Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity

The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH4) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH4 were readily oxidized by dissolved O-2 or H2O2. BH4 was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H2O2. Addition of 36 mumol/1 BH4 to 143 gmol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H2O2. In the presence of catalase, BH4 still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH4, most probably quininoid-BH2 (qBH(2)), could also stimulate ASC oxidation while itself being reduced back to BH4. ASC prevented the auto-oxidation of BH4 in a concentration-dependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 mumol/1 BH4. Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 umol/l BH4, and 0.5 mmol/l ASC enhanced the BH4-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH4 is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH4. Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH4 and may contribute to the stimulation of placental eNOS activity.