Systemic and vascular oxidation limits the efficacy of oral tetrahydrobiopterin treatment in patients with coronary artery disease

Abstract

Background: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease.

Methods and results: Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue.

Conclusions: Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states.

Trial registration: ClinicalTrials.gov NCT00423280.

Figures

Figure 1

Study scheme. CABG indicates coronary artery bypass graft surgery; MRI, magnetic resonance imaging; FMD, flow-mediated dilatation; BH4, tetrahydrobiopterin; and L-NAME, NG-nitro-l-arginine methyl ester.

Figure 2

Effect of oral tetrahydrobiopterin (BH4) treatment on plasma biopterins. Levels of plasma biopterin species were quantified at baseline and after treatment with oral BH4 or placebo. Treatment with oral BH4 400 or 700 mg/d resulted in a significant increase in plasma BH4 levels compared with placebo (A) but also a significant increase in plasma dihydrobiopterin (BH2; B) and biopterin (data not shown). Accordingly, total biopterins (tBio; the sum of BH4 and BH2 and biopterin) were significantly elevated by BH4 treatment (C), but there was no change in the ratio of reduced to oxidized biopterins, BH4/(BH2+biopterin) (D). Values are expressed as mean±SEM of log-transformed values. *P<0.001 vs placebo group for change from baseline; P values calculated with 2-factor ANOVA with time–by–treatment group interaction followed by Bonferroni post hoc test (2 comparisons per panel: BH4 400 and 700 mg/d vs placebo).

Figure 3

Effect of oral tetrahydrobiopterin (BH4) treatment on vascular biopterins. Samples of saphenous vein (SV) and internal mammary artery (IMA) were collected at the time of coronary artery bypass graft surgery for quantification of biopterin species. In SV, treatment with oral BH4 resulted in a significant increase in tissue levels of BH4 and dihydrobiopterin (BH2; A) and biopterin (data not shown) vs placebo. In IMA, there were no significant differences between treatment groups (A). In both vessel types, treatment with oral BH4 did not alter the tissue ratio of reduced to oxidized biopterins, BH4/(BH2+biopterin) (B). Values are expressed as mean±SEM. *P<0.001 vs placebo; 2-factor ANOVA showed a significant individual effect of vessel type and treatment group on both BH4 (P<0.0001 for both) and BH2 (P<0.001 for both), but there was no significant vessel type–by–treatment group interaction for either BH2 (P=0.380) or BH4 (P=0.053). This analysis was followed by Bonferroni post hoc test for individual between-group comparisons for each vessel type (SV or IMA) and each biopterin species [BH4 or BH2 or BH4/(BH2+biopterin) ratio] separately (2 comparisons per variable: BH4 400 and 700 mg/d vs placebo).

Figure 4

Effect of oral tetrahydrobiopterin (BH4) treatment on vascular superoxide production and eNOS coupling. Samples of saphenous vein (SV) and internal mammary artery (IMA) were collected at the time of coronary artery bypass graft surgery for quantification of superoxide production by lucigenin-enhanced chemiluminescence in the presence and absence of the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME). There was no effect of BH4 treatment on total vascular superoxide production in SV or IMA (A) and no effect on L-NAME–inhibitable superoxide (B). Values are expressed as median (25th to 75th percentiles) and range. Groups were compared by use of 1-way ANOVA of log-transformed values.

Figure 5

Effect of oral tetrahydrobiopterin (BH4) treatment on endothelial function. Isometric tension studies were performed on saphenous vein rings to quantify vasorelaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) ex vivo. Oral BH4 treatment had no significant effect on endothelium-dependent relaxation to ACh (A) or endothelium-independent relaxation to SNP (B). Endothelial function in vivo was determined with magnetic resonance imaging to quantify brachial artery flow-mediated dilatation (FMD). There was no significant effect of BH4 treatment on brachial FMD (C). Values are expressed as mean±SEM. Data in A and B were analyzed with 2-way ANOVA for repeated measures (examining the effect of ACh or SNP concentration–by–treatment group interaction on vasorelaxations) in a full factorial model. The data in C were analyzed by 2-factor ANOVA (examining the effect of time–by–treatment group interaction on brachial FMD).

Figure 6

Ex vivo incubation of saphenous vein rings with exogenous tetrahydrobiopterin (BH4) or dihydrobiopterin (BH2). A through D, Saphenous vein rings (n=6 subjects) were incubated for 30 minutes under the following experimental conditions: control, buffer alone; BH4, BH4 100 µmol/L; BH4+DTE, BH4 100 µmol/L plus dithioerythritol (antioxidant) 1 mmol/L; and BH2, BH2 100 µmol/L. Samples of incubation medium were stored at the beginning and end of the experiment. A, Exogenous BH4 in the incubation medium was totally depleted by the end of the experiment; the addition of DTE provided partial protection from oxidation. B and C, Incubation with BH4 significantly increased tissue BH2 levels compared with control but increased tissue BH4 levels only in the presence of DTE; incubation with BH2 resulted in a larger increase in tissue BH2 but no increase in tissue BH4. D, Incubation in exogenous BH4 resulted in a significant reduction in tissue BH4/(BH2+biopterin) ratio compared with control, even in the presence of DTE. *P<0.05 vs control; ¶P<0.05 vs BH4 alone; $P<0.05 vs BH4+DTE. E and F, In a further experiment, SV tissue was obtained from an additional 6 patients. Two rings from each subject were incubated with BH4 and DTE, with a further 2 control rings incubated without BH4/DTE. Endothelium was removed from one of each pair of rings. Endothelial denudation led to a reduction of vascular BH4 (E) and total biopterins (tBio; F) by ≈75%. In vessels incubated with BH4+DTE, vascular BH4 was significantly increased. After endothelium was removed from these vessels, BH4 was reduced by ≈85%, demonstrating that the majority of both endogenous and exogenous BH4 is present in the endothelium. *P<0.05 vs endo(+)/BH4(–); †P<0.05 vs endo(+)/BH4(+); ≠P<0.05 vs endo(–)/BH4(–). Values are expressed as mean±SEM. A, The levels of BH4 and BH2 in the medium before and after incubation were compared between the control, BH4, and BH4+DTE groups by use of 1-way ANOVA followed by Bonferroni post hoc test for individual between-group comparisons (3 comparisons). B through F, Variables were compared by use of 1-way ANOVA followed by Bonferroni post hoc test for individual between-group comparisons (for 6 comparisons in B–D and 4 comparisons in E and F).

Figure 7

Fate of endogenous vs exogenous tetrahydrobiopterin (BH4) in whole blood and plasma. Samples of whole blood (A) and plasma (B) from healthy volunteers (n=3) were incubated for 4 hours under the following experimental conditions: control, blood or plasma alone; BH4, supplementary BH4 50 nmol/L; BH4+DTE, supplementary BH4 50 nmol/L plus dithioerythritol (antioxidant) 1 mmol/L; and BH2, supplementary dihydrobiopterin 50 nmol/L. In the whole blood samples, plasma was separated at baseline and after 4 hours. Plasma biopterins were then quantified in all samples. In whole blood, there was no significant difference in endogenous BH4 or BH2 after 4 hours of incubation, whereas in plasma alone, endogenous BH4 was oxidized to BH2. Exogenous BH4 was completely oxidized in both whole blood and plasma after 4 hours; DTE afforded protection from oxidation at baseline but not after 4 hours. *P<0.05 vs control; ¶P<0.05 vs BH4 alone; $P<0.05 vs BH4+DTE; §P<0.05 vs baseline. Both preincubation and postincubation BH4 and BH2 levels were compared separately by use of 1-way ANOVA followed by Bonferroni post hoc test for individual between-group comparisons (6 comparisons). The change in BH4 before versus after incubation was tested only in the control groups by use of a paired t test.