doi: 10.1161/hq0701.092000
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Folate Improves Endothelial Function in Coronary Artery Disease
An Effect Mediated by Reduction of Intracellular Superoxide?
From the Departments of Pharmacology (S.N.D., D.L., M.B.K., M.J.L.), Biochemistry (I.F.W.M., S.J.M.), and Cardiology (J.G.), Cardiovascular Sciences Research Group, Wales Heart Research Institute, and the Department of Medical Computing and Statistics (R.G.N.), University of Wales College of Medicine, Heath Park, Cardiff, UK.
Correspondence to Dr J. Goodfellow, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. E-mail GoodfellowJ{at}cardiff.ac.uk
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Abstract |
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Abstract—Homocysteine is a risk factor for coronary artery disease (CAD). Folic acid lowers homocysteine and may improve endothelial function in CAD, although the mechanism is unclear. We investigated the effect of folic acid on endothelial function, homocysteine, and oxidative stress in patients with CAD. We also examined the acute effect of 5-methyltetrahydrofolate (5-MTHF), the principal circulating folate, on endothelial function in vivo and on intracellular superoxide in cultured endothelial cells. A randomized crossover study of folic acid (5 mg daily) for 6 weeks was undertaken in 52 patients with CAD. Ten further patients were given intra-arterial 5-MTHF. Endothelial function was assessed by flow-mediated dilatation (FMD). Folic acid increased plasma folate (P<0.001), lowered homocysteine by 19% (P<0.001), and improved FMD (P<0.001). FMD improvement did not correlate with homocysteine reduction. Malondialdehyde and total plasma antioxidant capacity, markers of oxidative stress, were unchanged. 5-MTHF acutely improved FMD (P<0.001) without altering homocysteine (P=0.47). In vitro, 5-MTHF abolished homocysteine-induced intracellular superoxide increase (P<0.001); this effect was also observed with folic acid and tetrahydrobiopterin. Our data support the beneficial effect of folic acid on endothelial function in CAD but suggest that the mechanism is independent of homocysteine. Reduction of intracellular endothelial superoxide may have contributed to the effect.
Key Words: folic acid • homocysteine • coronary artery disease • endothelial function • 5-methyltetrahydrofolate
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Introduction |
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Elevated total plasma homocysteine (tHcy) is associated with an increased risk of cardiovascular disease and appears to be largely independent of other conventional risk factors.1 However, whether the increased risk is mediated directly by homocysteine or whether it may simply be a marker remains controversial.2
The metabolism of folate and homocysteine are interrelated, and increasing folate intake augments remethylation of homocysteine, leading to a reduction of up to 25% in its plasma concentration.3 This effect occurs despite normal plasma folate and can be achieved by folic acid in doses of 400 µg to 5 mg/d.3 4 This has led to the proposal that folic acid treatment may reduce cardiovascular risk by reducing tHcy.
In homocystinuria, a rare inborn error with markedly elevated tHcy concentrations (>100 µmol/L), folic acid and pyridoxine (vitamin B6) lower tHcy and reduce cardiovascular events, the major cause of mortality.5 This benefit is observed despite residual tHcy concentrations that are often above the upper limit of "normal," which suggests a benefit from B group vitamins that is independent of homocysteine.6 However, in the general population, in which tHcy concentrations are much lower (5 to 15 µmol/L),7 there are, as yet, only limited data on the effect of folic acid treatment on cardiovascular outcome.8 9
Endothelial dysfunction is a key process in atherosclerosis10 and has been reported in chronic mild fasting hyperhomocysteinemia in subjects free of vascular disease11 and in experimental hyperhomocysteinemia, which is induced by methionine loading in normal subjects.12 Precisely how homocysteine may promote endothelial dysfunction is unclear; however, generation of reactive oxygen species is proposed to be an important mechanism.13 Recent studies suggest that B group vitamins enhance endothelial function in coronary artery disease (CAD) or hyperhomocysteinemia. However, the data are limited, and the mechanism underlying this improvement has not been established.14 15 16 17
5-Methyltetrahydrofolate (5-MTHF), the main circulating folate, can improve endothelial function in subjects with hypercholesterolemia who are free of vascular disease and not receiving lipid-lowering treatment.18 However, the effect of 5-MTHF in subjects with severe CAD on lipid-lowering treatment is unknown.
We sought to determine whether high-dose folic acid supplementation can improve endothelial dysfunction, a surrogate of cardiovascular risk, in patients with significant CAD on standard therapy, and we endeavored to correlate this effect, if any, with changes in plasma homocysteine, plasma folate, and oxidant stress. To further investigate mechanisms, we examined the acute effect of 5-MTHF on endothelial function in a similar group of 10 patients and its effect on intracellular levels of superoxide in cultured porcine aortic endothelial (PAE) cells.
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Methods |
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Subjects
Patients with CAD aged <70 years were recruited. We defined CAD as either angiographically proven coronary disease (
50% luminal stenosis) or a history of myocardial infarction
(creatinine kinase rise >2-fold normal with ECG changes).
Plasma homocysteine was not an entry criterion, and levels were not
made known to the investigators during the course of the study.
Patients were excluded if an acute coronary event had occurred
<3 months before entry or if there was diabetes mellitus, uncontrolled
hypertension, fasting plasma cholesterol >6.5 mmol/L,
impaired renal function (creatinine >120 µmol/L), or
clinically significant heart failure. Patients actively smoking or who
had recently ceased smoking (<6 months), patients taking antioxidant
vitamins (E or C), folic acid, or fish oils, and women on hormone
replacement were also excluded.
The present study was designed to recruit a minimum of
46 subjects, to achieve 80% power to detect an improvement in
flow-mediated dilatation (FMD) from 40 µm on placebo to 80
µm on folic acid at the 
=0.05 level. Of 534 patients screened, 52
were eligible for entry to the folic acid study, and 10 similar
subjects were identified for the intra-arterial study.
All selected subjects were tested to exclude vitamin
B12 deficiency, which precludes folic acid
treatment, before entry.
Study Design
Folic Acid Study
The study was a randomized, double-blind,
placebo-controlled crossover design. It involved two 6-week treatment
periods of folic acid (5 mg daily) or matched placebo separated by a
washout period of 4 months.
Intra-Arterial 5-MTHF Study
The study was open label and investigated the acute
effects of 5-MTHF on brachial artery FMD and its NO
component.
All patients gave written informed consent, and both protocols were approved by the Local Research Ethics Committee.
Study Protocol
Folic Acid Study
Each patient was studied at week 0, week 6, week 22,
and week 28. At each visit, venous blood was collected into
Vacutainers. Lipids, glucose, and creatinine were
analyzed on the day of sampling; other samples were separated;
and the serum/plasma was stored at -70°C until analysis.
Vascular studies were performed by a single experienced operator in a
temperature-controlled room (21°C to 24°C) at the same time of day
on patients fasted overnight. Medications were omitted on the morning
of the visit, and nitrates were withheld for 24 hours before the
studies. At each visit, FMD was measured. Vascular measurements were
made at baseline, at 1-minute intervals for 6 minutes, and at 8 and 10
minutes after cuff release to establish the time course of vessel
diameter change. The nitroglycerin (NTG)
response was then recorded.
Intra-Arterial 5-MTHF Study
After baseline venous blood sampling, baseline
vascular measurements and the NTG response were recorded, and a
period of at least 45 minutes was allowed to elapse. A 27-gauge needle
was then inserted into the brachial artery of the nondominant arm.
Normal saline was infused at 0.5 mL/min, and this infusion rate was
kept constant throughout. The brachial artery was imaged 7- to 10-cm
distal to the puncture site, and FMD was measured. After this, 5-MTHF
(Sigma Chemical Co) was then infused at 50
µg/min to achieve a plasma concentration of at least 457 µg/L (1
µmol/L), which has been shown to improve endothelial
function in untreated
hypercholesterolemia.18
After 30 minutes of infusion, venous blood samples were drawn from the
ipsilateral antecubital vein, and FMD was reassessed.
NG-Monomethyl-L-arginine
(L-NMMA), an inhibitor of endothelial NO
synthase (eNOS), was then coinfused (3 mg/min) with 5-MTHF to assess
the NO component of the observed FMD. The L-NMMA infusion was then
stopped, and during infusion with 5-MTHF, the response to NTG was
reassessed.
Noninvasive Measurement of
Endothelial Function
FMD was measured by using high-resolution ultrasound
and wall tracking, as previously described by
us19 in response to
increased flow in the brachial artery induced by release of a cuff
placed at the wrist inflated for 5 minutes at 250 mm Hg. FMD was
taken as the greatest absolute increase in vessel
end-diastolic diameter (EDD) during the first 3 minutes
after cuff release. Endothelium-independent dilatation
in response to NTG (400 µg) was measured after return of the vessel
diameter to baseline and reported as the greatest absolute increase in
EDD. Blood pressure was measured continuously in the study arm by using
photoplethysmography (Finapres). Blood flow was calculated as the
product of the Doppler time-velocity integral, heart rate, and
brachial artery diameter measured by wall tracking at that
time.
Biochemical Assays
Lipids, glucose, and creatinine were
assayed routinely. tHcy was measured by enzymatic immunoassay (Abbot
IMx, Abbot Diagnostics). Plasma malondialdehyde (MDA) and
5-MTHF were measured by high-performance liquid
chromatography.20 21
Plasma total antioxidant capacity (TAOC) was measured by using a
commercially available kit (Randox Laboratories) according to the
method of Miller et al.22
Vitamin B12 and folate were measured by
competitive protein binding assays on an Elecys 2010 analyzer
(Roche Diagnostics).
Effect of 5-MTHF, Folic Acid, and
BH4 on Intracellular Superoxide In Vitro
Experiments were performed on cultured porcine
endothelial cells to assess the effects of 5-MTHF,
folic acid, and tetrahydrobiopterin (BH4) on
intracellular superoxide in cells exposed to homocysteine. Free reduced
L-homocysteine was prepared
from L-homocysteine
thiolactone, as described
previously.23 PAE cells were
isolated and cultured as previously
described,24 and all
experiments were carried out on first-passage cells. PAE cells were
incubated at 37°C for 24 hours with buffer or homocysteine alone
(1 mmol/L) or were coincubated with homocysteine (1 mmol/L)
and 5-MTHF, folic acid, or BH4 (all at 0.5
mmol/L). Intracellular PAE cell superoxide levels were then measured as
previously described.25
Briefly, cells were washed with sterile saline (0.9% [wt/vol])
before being trypsin (0.05% [wt/vol])–digested and isolated. The
resulting cell pellet was resuspended in HEPES-buffered
physiological saline, and the cell number measured
with a Coulter Counter. Cells were added to an aliquot of buffer, to
which lucigenin was added to a final concentration of 500 µmol/L.
Cells were then placed into the warmed chamber of a luminometer with
output measured in millivolts. Intracellular superoxide was measured
after the addition of a lysing agent (Triton
X-100, 1% [vol/vol]), calculated from the integral for the response,
and normalized for cell number.
Withdrawals, Medication Changes, and
Compliance
Two subjects were withdrawn from the folic acid
study: 1 with a nonfatal myocardial infarction and 1 with atrial
fibrillation. During the study, all efforts were made to hold
medication constant, but clinical considerations forced changes in 6
patients. Treatment with folic acid was well tolerated, and no side
effects were reported. Compliance assessed by a tablet count was
96%.
Statistical Analysis
The main statistical analyses of the folic
acid study results were based on 50 subjects after removal of the 2
withdrawals. Prefolate to postfolate changes in biochemical and
vascular measurements were compared with corresponding preplacebo to
postplacebo changes by using the Hills-Armitage
method.26 The relationships
between changes in FMD, homocysteine, and other parameters
on folate administration were characterized by the Spearman rank
correlation, with 95% CIs calculated by the
tanh-1 method. In the
intra-arterial study, paired
t tests were used. The main
outcome variables of FMD, folate, and homocysteine were little
changed after removal of the 6 subjects in whom medication changes took
place.
For the in vitro experiments, data are expressed as mean±SEM (n=10) and compared by ANOVA, followed by an appropriate multiple range test. All differences were considered significant at P<0.05.
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Results |
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Baseline Characteristics
The folic acid study group consisted of 52 patients aged 57±8 years (males 57±8 years, females 58±8 years). The 5-MTHF study group consisted of 10 similar subjects, aged 55±9 years (all male). Baseline characteristics were similar in the 2 populations (Table 1
).
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Folic Acid Study
Effects on Biochemical
Parameters
Biochemical parameters are shown in
Table 2. Folic acid significantly decreased tHcy (9.3±2.4
versus 10.8±2.4 [placebo] µmol/L,
P<0.001) and markedly
increased plasma folate (310±234 versus 9.1±3.4 [placebo] µg/L,
P<0.001). MDA and plasma TAOC
were not significantly altered by folic acid. Vitamin
B12, lipids, glucose, and creatinine
were unchanged by folic acid compared with
placebo.
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Effects on Flow-Mediated Dilatation and
Vascular Measurements
Vascular data involving folic acid treatment are shown
in
Table 3. The coefficient of variation for the measurement
of FMD in our laboratory was 5.6%. FMD was impaired in the folic acid
group at baseline compared with published normal values (50±33 µm
and 1.2±0.97% baseline
EDD).27 FMD significantly
improved after folic acid compared with placebo (110±43 versus 47±35
µm, respectively; P<0.001;
Figure 1), and in addition, the time course of vessel
diameter change after cuff release was significantly altered
(Figure 2). Heart rate, blood pressure, baseline brachial
artery EDD, peak hyperemic flow, and NTG response did not
differ significantly after folic acid.
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EDD, mean±SEM) after cuff release, before and after placebo and folic acid.
Intra-Arterial 5-MTHF Study
Effects on Biochemical
Parameters
After 30 minutes of infusion, plasma 5-MTHF markedly
increased (from 20.2 to 1595 µg/L,
P<0.001), whereas no change in
plasma homocysteine was observed (from 10.50±2.46 to 10.53±2.52
µmol/L,
P=0.47).
Effects on Flow-Mediated Dilatation and
Vascular Measurements
Vascular data during control, 5-MTHF infusion, and
5-MTHF/L-NMMA coinfusion are shown in
(Table 4). FMD was impaired in the 10 subjects at baseline
compared with published normal values (FMD 43±15 µm, 0.96±0.34%
baseline EDD).27 5-MTHF
acutely improved FMD compared with control (80±20 versus 43±15 µm,
respectively; P=<0.001), an
effect that was completely suppressed by coinfusion with L-NMMA
(Figure 3). Heart rate, blood pressure, baseline brachial
artery EDD, peak hyperemic flow, and NTG response did not
differ significantly during 5-MTHF infusion compared with
control.
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Comparing 5-MTHF/L-NMMA coinfusion vs 5-MTHF alone. 
Comparing control vs 5-MTHF/L-NMMA coinfusion. All comparisons were made by 2-tailed paired t test.
In Vitro Effect of 5-MTHF, Folic Acid, and
BH4 on Intracellular Superoxide Levels
Intracellular superoxide content was 21.0±2.4 mV · s
per 106 cells in the control. Exposure to
homocysteine resulted in a significant
(P<0.001) increase in
superoxide levels to 53.4±2.9 mV · s per
106 cells. This increase was abolished
(P<0.001) by coincubation with
either 5-MTHF, folic acid, or BH4
(Figure 4).
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Correlates of Improved FMD
Although homocysteine was significantly decreased by
folic acid, a positive correlation between improvement in FMD and
reduction in plasma homocysteine was not found. To the contrary, there
was a trend toward a negative correlation
(rs=-0.21,
P=0.15, 95% CI -0.46 to
0.08). FMD improvement was weakly and negatively correlated with
baseline homocysteine
(rs=-0.23,
P=0.11, 95% CI -0.48 to
0.06). No correlation was found between improvement in FMD and increase
in plasma folate
(rs=0.09,
P=0.54, 95% CI -0.20 to
0.36) or with changes in MDA or TAOC.
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Discussion |
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The present study demonstrated a significant improvement in endothelial function after 6 weeks of treatment with folic acid (5 mg daily) in subjects with significant CAD on standard therapy and with good lipid control (mean cholesterol 4.7 mmol/L). The majority (88%) of patients were taking statins, which have previously been shown to improve endothelial function,28 indicating additive benefit.
The oral folate study, statistically the most powerful to our knowledge, confirms and extends the findings of recent parallel group studies of folic acid (5 mg daily) alone or in combination with other B group vitamins on endothelial function in CAD.14 15 In previous studies,14 15 mean baseline tHcy concentration was higher than that in the present study: 13 µmol/L14 and 12.3 µmol/L15 compared with 11.2 µmol/L in the present study. Furthermore, improvement in endothelial function in the present study was not confined to those with higher baseline homocysteine concentrations. Indeed, there was a trend toward greater FMD enhancement in patients with tHcy <9 µmol/L (n=14) compared with tHcy >9 µmol/L (n=36); change in FMD was 73±43 versus 47±43 µm, respectively (P=0.07). This suggests that the benefit is independent of pretreatment tHcy level.
We found no correlation between tHcy reduction and
improvement in endothelial function and, indeed,
observed a trend toward the reverse. This contrasts with the findings
of a recent study in which a significant positive correlation between
tHcy reduction and improvement in endothelial function
was reported.15 It has
recently been suggested that improved endothelial
function seen with B group vitamins (which included folic acid) in CAD
is mediated by a reduction in free (unbound) but not total
homocysteine, in accordance with the observation that FMD improvement
is correlated with a reduction in free but not total
homocysteine.14 A number of
factors would appear to argue against this correlation being a causal
relationship. First, all of the observational data linking homocysteine
and cardiovascular risk has been based on total, not
free, homocysteine. Second, of the free component, only 
1% is in
the reduced form (ie, possesses a free sulfhydryl group), which can
support auto-oxidation and therefore superoxide generation, with the
remaining component being
oxidized.29 30
The pathological significance of such low levels of free reduced
homocysteine on oxidative burden in the plasma has been questioned
recently.31
The lack of a positive correlation between the enhancement in endothelial function and tHcy reduction suggests that the beneficial effect of folic acid is unlikely to be mediated principally via tHcy lowering. This proposal is supported by the intra-arterial study, which demonstrated acute improvement in endothelial function after 30 minutes of infusion with 5-MTHF independent of a change in tHcy. This effect has not been previously reported in subjects with CAD. The improvement was abolished by coinfusion with the NO synthase inhibitor L-NMMA, indicating that it was mediated by an increase in NO bioavailability.
Generation of reactive oxygen species is proposed to be an
important mechanism of homocysteine-induced endothelial
injury.13 This view is
supported by methionine loading in normal subjects, which raises tHcy
to 30 to 35 µmol/L and which acutely impairs
endothelial
function.12 The mechanism is
believed to involve oxidant
stress32 and is abrogated by
treatment with antioxidant
agents.33 34
However, it cannot be assumed that moderately elevated homocysteine
levels in the general population exert oxidative stress, because
methionine loading results in large unphysiological
increases in free reduced homocysteine and
methionine.35 Although some
observational studies have shown a correlation between tHcy levels and
markers of oxidant stress in the general
population,36 37
a causal relationship has not been established. In the present
study, although tHcy was significantly reduced by 19%, there was no
reduction in MDA or any increase in TAOC, a marker of the antioxidant
capacity of plasma. This suggests either that homocysteine does not
directly exert oxidative stress in the plasma or that measurement of
MDA and TAOC are not sensitive enough indicators. However, MDA and TAOC
were selected because methionine loading in normal subjects is
associated with an increase in MDA and decrease in TAOC, indicating
that under these conditions, they can detect changes in plasma oxidant
stress.38 39
The in vitro study used concentrations of homocysteine that
were far higher than those experienced in vivo (1 mmol/L versus 10
to 15 µmol/L, respectively). The high concentration of homocysteine
used was similar to previously published reports of in vitro work.
Higher pharmacological concentrations are sometimes required in vitro
to reproduce the in vivo situation, in part, because the exposure times
used are much shorter than those experienced in hyperhomocysteinemia in
humans. The main aim of the present study was to establish
homocysteine-induced endothelial dysfunction and
investigate possible mechanism(s) by which folate may reverse this and
not to mimic the in vivo situation exactly. We have recently reported
that exposure of cultured endothelial cells to
homocysteine (30 µmol/L) stimulates intracellular generation of
superoxide.25 The in vitro
study confirmed this finding and further revealed that 5-MTHF can
reduce levels of intracellular superoxide, suggesting a possible
explanation for improvement in FMD observed in the human study. A
number of mechanisms may account for this: (1) intracellular
homocysteine was decreased, (2) 5-MTHF scavenged superoxide directly,
and/or (3) 5-MTHF reduced superoxide production. Given that it
takes some weeks before levels of homocysteine fall significantly with
folic acid, a reduction in homocysteine after 24 hours seems unlikely,
and other direct actions of 5-MTHF more likely explain this
observation. In vitro, 5-MTHF has recently been demonstrated to be
capable of directly scavenging superoxide, increasing NO
production by eNOS, and also reducing superoxide generation by
eNOS.40
The in vitro study also demonstrated that BH4 is capable of inhibiting the homocysteine-induced increases in endothelial superoxide. BH4 is an essential cofactor for eNOS, and its depletion results in uncoupling of eNOS activity and a switch from production of NO, from L-arginine, to generation of superoxide.41 5-MTHF is essential in the redox cycling of the inactive quinonoid BH2 (qBH2) back to the active form, BH4.42 Furthermore, oral BH4 supplementation can improve endothelial function in CAD.43 Thus, the ability of 5-MTHF to regenerate intracellular BH4 from qBH2 may, at least in part, explain the improvement in NO bioavailability observed in the human studies.
Implications of the Present Study
The oral folic acid study supports the finding that
high-dose supplementation improves endothelial function
in patients with CAD. Furthermore, this improvement is observed in
subjects already treated with statins and is independent of baseline
tHcy or its reduction. In contrast to earlier reports, the present
data do not support the view that improvement is mediated by tHcy
reduction but point rather to direct actions of folic acid, possibly
mediated by reduction in intracellular but not plasma oxidant stress.
The dose of folic acid used was pharmacological, with plasma levels far
in excess of the normal range, and it cannot be assumed that the
effects demonstrated will apply to low-dose folic acid (400 µg/d) or
improved dietary intake. In conclusion, folic acid is safe and offers a
simple, well-tolerated, and inexpensive therapeutic option for
improving endothelial function in subjects with
ischemic heart
disease.
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Acknowledgments |
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Dr S.N. Doshi is supported by a Junior Research Fellowship from the British Heart Foundation. We wish to thank J. Chapman for laboratory assistance, Z.E. Clark for homocysteine assays, and R.D. Ellis for folate assays. We gratefully acknowledge the donation of folic acid and matched placebo by Dr A.S. Burbage of Peter Black Healthcare Ltd.
Received January 29, 2001; accepted February 22, 2001.
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