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(Circulation Research. 2000;86:1129.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Folic Acid Reverts Dysfunction of Endothelial Nitric Oxide Synthase

E. S. G. Stroes, E. E. van Faassen, M. Yo, P. Martasek, P. Boer, R. Govers, T. J. Rabelink

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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Abstract—5-Methyltetrahydrofolate (MTHF), the active form of folic acid, has been reported to restore NO status in hypercholesterolemic patients. The mechanism of this effect remains to be established. We assessed the effects of L- and D-MTHF on tetrahydrobiopterin (BH₄)–free and partially BH₄-repleted endothelial NO synthase (eNOS). Superoxide production of eNOS and the rate constants for trapping of superoxide by MTHF were determined with electron paramagnetic resonance using 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin trap for superoxide. NO production was measured with [³H]arginine–citrulline conversion or nitrite assay. The rate constants for scavenging of superoxide by L- and D-MTHF were similar, 1.4x10⁴ ms^–1. In BH₄-free eNOS, L- and D-MTHF have no effect on enzymatic activity. In contrast, in partially BH₄-repleted eNOS, we observe a 2-fold effect of MTHF on the enzymatic activity. First, superoxide production is reduced. Second, NO production is enhanced. In cultured endothelial cells, a similar enhancement of NO production is induced by MTHF. In the present study, we show direct effects of MTHF on the enzymatic activity of NO synthase both in recombinant eNOS as well as in cultured endothelial cells, which provides a plausible explanation for the previously reported positive effects of MTHF on NO status in vivo.

Key Words: folic acid • nitric oxide • nitric oxide synthase • tetrahydrobiopterin • electron spin resonance

	Introduction

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Impaired availability of endothelium-derived NO has been identified as both a mediator of atherosclerosis and therapeutic target in cardiovascular prevention trials.^{1 2 3 4 5} Several factors can contribute to reduced bioavailability of NO, ranging from impaired production to increased degradation, depending on the risk factors involved.^{6 7} NO is synthesized by dimers of the 130-kDa enzyme endothelial NO synthase (eNOS) in a reaction in which L-arginine is oxidized to NO and L-citrulline. Recently, eNOS has been shown to produce superoxide radicals as well as NO.^{8 9} Under physiological conditions, eNOS predominantly produces NO, controlled by the regulatory coenzyme calmodulin, the substrate L-arginine, and the cofactor tetrahydrobiopterin (BH₄).¹⁰ Under pathophysiological conditions, such as dyslipidemia, production shifts from NO to superoxide.¹¹ The exact mechanism behind this "dysfunction" has been a matter of debate. Clinical studies have shown impaired NO bioavailability in patients with (risk factors for) atherosclerosis.^{12 13 14 15 16 17 18 19 20} Evidence has accumulated showing that increased degradation of NO by superoxide, rather than impaired formation of NO, is the predominant cause of impaired NO bioavailability in early atherosclerosis.^{21 22 23} These observations indicate that atherogenesis is linked to a pathological imbalance between NO and superoxide, rather than to reduced NO production per se.

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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Chemicals
BH₄-free bovine eNOS was obtained through expression of eNOS in Escherichia coli as described previously.²⁶ L-MTHF and its stereoisomer D-MTHF (purity >99.8%) were a kind gift from Eprova (Schaffhausen, Switzerland). The spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was purchased from Calbiochem-Novabiochem GmbH. All other chemicals were obtained from Sigma.

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.

View larger version (16K): [in this window] [in a new window]   Figure 3. CST by L- and D-MTHF in eNOS. The slope of the curve is much steeper for pterin-repleted (•) than for pterin-free () eNOS, both for L- and for D-MTHF (P<0.05 pterin-repleted vs pterin-free eNOS). This shows that MTHF interferes with enzymatic superoxide production by pterin-repleted eNOS.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of BH4 on superoxide production by eNOS in presence and absence of L-MTHF BH4 induces a dose-dependent decrease in radical-adduct formation (). Preincubation with L-MTHF (25 µmol/L) significantly enhances the BH4-associated decrease in radical adduct formation by eNOS (•). *P<0.05 L-MTHF vs saline.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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XO Activity
Assessment of urate levels (assessed with a uricase–hydrogen peroxide assay) is a standard method to determine enzymatic activity of XO. However, reductive substances, such as vitamin C or MTHF, are known to interfere with the urate determination. Basal urate levels after 60 minutes of incubation (12.5 mU/mL XO, 0.5 mmol/L hypoxanthine in phosphate buffer, pH 7.4, 37°C) were 128±20 µmol/L. Addition of 50 µmol/L L-MTHF at the beginning of the incubation period resulted in a significantly lower urate level of 48.7±1.7 µmol/L. Addition of 50 µmol/L L-MTHF at the end of the incubation period (just before urate assessment) resulted in a similar urate level (49.8±1.9 µmol/L). These data show that L-MTHF interferes with the quantification of urate, rather than the urate production itself. We interpret these results as showing that MTHF does not affect the rate of urate production by XO.

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 k_L-MTHF/k_DEPMPO=k_D-MTHF/k_DEPMPO=175±13 (at 37°C; pH 7.4). Using the literature value of k_DEPMPO=80 ms^–1 (see Note), we find the following rate constants for scavenging of superoxide: k_L-MTHF = k_D-MTHF=1.4x10⁴ ms^–1. For comparison, the scavenging rate of the potent antioxidant ascorbic acid is 2.7x10⁵ ms^–1 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.

View larger version (14K): [in this window] [in a new window]   Figure 1. Time curve of EPR intensity for HX/XO.

View larger version (16K): [in this window] [in a new window]   Figure 2. CST by L- and D-MTHF in HX/XO. From the slope of the curves, it follows that the superoxide trapping rates for L-MTHF (•) and D-MTHF () are similar and 175 times the superoxide trapping rate of DEPMPO. The temperature is 37°C; pH=7.4.

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).

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of L-/D-MTHF and BH4 on NO production. Pterin-free eNOS produces no NO. Addition of BH4 results in significant NO production. Both L- and D-MTHF cause a further increase in NO production by pterin-repleted eNOS, whereas MTHF has no effect on pterin-free eNOS.

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).

View larger version (14K): [in this window] [in a new window]   Figure 6. Effect of L-MTHF and sepiapterin on acetylcholine-stimulated nitrite production by endothelial cells. Preincubation with MTHF (1 and 10 µmol/L) or sepiapterin (100 µmol/L) significantly enhances acetylcholine-induced nitrite production from endothelial cells. *P<0.05 vs saline.

	Discussion

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Our in vitro data reveal the following 3 characteristic properties of MTHF. (1) Both isomers of MTHF have a substantial capacity to scavenge superoxide in solution. (2) In pterin-free eNOS, neither isomer affects the enzymatic activity, either with regard to NO or to superoxide production. (3) In partially pterin-repleted eNOS, both isomers have the same strong effect on the activity of the enzyme; ie, they enhance NO production concomitant with a decreased production of superoxide. The MTHF-induced increase in NO production was confirmed in cultured endothelial cells, thus supplying a new mechanism by which MTHF may affect vascular function in vivo.

MTHF–Direct 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.

MTHF–Pterin-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.

MTHF–Pterin-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.

MTHF–Pterin 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

MTHF–Endothelial 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

MTHF–Clinical 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 ms^–1 (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 k_DEPMPO=80 ms^–1 for trapping of superoxide by DEPMPO at pH of 7.4 and temperature of 37°C.

	Acknowledgments

 
P.M. is supported by the Grant Agency of Czech Republic (GACR 306/99/0054). E.S.G. Stroes is a fellow of the Dutch Heart Foundation (D 97/023). We thank F. de Ruijter for her expert technical assistance, Dr C.S. Raman (University of California, Irvine, Calif) for his valuable discussions, Dr Alan Schwartz (Washington University, St. Louis, Mo) for kindly providing the bEND3 cell line, and Dr B.S.S. Masters (University of Texas Health Science Center, San Antonio, Tex) for critical reading of the manuscript.

Received March 16, 2000; accepted April 5, 2000.

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