Molecular Medicine |
From the Departments of Vascular Medicine (E.S.G.S., M.Y., R.G., T.J.R.) and Nephrology (P.B.), University Hospital Utrecht, The Netherlands; Debye Institute (E.E.v.F.), University Utrecht, The Netherlands; and Department of Pediatrics, First Faculty of Medicine (P.M.), Charles University, Prague, Czech Republic.
Correspondence to Dr E.S.G. Stroes, Department of Vascular Medicine, F 03.226, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail e.stroes{at}digd.azu.nl
| Abstract |
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Key Words: folic acid nitric oxide nitric oxide synthase tetrahydrobiopterin electron spin resonance
| Introduction |
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We recently demonstrated that folic acid and its active form 5-methyltetrahydrofolate (MTHF) restore impaired NO bioavailability in dyslipidemic conditions, probably by an ameliorative effect on eNOS uncoupling.24 25 In this study, we address the following 4 questions: (1) whether the effect of MTHF can be explained by its scavenging of superoxide or whether MTHF directly interacts with eNOS itself; (2) whether the action of MTHF affects enzymatic synthesis of superoxide, NO, or both; (3) whether this interference with enzymatic activity is direct or involves intermediary substances such as the pterin cofactor BH4; and (4) whether the observations on purified recombinant enzyme are representative for the situation as encountered in endothelial cells. First, we determined the reaction rates for the direct scavenging of superoxide by L-MTHF and its stereoisomer D-MTHF. These reaction rates allowed us to discriminate between direct scavenging and interference with enzymatic activity of eNOS. Subsequent experiments show that, in the presence of small amounts of BH4, both stereoisomers affect the enzymatic activity of eNOS.
| Materials and Methods |
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Electron Paramagnetic Resonance (EPR) Measurements
The EPR spectra were recorded at 37°C on a modified Bruker
ESP 300 spectrometer, as described previously.9 Spin trap
experiments were performed with both hypoxanthine/xanthine oxidase
(HX/XO) and eNOS. For HX/XO the solution contained 0.5 mmol/L
hypoxanthine, 12.5 mU/mL xanthine oxidase, and 50 mmol/L DEPMPO in
phosphate buffer (pH 7.4). The eNOS assay contained 250 nmol/L
eNOS dimers (0.065 mg protein/mL); 10 µmol/L
L-arginine, 300 U/mL calmodulin; and (in
mmol/L) NADPH 0.5, CaCl2 1, and DEPMPO 50 in
phosphate buffer (pH 7.4).
Determination of Superoxide Trapping Rates by Competitive
Superoxide Trapping (CST)
The trapping rates for superoxide were determined at 37°C by
comparing the trapping efficiency of L- and D-MTHF with the known
trapping efficiency of the spin trap DEPMPO27 (CST). Using
HX/XO as a superoxide-generating system, the presence of other
compounds (such as MTHF) will result in less generation of DEPMPO spin
adducts. Reaction channels other than with DEPMPO or MTHF can be
neglected, as the adduct yield does not increase further if DEPMPO
concentrations higher than 50 mmol/L are used (data not
shown).
Determination of NO Production by eNOS
NOS activity was determined by quantifying the conversion of
L-[2,3,4,5-3H]arginine into
L-[2,3,4,5-3H]citrulline, mainly as
described previously.9 Briefly, 2 µg eNOS
(BH4-free or -repleted) was incubated for 5
minutes at 37°C in 100 µL HEPES buffer (pH 7.4) containing (in
mmol/L) diethylenetriamine penta-acetic acid 0.1,
CaCl2 0.2, and NADPH 0.5; (in µmol/L),
flavin mononucleotide 1, flavin adenine
dinucleotide 1, glutathione 100, and L-arginine
100; calmodulin (20 µg/mL); BSA (200 µg/mL); and
L-[2,3,4,5-3H]arginine (3.7 KBq).
All measurements were performed in triplicate. After correction for
nonspecific activity, eNOS activity was calculated from the percentage
conversion of [3H]arginine into
[3H]citrulline and expressed as nmol per mg
protein/min.
Cell Cultures
Microvascular endothelial bEND3
cells28 were cultured to confluence in 6-well culture
plates for determination of nitrite or in 15-cm dishes for electron
spin resonance experiments. After the cells reached confluence, the
medium was changed to M-199 (Sigma) supplemented with 0.1% BSA, 5
mmol/L L-glutamine, antibiotics, and MTHF (0, 1, and
10 µmol/L) or sepiapterin (100 µmol/L), respectively, for
24 hours.
Determination of NO Production by Endothelial
Cells
The NO production by endothelial cells
was assessed by quantification of the nitrite content in the
supernatant with a commercially available fluorimetic kit (Cayman
Chemicals).29 Acetylcholine-induced NO production
is presented as the difference between stimulated minus the
unstimulated nitrite content.
Statistical Analysis
Changes in NO production were tested with an unpaired
t test. Changes in radical adduct formation by
BH4 (Figure 3
) or L-MTHF (Figure 4
)
were tested with ANOVA. If variance ratios reached statistical
significance, differences between the means were analyzed with
the Student-Newman-Keuls test for P<0.05.
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An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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Superoxide Trapping by MTHF
The time curves of the EPR intensity in the HX/XO system were
described by single exponentials with a time constant,
t=10±0.5 minutes (Figure 1
).
Plots of the steady-state limits as a function of L- and D-MTHF
concentration in the HX/XO system are given in Figure 2
(). Both isomers show the same
linear concentration dependence. From the slopes we find
kL-MTHF/kDEPMPO=kD-MTHF/kDEPMPO=175±13
(at 37°C; pH 7.4). Using the literature value of
kDEPMPO=80 ms1 (see
Note), we find the following rate constants for scavenging of
superoxide: kL-MTHF =
kD-MTHF=1.4x104
ms1. For comparison, the scavenging rate of the
potent antioxidant ascorbic acid is 2.7x105
ms1 at pH 7.4 and 37°C. Hence, the scavenging
potency of both L- and D-MTHF is 20 times less than that of ascorbic
acid.
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Superoxide Production by Pterin-Free eNOS
Previous clinical studies have demonstrated that L-MTHF improves
NO bioavailability in vivo in hypercholesterolemic
patients.24 30 To elucidate whether this may relate to a
direct effect of L-MTHF on eNOS, we repeated the CST experiments using
eNOS as a superoxide-generating system. The effect of L- and D-MTHF on
the pterin-free eNOS (Figure 3
, )
coincides with the data from the HX/XO experiments (compare Figure 2
). It demonstrates that for pterin-free eNOS, impaired
formation of spin adducts can be fully accounted for by the capacity of
L- and D-MTHF to scavenge superoxide in a bimolecular scavenging
reaction. In particular, we conclude that the presence of L- or D-MTHF
does not affect the rate of superoxide production by
pterin-free eNOS.
Superoxide Production by eNOS: Pterin-Repleted
Case
As far as pterin-repleted eNOS is concerned (Figure 3
,
), addition of L- or D-MTHF results in a very strong reduction in
the rate of DEPMPO adduct formation (as it manifests itself from a much
steeper slope). This reduction by far exceeds the reduction observed in
pterin-free eNOS. This observation cannot be fully explained by the
capacity of L- and D-MTHF to scavenge superoxide. We conclude that the
presence of MTHF reduces the superoxide production by eNOS in a
concentration-dependent way, with both stereoisomers of MTHF having the
same potency. To evaluate whether MTHF only affects eNOS after
preincubation with BH4, we added increasing
amounts of BH4 to pterin-free eNOS in the
presence and absence of L-MTHF 25 µmol/L (Figure 4
). As expected, in the absence of MTHF,
addition of BH4 caused a dose-dependent decrease
in superoxide production. In pterin-free eNOS, addition of
25 µmol/L L-MTHF does not cause significant changes in radical
adduct formation (Figure 4
). In contrast, addition of MTHF to
partially repleted eNOS (still BH4 deficient)
causes a substantial reduction in the amount of superoxide adducts
(Figure 4
).
NO Production by eNOS
The NO production by pterin-free eNOS is located at the
detection limit of the arginine-citrulline conversion assay (Figure 5
). Addition of L- or D-MTHF (100
µmol/L final) to pterin-free eNOS has no significant effect on NO
production (Figure 5
). In contrast, in pterin-repleted
eNOS, 2 significant differences arise, as follows. (1) A clear basal NO
production is observed (Figure 5
). (2) The addition of
both L- or D-MTHF (100 µmol/L final) causes a significant
increase in NO production (Figure 5
; P<0.05
versus BH4 alone).
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NO Production by Endothelial Cells
Preincubation with MTHF did not affect nitrite release in
unstimulated endothelial cells. Acetylcholine
stimulation caused a significant increase in nitrite release (Figure 6
). Preincubation of
endothelial cells with L-MTHF and sepiapterin resulted
in a significant further increase in acetylcholine-induced nitrite
production (Figure 6
).
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| Discussion |
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MTHFDirect Superoxide Scavenging
At 37°C, the scavenging potency of L- and D-MTHF is
20-fold
lower than the effect of the well-known antioxidant vitamin C. This
raises the question whether this antioxidant potency of MTHF may be
relevant in vivo. The usual plasma concentration for folate is in the
low nanomolar range.25 On oral supplementation, the level
of folic acid may be raised to micromolar range,30 which
is still below the vitamin C levels of 30 to 50 µmol/L observed
in vivo.31 We therefore argue that the superoxide
scavenging capacity of folate is not relevant in vivo, where
antioxidant mechanisms such as vitamin C or superoxide dismutase have
far higher capacity for removal of superoxide. This implies that the
scavenging properties of MTHF are irrelevant for the observed
restoration of impaired NO bioavailability in
hypercholesterolemic patients.25 30
Instead, it is suggested that MTHF exerts its beneficial effects
through modulation of the enzymatic activity of eNOS.
MTHFPterin-Free eNOS
In pterin-free eNOS, MTHF by itself is not able to induce NO
synthesis. This shows that folates cannot substitute for
BH4 as a cofactor for eNOS. Given the bulky
carbon moiety of folates, it seems unlikely that folates are able to
fit into the BH4 pocket of eNOS.32
However, both L- and D-MTHF significantly reduce the formation of
DEPMPO superoxide adducts. The degree of reduction in superoxide
activity by MTHF is equivalent to that observed in the HX/XO system. It
shows that for pterin-free eNOS, MTHF exerts its effects through pure
scavenging only, without interfering with enzymatic activity. Combining
the results for NO and superoxide, pterin-free eNOS is seen to be
completely oblivious to the presence of MTHF.
MTHFPterin-Repleted eNOS
The pterin-repleted eNOS used in our studies shows substantial
basal production of both NO and superoxide and therefore should
be considered as BH4 deficient, ie, partially
uncoupled. Under these conditions, addition of either L- or D-MTHF
increases NO production. Interestingly, both D- and
L-isomers have the same effect. At the same time, the
formation of DEPMPO superoxide adducts is strongly reduced by both L-
and D-MTHF. The reduction of adduct formation caused by MTHF by far
exceeds that observed in the HX/XO system. This shows that the major
impact of MTHF must be a direct interference with the enzymatic
superoxide production by the pterin-repleted eNOS. Again, L-
and D-MTHF have comparable effects. Combining the results for NO and
superoxide, the enzymatic activity of pterin-repleted eNOS is highly
sensitive to the presence of either stereoisomer. The overall effect is
a substantial shift from superoxide production toward NO
production. From Figure 2
, we estimate that superoxide
production by eNOS is reduced by a factor of 2 at a
concentration of [MTHF]= 50 µmol/L, ie,
200 MTHF molecules
per eNOS dimer. Similar molecular ratios of MTHF versus eNOS can be
achieved in vivo on oral supplementation with folate.
MTHFPterin Interaction
The exact role of BH4 in enzyme function of
eNOS is still unknown.33 BH4 is
tightly bound to the haem domain of eNOS.33 Just recently,
it has been shown that BH4 does not act as a
redox-active cofactor during NO biosynthesis.34 Previous
work revealed that eNOS-derived superoxide production in vitro
is reduced significantly on addition of
BH4.9 35 In line with these
observations, BH4 infusion in vivo improves
impaired NO availability in patients with
hypercholesterolemia.36 These data
imply that, under pathophysiological conditions,
eNOS should be considered deficient in BH4,
denoting that the occupancy of an eNOS dimer is significantly lower
than 2. Our experiments show that MTHF requires
BH4 before it can affect the enzymatic activity
of eNOS. This suggests that MTHF somehow supports the action as a
cofactor of BH4. Two possible mechanisms can be
envisaged, as follows: (1) by improving the redox state of the
BH4 and (2) by enhancing the binding of
BH4 to eNOS. On closer scrutiny, both
alternatives seem implausible to us, as explained below.
With regard to the first option of improved redox state, it has been
suggested that MTHF may increase regeneration of
BH4 from
qBH2.37 However, we have previously
found that biopterin levels in hypercholesterolemic
patients are not significantly different from levels found in healthy
controls.25 Moreover, folate therapy did not show any
effect on biopterin levels in vivo,30 thus ruling out the
first option. Let us now consider the second option of enhanced binding
of BH4 to eNOS. By selecting
[BH4]=75 and 250 nmol/L dimers32
of eNOS, a highly BH4-deficient mixture is
obtained, with the majority of dimers having either zero or single
occupancy with BH4 and a minute fraction being
doubly occupied. Assuming that the dissociation constant between
BH4 and neuronal NO synthase (k=40
nmol/L)38 also holds for eNOS, we estimate that
<10% of the total BH4 remains free in solution.
Under these conditions, there is not enough free
BH4 available to improve the occupancy of (singly
occupied) eNOS significantly. However, in such a mixture, addition of
MTHF still causes a substantial decrease in the production of
superoxide (Figure 4
). Thus, we conclude that it is unlikely
that MTHF exerts its effect via enhanced binding of
BH4 to eNOS.
If improved redox state and/or affinity of BH4 for eNOS are not involved, what explains the increased "effectiveness" of BH4 on eNOS uncoupling during MTHF supplementation? Recent elucidation of the crystal structure of the haem-eNOS domain lends mechanistic support for the involvement of the BH4 radical in the catalysis of NO formation by eNOS.32 In support, Bec et al39 have recently described a model showing that the BH4 radical is of crucial importance for electron flux in the haem domain of neuronal NO synthase. We hypothesize that MTHF could act as facilitator of the 1-electron oxidation of BH4 to the BH4 radical. Of note, the potent antioxidant ascorbic acid has recently also been shown to amplify stimulated NO production in cultured endothelial cells in a dose-dependent fashion.40 This potentiating effect disappeared after BH4 supplementation, again suggesting a direct beneficial effect of a reducing substance on the uncoupled state of eNOS.40
MTHFEndothelial Cells
Our data show that the effects of MTHF on endogenous
eNOS in endothelial cells are compatible with the
findings on the recombinant enzyme. In particular, we show an enhanced
NO status in cultured endothelial cells on MTHF
suppletion. We could not verify changes in superoxide
production. Several factors can combine to keep superoxide
levels and/or spin adduct levels in endothelial cells
below the detection limit, including the relatively low affinity of the
spin traps for superoxide in comparison to, eg, NO and superoxide
dismutase,41 the finite lifetime preventing accumulation
of the adducts,42 and the innate cellular capacity for
metabolism of spin adducts.43 Other
techniques, such as lucigenin, seem less reliable, as it has been
reported that this compound induces a large artificial superoxide
release from NO synthase.44
MTHFClinical Perspective
Uncoupling of eNOS is a major cause of endothelial
dysfunction in dyslipidemic patients.11 45 Our
present observations show that, on a molecular level,
administration of MTHF, the active form of folic acid, has a 2-fold
effect on the enzymatic activity of "uncoupled" eNOS; it reduces
superoxide production and enhances NO synthesis by the enzyme.
The relevance of this finding for the NO status in vivo is underscored
by the increased NO production in cultured
endothelial cells on MTHF suppletion. This direct,
2-fold action of MTHF on eNOS provides a plausible explanation for the
increased NO bioavailability in humans on MTHF suppletion during
dyslipidemia.25 30
Note
There is considerable uncertainty in the literature as to the
absolute value of the rate constant for superoxide trapping by DMPO,
and as a consequence, by DEPMPO. Certainly, the protonated form
HO2 · is trapped by DMPO with a far higher rate than superoxide
itself, so that pH is a very important parameter. At pH 7.4
we consider the DMPO trapping rate of 26 ms1
(at 25°C)46 to be the most reliable. As DEPMPO trapping
rates are a factor 1.5 times higher,27 and a temperature
increase of 12°C degrees will increase the rate by a further
factor of 2, we estimate kDEPMPO=80
ms1 for trapping of superoxide by DEPMPO at pH
of 7.4 and temperature of 37°C.
| Acknowledgments |
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Received March 16, 2000; accepted April 5, 2000.
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M. L. Ojeda, M. J. Delgado-Villa, R. Llopis, M. L. Murillo, and O. Carreras Lipid Metabolism in Ethanol-Treated Rat Pups and Adults: Effects of Folic Acid Alcohol Alcohol., September 1, 2008; 43(5): 544 - 550. [Abstract] [Full Text] [PDF] |
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C. Antoniades, C. Shirodaria, T. Van Assche, C. Cunnington, I. Tegeder, J. Lotsch, T. J. Guzik, P. Leeson, J. Diesch, D. Tousoulis, et al. GCH1 Haplotype Determines Vascular and Plasma Biopterin Availability in Coronary Artery Disease: Effects on Vascular Superoxide Production and Endothelial Function J. Am. Coll. Cardiol., July 8, 2008; 52(2): 158 - 165. [Abstract] [Full Text] [PDF] |
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A. L. Moens, C. J. Vrints, M. J. Claeys, J.-P. Timmermans, H. C. Champion, and D. A. Kass Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H1971 - H1977. [Abstract] [Full Text] [PDF] |
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A. L. Moens, H. C. Champion, M. J. Claeys, B. Tavazzi, P. M. Kaminski, M. S. Wolin, D. J. Borgonjon, L. Van Nassauw, A. Haile, M. Zviman, et al. High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury Circulation, April 8, 2008; 117(14): 1810 - 1819. [Abstract] [Full Text] [PDF] |
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A. de Bree, L. A van Mierlo, and R. Draijer Folic acid improves vascular reactivity in humans: a meta-analysis of randomized controlled trials Am. J. Clinical Nutrition, September 1, 2007; 86(3): 610 - 617. [Abstract] [Full Text] [PDF] |
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R. R. Lamberts, E. Caldenhoven, M. Lansink, G. Witte, R. J. Vaessen, J. A. St Cyr, and G. J. M. Stienen Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1869 - H1876. [Abstract] [Full Text] [PDF] |
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A. S. Pena, E. Wiltshire, R. Gent, L. Piotto, C. Hirte, and J. Couper Folic Acid Does Not Improve Endothelial Function in Obese Children and Adolescents Diabetes Care, August 1, 2007; 30(8): 2122 - 2127. [Abstract] [Full Text] [PDF] |
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P. Leeson Pediatric Prevention of Atherosclerosis: Targeting Early Variation in Vascular Biology Pediatrics, June 1, 2007; 119(6): 1204 - 1206. [Full Text] [PDF] |
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C. Shirodaria, C. Antoniades, J. Lee, C. E. Jackson, M. D. Robson, J. M. Francis, S. J. Moat, C. Ratnatunga, R. Pillai, H. Refsum, et al. Global Improvement of Vascular Function and Redox State With Low-Dose Folic Acid: Implications for Folate Therapy in Patients With Coronary Artery Disease Circulation, May 1, 2007; 115(17): 2262 - 2270. [Abstract] [Full Text] [PDF] |
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C. Antoniades, C. Shirodaria, N. Warrick, S. Cai, J. de Bono, J. Lee, P. Leeson, S. Neubauer, C. Ratnatunga, R. Pillai, et al. 5-Methyltetrahydrofolate Rapidly Improves Endothelial Function and Decreases Superoxide Production in Human Vessels: Effects on Vascular Tetrahydrobiopterin Availability and Endothelial Nitric Oxide Synthase Coupling Circulation, September 12, 2006; 114(11): 1193 - 1201. [Abstract] [Full Text] [PDF] |
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T. J. Anderson, Y.-H. Sun, J. Hubacek, M. E. Hyndman, S. Verma, L. Shewchuk, and N. Scott-Douglas Effects of folinic acid on forearm blood flow in patients with end-stage renal disease Nephrol. Dial. Transplant., July 1, 2006; 21(7): 1927 - 1933. [Abstract] [Full Text] [PDF] |
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K. E. MacKenzie, E. J. Wiltshire, R. Gent, C. Hirte, L. Piotto, and J. J. Couper Folate and Vitamin B6 Rapidly Normalize Endothelial Dysfunction In Children With Type 1 Diabetes Mellitus Pediatrics, July 1, 2006; 118(1): 242 - 253. [Abstract] [Full Text] [PDF] |
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L. M Title, E. Ur, K. Giddens, M. J McQueen, and B. A Nassar Folic acid improves endothelial dysfunction in type 2 diabetes - an effect independent of homocysteine-lowering Vascular Medicine, May 1, 2006; 11(2): 101 - 109. [Abstract] [PDF] |
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C. Torrens, L. Brawley, F. W. Anthony, C. S. Dance, R. Dunn, A. A. Jackson, L. Poston, and M. A. Hanson Folate Supplementation During Pregnancy Improves Offspring Cardiovascular Dysfunction Induced by Protein Restriction Hypertension, May 1, 2006; 47(5): 982 - 987. [Abstract] [Full Text] [PDF] |
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J. D. Symons, U. B. Zaid, C. N. Athanassious, A. E. Mullick, S. R. Lentz, and J. C. Rutledge Influence of Folate on Arterial Permeability and Stiffness in the Absence or Presence of Hyperhomocysteinemia Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 814 - 818. [Abstract] [Full Text] [PDF] |
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J. D. Symons, J. C. Rutledge, U. Simonsen, and R. A. Pattathu Vascular dysfunction produced by hyperhomocysteinemia is more severe in the presence of low folate Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H181 - H191. [Abstract] [Full Text] [PDF] |
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T. Munzel, A. Daiber, and A. Mulsch Explaining the Phenomenon of Nitrate Tolerance Circ. Res., September 30, 2005; 97(7): 618 - 628. [Abstract] [Full Text] [PDF] |
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S. Dayal, A. M. Devlin, R. B. McCaw, M.-L. Liu, E. Arning, T. Bottiglieri, B. Shane, F. M. Faraci, and S. R. Lentz Cerebral Vascular Dysfunction in Methionine Synthase-Deficient Mice Circulation, August 2, 2005; 112(5): 737 - 744. [Abstract] [Full Text] [PDF] |
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M. A. Mansoor, O. Kristensen, T. Hervig, J. A. Stakkestad, T. Berge, P. A. Drablos, S. Rolfsen, and T. Wentzel-Larsen Relationship between Serum Folate and Plasma Nitrate Concentrations: Possible Clinical Implications Clin. Chem., July 1, 2005; 51(7): 1266 - 1268. [Full Text] [PDF] |
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A.M.W. Spijkerman, Y.M. Smulders, P.J. Kostense, R.M.A. Henry, A. Becker, T. Teerlink, C. Jakobs, J.M. Dekker, G. Nijpels, R.J. Heine, et al. S-Adenosylmethionine and 5-Methyltetrahydrofolate Are Associated With Endothelial Function After Controlling for Confounding by Homocysteine: The Hoorn Study Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 778 - 784. [Abstract] [Full Text] [PDF] |
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J. P. Forman, E. B. Rimm, M. J. Stampfer, and G. C. Curhan Folate Intake and the Risk of Incident Hypertension Among US Women JAMA, January 19, 2005; 293(3): 320 - 329. [Abstract] [Full Text] [PDF] |
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S. J. Moat, S. N. Doshi, D. Lang, I. F. W. McDowell, M. J. Lewis, and J. Goodfellow Treatment of coronary heart disease with folic acid: is there a future? Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H1 - H7. [Full Text] [PDF] |
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S. Hirsch, A. M. Ronco, M. Vasquez, M. P. de la Maza, A. Garrido, G. Barrera, V. Gattas, A. Glasinovic, L. Leiva, and D. Bunout Hyperhomocysteinemia in Healthy Young Men and Elderly Men with Normal Serum Folate Concentration Is Not Associated with Poor Vascular Reactivity or Oxidative Stress J. Nutr., July 1, 2004; 134(7): 1832 - 1835. [Abstract] [Full Text] |
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G. Paradisi, F. Cucinelli, M. C. Mele, A. Barini, A. Lanzone, and A. Caruso Endothelial function in post-menopausal women: effect of folic acid supplementation Hum. Reprod., April 1, 2004; 19(4): 1031 - 1035. [Abstract] [Full Text] [PDF] |
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N. J. Alp, M. A. McAteer, J. Khoo, R. P. Choudhury, and K. M. Channon Increased Endothelial Tetrahydrobiopterin Synthesis by Targeted Transgenic GTP-Cyclohydrolase I Overexpression Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 445 - 450. [Abstract] [Full Text] |
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T. J. Rabelink Cardiovascular risk in patients with renal disease: treating the risk or treating the risk factor? Nephrol. Dial. Transplant., January 1, 2004; 19(1): 23 - 26. [Full Text] [PDF] |
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K.-i. Sasaki, J. Duan, T. Murohara, H. Ikeda, S. Shintani, T. Shimada, T. Akita, K. Egami, and T. Imaizumi Rescue of hypercholesterolemia-related impairment of angiogenesis by oral folate supplementation J. Am. Coll. Cardiol., July 16, 2003; 42(2): 364 - 372. [Abstract] [Full Text] [PDF] |
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R. J. Bisoendial, G. K. Hovingh, J. H.M. Levels, P. G. Lerch, I. Andresen, M. R. Hayden, J. J.P. Kastelein, and E. S.G. Stroes Restoration of Endothelial Function by Increasing High-Density Lipoprotein in Subjects With Isolated Low High-Density Lipoprotein Circulation, June 17, 2003; 107(23): 2944 - 2948. [Abstract] [Full Text] [PDF] |
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J. D. Horowitz Amelioration of nitrate tolerance: matching strategies with mechanisms J. Am. Coll. Cardiol., June 4, 2003; 41(11): 2001 - 2003. [Full Text] [PDF] |
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K. S. Brown, L. A.J. Kluijtmans, I. S. Young, J. Woodside, J. W.G. Yarnell, D. McMaster, L. Murray, A. E. Evans, C. A. Boreham, H. McNulty, et al. Genetic Evidence That Nitric Oxide Modulates Homocysteine: The NOS3 894TT Genotype Is a Risk Factor for Hyperhomocystenemia Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1014 - 1020. [Abstract] [Full Text] [PDF] |
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A. De Bree, W. M. M. Verschuren, D. Kromhout, L. A. J. Kluijtmans, and H. J. Blom Homocysteine Determinants and the Evidence to What Extent Homocysteine Determines the Risk of Coronary Heart Disease Pharmacol. Rev., December 1, 2002; 54(4): 599 - 618. [Abstract] [Full Text] [PDF] |
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A. Warnholtz, H. Mollnau, T. Heitzer, A. Kontush, T. Moller-Bertram, D. Lavall, A. Giaid, U. Beisiegel, S. L. Marklund, U. Walter, et al. Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits J. Am. Coll. Cardiol., October 2, 2002; 40(7): 1356 - 1363. [Abstract] [Full Text] [PDF] |
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M. E. Hyndman, S. Verma, R. J. Rosenfeld, T. J. Anderson, and H. G. Parsons Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2167 - H2172. [Abstract] [Full Text] [PDF] |
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J. D. Symons, A. E. Mullick, J. L. Ensunsa, A. A. Ma, and J. C. Rutledge Hyperhomocysteinemia Evoked by Folate Depletion: Effects on Coronary and Carotid Arterial Function Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 772 - 780. [Abstract] [Full Text] [PDF] |
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R. W. van Etten, E. J.P. de Koning, M. L. Honing, E. S. Stroes, C. A. Gaillard, and T. J. Rabelink Intensive Lipid Lowering by Statin Therapy Does Not Improve Vasoreactivity in Patients With Type 2 Diabetes Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 799 - 804. [Abstract] [Full Text] [PDF] |
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M.C. Verhaar, E. Stroes, and T.J. Rabelink Folates and Cardiovascular Disease Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 6 - 13. [Abstract] [Full Text] [PDF] |
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S. N. Doshi, I. F.W. McDowell, S. J. Moat, N. Payne, H. J. Durrant, M. J. Lewis, and J. Goodfellow Folic Acid Improves Endothelial Function in Coronary Artery Disease via Mechanisms Largely Independent of Homocysteine Lowering Circulation, January 1, 2002; 105(1): 22 - 26. [Abstract] [Full Text] [PDF] |
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C. H. Pullin, P. A. L. Ashfield-Watt, M. L. Burr, Z. E. Clark, M. J. Lewis, S. J. Moat, R. G. Newcombe, H. J. Powers, J. M. Whiting, and I. F. W. McDowell Optimization of dietary folate or low-dose folic acid supplements lower homocysteine but do not enhance endothelial function in healthy adults, irrespective of the methylenetetrahydrofolate reductase (C677T) genotype J. Am. Coll. Cardiol., December 1, 2001; 38(7): 1799 - 1805. [Abstract] [Full Text] [PDF] |
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J. Loscalzo Folate and Nitrate-Induced Endothelial Dysfunction: A Simple Treatment for a Complex Pathobiology Circulation, September 4, 2001; 104(10): 1086 - 1088. [Full Text] [PDF] |
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T. Gori, J. M. Burstein, S. Ahmed, S. E.S. Miner, A. Al-Hesayen, S. Kelly, and J. D. Parker Folic Acid Prevents Nitroglycerin-Induced Nitric Oxide Synthase Dysfunction and Nitrate Tolerance: A Human In Vivo Study Circulation, September 4, 2001; 104(10): 1119 - 1123. [Abstract] [Full Text] [PDF] |
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S. N. Doshi, I. F. W. McDowell, S. J. Moat, D. Lang, R. G. Newcombe, M. B. Kredan, M. J. Lewis, and J. Goodfellow Folate Improves Endothelial Function in Coronary Artery Disease : An Effect Mediated by Reduction of Intracellular Superoxide? Arterioscler Thromb Vasc Biol, July 1, 2001; 21(7): 1196 - 1202. [Abstract] [Full Text] [PDF] |
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A. A Brown and F. B Hu Dietary modulation of endothelial function: implications for cardiovascular disease Am. J. Clinical Nutrition, April 1, 2001; 73(4): 673 - 686. [Abstract] [Full Text] [PDF] |
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M. E. Hyndman, S. Verma, R. J. Rosenfeld, T. J. Anderson, and H. G. Parsons Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2167 - H2172. [Abstract] [Full Text] [PDF] |
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S. Dayal, T. Bottiglieri, E. Arning, N. Maeda, M. R. Malinow, C. D. Sigmund, D. D. Heistad, F. M. Faraci, and S. R. Lentz Endothelial Dysfunction and Elevation of S-Adenosylhomocysteine in Cystathionine {beta}-Synthase-Deficient Mice Circ. Res., June 8, 2001; 88(11): 1203 - 1209. [Abstract] [Full Text] [PDF] |
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