 Results
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As shown in Table 1Go, plasma homocysteine concentrations were significantly higher at the end than at the start of the washout (50.9±9.99 vs 42.4±9.01 µmol/l; P<0.01) and fell after 5-MTHF treatment to 23.02±2.33 µmol/l (P<0.004). When vitamin B12 was combined with 5-MTHF, plasma homocysteine showed a further decrease to 17.4±2.04 µmol/l (P<0.0002) (Figure 2Go).


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Table 1.  Patient characteristics

 


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Fig. 2.  Effect of treatment with 5-MTHF and vitamin B12 on plasma homocysteine concentration in each patient (a) and on mean plasma homocysteine concentration (b) at various study times. *P<0.01.

 
Serum folate levels decreased significantly during the washout period from 9.2±0.99 to 5.9±0.59 ng/ml (P<0.002). They rose significantly after 5-MTHF treatment to 51±1.3 ng/ml (P<0.001), and were unchanged after combined vitamin B12 with 5-MTHF (48.7±0.85 vs 51.0±1.3 ng/ml).

Erythrocyte folate concentrations did not change from T0 to T1 but rose significantly after 5-MTHF treatment (622.1±72.65 to 2168±118.9 nmol/l; P<0.001). They were not altered by the addition of vitamin B12.

Vitamin B12 plasma levels did not change and were within the normal range at T0, T1 and T2, but rose significantly after the addition of vitamin B12 to 5-MTHF treatment (517.5±50.63 vs 1933.9± 56.7 pg/ml; P<0.0001) (Table 1Go).

The per cent reduction in plasma homocysteine was significantly greater in both t-MTHFR homozygotes (P<0.05) and heterozygotes (P<0.05) compared with patients with normal genotypes (-65.6±11.01; and -54.1±7.5 vs -25.9±6.1, respectively).

At T1, T2 and T3, endothelial function was evaluated by determining the vasodilatory response of the brachial artery to reactive hyperaemia and isosorbide dinitrate. The absolute and percentage variation in vessel diameter induced by hyperaemia increased significantly after 5-MTHF treatment (5.17±0.57 mm vs 4.91±0.74 mm, P<0.05, and 12.5±2.47% vs 7.03±1.65%, P<0.05, respectively) but was unaltered after pharmacological dilation. Supplementation with vitamin B12 did not further modify vasodilatory responses to reactive hyperaemia or to pharmacological dilation (Figures 3Go and 4Go).



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Fig. 3.  Endothelium-dependent vasodilation (FMD) at T1, T2 and T3, expressed as per cent variation of brachial artery diameter after ischaemia induced by sphygmomanometer cuff. *P<0.05.

 


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Fig. 4.  Endothelium-independent vasodilation (NMD) at T1, T2 and T3, expressed as per cent variation of brachial artery diameter after sublingual administration of isosorbide dinitrate. *P<0.05.

 



   Discussion
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 Subjects and methods
 Results
 Discussion
 References
 
In this study, plasma homocysteine decreased by 47% after 8 weeks of i.v. 5-MTHF, and by 60.6% after 2 additional weeks of combined s.c. vitamin B12 with 5-MTHF. Other authors reported that folic acid administered orally at doses varying from 1 to 60 mg produced reductions in homocysteine levels that varied from 30 to 40% [3,4,17], with maximumal responses obtained at 15 mg/week [17].

Normal subjects have endogenous pools of folates derived from the diet that are stored in tissues and then used to maintain normal levels of plasma folate. During folate deficiency, the enterohepatic circulation supplies an extracellular folate pool that is readily available for distribution to tissues. If the deficiency persists, a progressive reduction in the hepatic reserve occurs while folates from metabolically inactive red blood cells partially compensate for the deficiency, delivering these to the hepatic tissue through a metabolic support pathway [18].

Folate of dietary origin, introduced in the form of polyglutamate, requires the presence of glutamyl carboxypeptidase for its transformation to monoglutamate in the intestinal wall. This passes via the portal vein to the liver where it undergoes transformation first to dihydrofolate, then to tetrahydrofolate, and finally to 5-10-MTHF. This latter compound is reduced to 5-MTHF by the enzyme MTHFR. 5-MTHF returns to the small intestine through the enterohepatic circulation, and after absorption is distributed to the tissues [19].

In uraemia, experimental and clinical data suggest the presence of plasma inhibitors that limit the activity of the conjugases responsible for the transformation of polyglutamate to monoglutamate [5], for transmembrane transport of folic acid [7], and for MTHF absorption [20]. These data, although fragmentary, suggest that treatment with active metabolites of folic acid, both oral and intravenous, is more efficacious than the use of folic acid itself [6]. After a 2-month oral treatment with 5-MTHF, Perna et al. [9] observed a 70% reduction in plasma homocysteine in 14 haemodialysed patients, with normalization in five patients. Touam et al. [8], administering 50 mg i.v. folinic acid once a week at the end of dialysis sessions combined with 250 mg i.v. pyridoxine three times weekly (plus 1 mg/day vitamin B12 in two patients) for ~1 year, reported a 67% reduction in plasma homocysteine compared with baseline, and normalization was achieved in 78% of the patients. In a cross-sectional study from our dialysis population, 27 of 55 patients given 0.9 mg i.v. folinic acid plus 1.5 mg hydroxycobalamin and 0.5 mg cyanocobalamin for macrocytosis at the end of each dialysis session for at least 6 months had reduced homocysteine levels compared with non-treated patients [21]. In contrast, two recent papers [22,23] found a preponderance of haemodialysis patients exhibiting mild hyperhomocysteinaemia that was refractory to treatment with folic acid or 5-MTHF. In addition, these patients had a similar lowering of plasma homocysteine to folic acid, folinic acid or 5-MTHF. The mild hyperhomocysteinaemia was probably related to the fact that folate fortification in food was introduced in the US some years ago.

It has been reported that plasma folate levels are persistently elevated even 4 months after suspension of folic acid supplementation [24]. In the present study, we suspended folate supplementation for 12 months to ensure a state of folate deficiency. After this, addition of the active metabolite, 5-MTHF, reduced plasma homocysteine levels by 47%, and vitamin B12 with 5-MTHF caused a further reduction of 13.6%.

Parenterally administered vitamin B12 efficaciously reduces plasma homocysteine levels in diabetic [25] and haemodialysed patients [26]. However, this lowering effect of vitamin B12 was less clear in studies that administered it in combination with folic acid and pyridoxine to dialysed patients by oral [3] or intravenous routes [8]. The notion that vitamin B12 modulates homocysteinaemia was based on the observation that, during remethylation, vitamin B12 acquires a methyl group from 5-MTHF or betaine to form methionine. Although this reaction occurs in all tissues and is vitamin B12-dependent, the reaction with betaine is vitamin B12-independent.

Two factors appear to be important for reducing plasma homocysteine concentrations: the availability of the active metabolite of folic acid or its immediate precursor, and the presence of vitamin B12. The intravenous route, which bypasses intestinal folate metabolism, is inhibited by the uraemic milieu, producing an improved response even though an equivalent response seems to be obtained with oral administration of the active metabolite at elevated doses for 8 weeks 9]. In addition, the percentage of homocysteine is extremely reduced in the dialysate, and convective treatments such as those given to our patients remove uraemic toxins that develop inhibitory activities against the transmethylation and transsulfuration pathways [27].

In our patients, reductions in plasma homocysteine were significantly greater in t-MTHFR homozygotes and heterozygotes than in subjects with normal genotypes. This is probably related to higher substrate concentrations in the former two subgroups than in the third, which confirms the observation of Tremblay et al. [3].

Numerous in vitro and in vivo studies suggest that the primary mechanism of atherogenesis consists of endothelial dysfunction, probably mediated by homocysteine induced increases in oxidative stress. In healthy subjects, administration of folic acid prevents the endothelial dysfunction produced by acute [12] or persistent hyperhomocysteinaemia [13]. A similar improvement of endothelium-dependent flow-mediated vasodilation is observed in patients with familial hypercholesterolemia [14], but not in uraemic patients studied both before [15] and after the start of dialysis treatment [16].

Several characteristics of our dialysis population may explain differences between our results and those of another study, also with haemodialysis patients [16]. For instance, our patients had very low folate levels in comparison with normal levels in the study by van Guldener et al. [16]. Furthermore, oral folic acid was given in the latter study rather than intravenous administration of the metabolically active form. Finally, our patients were on convective haemodialysis.

Before starting 5-MTHF administration, we measured flow-mediated dilation in five patients before and after a dialysis session. Before dialysis, endothelial responses of the patients were markedly reduced compared with 90 healthy volunteers (3.4±1.4 vs 12.6±1.9%). After 4 h of dialysis, flow-mediated dilation recovered (14.3±1.9%) but deteriorated rapidly over the next 6–12 h. Similarly, in a recent study that investigated the variation of the interdialysis curve of plasma homocysteine in six patients, plasma homocysteine was increased 8 h after the session [27]. In the present series, we demonstrated that 5-MTHF supplementation for 2 months significantly improved endothelium-dependent flow-mediated vasodilation, whereas maximum endothelium-independent vasodilation did not alter significantly.

At the endothelial level, endogenous nitric oxide in the presence of homocysteine is transformed to S-nitrous homocysteine, neutralizing its potential toxicity. When homocysteine concentrations are elevated, nitric oxide is no longer able to control this reaction. Following this, there are reductions in nitric oxide production, and homocysteine causes further vascular damage [11].

As seen in familial hypercholesterolaemia and coronary disease [2830], 5-MTHF administered to our uraemic patients probably had a positive effect on nitric oxide availability at the endothelial level by modulating its production or reducing its catabolism.

The main limitation of our prospective study was the lack of an appropriate control group. This was caused by the difficulty in finding appropriate numbers of patients undergoing acetate-free biofiltration without folate and vitamin B12 supplementation. Our study also did not determine the minimal dose of folate or the reduction in homocysteinaemia necessary to modulate the endothelial response.



   Notes
 
Correspondence and offprint requests to: Prof. G. Buccianti, Azienda Ospedaliera S. Gerardo, Presidio E. Bassini, Via Gorki 50, CAP 20092, Cinisello Balsamo, Milano, Italy. Email: ghbucci{at}hotmail.com Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 7.01
Accepted in revised form: 29.11.01