Nephrol Dial Transplant (2002) 17: 857–864
Original Article
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,
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 4Clinical Chemistry and
Haematology Laboratory, Hospital Niguarda Cà Granda, Milan, Italy
2
Abstract
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 mguweek i.v. for 10 weeks, combined
during the last 2 weeks with vitamin B12, 500 mg 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. Flowmediated 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.
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@
hotmail.com
#
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
Hyperhomocysteinaemia constitutes an independent
risk factor for the development of atherosclerosis in
patients with chronic renal failure w1x and is frequently
associated with modifications of endothelial function w2x. Oral treatment with folic acid at doses varying from 5 to 60 mg reduces homocysteine plasma
concentrations, although not to normal levels w3,4x.
Folate metabolism is abnormal in patients with
chronic renal failure. Plasma folate activities are
reduced by plasma inhibitors w5x, which probably alter
the transformation of folate polyglutamate forms to
monoglutamate w6x. Moreover, the anions retained
in uraemia inhibit transmembrane transport of folate
w7x. Abnormal folate metabolism probably results in
an insufficient intracellular concentration of 5-methyltetrahydrofolate (5-MTHF), a metabolically active
compound w6x.
Intravenous administration of 50 mguweek 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 w8x, whereas high-dose 5-MTHF (105 mguweek),
which bypasses the transformation mechanisms of
the various metabolites at the intestinal level, reduced
2002 European Renal Association–European Dialysis and Transplant Association
858
homocysteine by 70% in another series, normalizing
the values in five patients w9x.
The vascular endothelium opposes the atherosclerotic process by various mechanisms including the
production of nitric oxide w10x. 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 w11x.
In healthy subjects, administration of folic acid prevents the endothelial dysfunction produced by acute
w12x or persistent hyperhomocysteinaemia w13x. A similar improvement of endothelium-dependent, flowmediated vasodilation is observed in patients with
familial hypercholesterolaemia w14x but not in uraemic
patients studied either before w15x or after the start
of dialysis treatment w16x.
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
G. Buccianti et al.
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 mlumin; dialysate flow rate, 500 mlumin;
reinfusion rate in post dilution, 2000 mluh; Na, K and Ca2q
in the dialysate, 150, 2–3 and 4 mEqul, respectively. All
patients gave informed consent to participate in the study.
Study design
According to the study design (Figure 1), 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 mg 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).
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.
Patients
Laboratory methods
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
Fig. 1. Study protocol.
From each patient, two predialysis blood specimens were
collected and placed into light-protected tubes, either plain
5-MTHF restores endothelial function in uraemia
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 208C 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
859
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:
(Maximum diameter between T30, T90, T150, T210) mean
(B0, B1, B2) 3 100uMean (B0, B1, B2)
NMD was calculated by:
DNA extraction was performed in all 15 patients. DNA
was obtained from 50 ml 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 mM of each
primer, 0.05 mM FAM probe, 0.15 mM TET probe, 2.5 U
TaqGold (Perkin Elmer Cetus, Norwalk, CT, USA), 0.05 U
UNG, 5 ml of DNA (100 mg) in a 25-ml total volume.
The primers were: TMTHFR for 59-CACAAAGCAAGAATGTGTCA-39
and
TMTHFR
rev-59-GACCTGAAGCACTTGGAGAA-39; and the probes were 59
FAM-ATGATGAAATCGACTCCCGCAG and 59 TETATGATGAAATCGACTCCCGACA.
PCR conditions were as follows: one cycle at 508C for
2 min; a hot start at 948C for 10 min; 45 cycles of
denaturation for 15 s each and annealing at 608C 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"2.38C).
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
(Maximum diameter between ISDN1, ISDN2, ISDN3)
mean (B3, B4, B5) 3 100u(Mean B3, B4, B5)
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
As shown in Table 1, 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 mmolul;
P-0.01) and fell after 5-MTHF treatment to
23.02"2.33 mmolul (P-0.004). When vitamin B12
was combined with 5-MTHF, plasma homocysteine
showed a further decrease to 17.4"2.04 mmolul
(P-0.0002) (Figure 2).
Serum folate levels decreased significantly during
the washout period from 9.2"0.99 to 5.9"0.59 nguml
(P-0.002). They rose significantly after 5-MTHF
treatment to 51"1.3 nguml (P-0.001), and were
unchanged after combined vitamin B12 with 5-MTHF
(48.7"0.85 vs 51.0"1.3 nguml).
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 nmolul;
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
860
G. Buccianti et al.
Table 1. Patient characteristics
Sex (MuF)
Age (years)
Dialytic duration (months)
BMI
Plasma homocysteine (n.v. -11 mmolul)
Erythrocyte folates (n.v. 540–1464 nmolul)
Serum folate (n.v. 7–40 nguml)
KTuV (SPVV)
Vitamin B12 (n.v. 200–700 pguml)
Albumin (n.v. 3.5–5 gudl)
T0
T1
T2
T3
7u8
61"3.2
128"20.8
23.5"0.5
42.3"9.01
663.8"79.25
9.26"0.99
1.2"0.05
604.2"117.5
3.5"0.1
7u8
62.3"3.19
140"22.3
23.5"0.52
50.9"9.99a
622.1"72.65
5.84"0.59a
1.26"0.03
409.9"47.77a
3.93"0.09
7u8
62.3"3.19
142"22.3
23.5"0.5
23.1"2.33b,c
2168.0"118.9b,c
51.0"1.3b,c
1.2"0.06
517.5"50.63
3.86"0.15
7u8
62.3"3.19
142"22.3
23.5"0.5
17.4"2.04d,e,f
2027.0"101.4d,e,f
48.7"0.85d,e,f
1.24"0.05
1933.9"56.7d,e,f
3.95"0.1
a
P-0.01 T1 vs T0; bP-0.01 T2 vs T1; cP-0.01 T2 vs T0; dP-0.05 T3 vs T2; eP-0.01 T3 vs T1; fP-0.01 T3 vs T0.
n.v., normal value.
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.
rose significantly after the addition of vitamin B12
to 5-MTHF treatment (517.5"50.63 vs 1933.9"
56.7 pguml; P-0.0001) (Table 1).
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 3 and 4).
Discussion
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% w3,4,17x, with maximumal responses
obtained at 15 mguweek w17x.
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.
5-MTHF restores endothelial function in uraemia
861
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.
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.
862
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 w18x.
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 w19x.
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 w5x, for transmembrane transport of folic acid w7x, and for MTHF
absorption w20x. 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 w6x. After a 2-month oral
treatment with 5-MTHF, Perna et al. w9x observed a
70% reduction in plasma homocysteine in 14 haemodialysed patients, with normalization in five patients.
Touam et al. w8x, 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 mguday 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 w21x. In contrast, two recent
papers w22,23x 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 w24x. 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%.
G. Buccianti et al.
Parenterally administered vitamin B12 efficaciously
reduces plasma homocysteine levels in diabetic w25x and
haemodialysed patients w26x. 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 w3x or intravenous routes w8x. 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
9x. 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 w27x.
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. w3x.
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 w12x
or persistent hyperhomocysteinaemia w13x. A similar
improvement of endothelium-dependent flow-mediated
vasodilation is observed in patients with familial
hypercholesterolemia w14x, but not in uraemic patients
studied both before w15x and after the start of dialysis
treatment w16x.
Several characteristics of our dialysis population
may explain differences between our results and those
of another study, also with haemodialysis patients
w16x. For instance, our patients had very low folate
levels in comparison with normal levels in the
study by van Guldener et al. w16x. 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
5-MTHF restores endothelial function in uraemia
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 w27x. 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 w11x.
As seen in familial hypercholesterolaemia and coronary disease w28–30x, 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.
863
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20.
21.
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Received for publication: 23.7.01
Accepted in revised form: 29.11.01