ascorbic-acid and 7-8-dihydrobiopterin

Ascorbate (Asc) has been shown to increase nitric oxide (NO) bioavailability and thereby improve endothelial function in patients showing signs of endothelial dysfunction. Tetrahydrobiopterin (BH₄) is a co-factor of endothelial nitric oxide synthase (eNOS) which may easily become oxidized to the inactive form dihydrobiopterin (BH₂). Asc may increase NO bioavailability by a number of mechanisms involving BH₄ and eNOS. Asc increases BH₄ bioavailability by either reducing oxidized BH₄ or preventing BH₄ from becoming oxidized in the first place. Asc could also increase NO bioavailability in a BH₄-independent manner by increasing eNOS activity by changing its phosphorylation and S-nitrosylation status or by upregulating eNOS expression. In this review, we discuss the putative mechanisms by which Asc may increase NO bioavailability through its interactions with BH₄ and eNOS.

Topics: Animals; Ascorbic Acid; Biomarkers; Biopterins; Cardiovascular Diseases; Guinea Pigs; Humans; Mice; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrogen; Oxygen; Phosphorylation; Rats; Reactive Oxygen Species; Risk; Vasodilation

Tetrahydrobiopterin (BH₄) is an essential co-factor of nitric oxide synthases and is easily oxidized to dihydrobiopterin (BH₂) which promotes endothelial nitric oxide synthase uncoupling and deleterious superoxide production. Vitamin C has been shown to improve endothelial function by different mechanisms, some involving BH₄. The hypothesis of the present study was that vitamin C status, in particular low levels, influences biopterin redox status in vivo. Like humans, the guinea pig lacks the ability to synthesize vitamin C and was therefore used as model. Seven day old animals (n = 10/group) were given a diet containing 100, 250, 500, 750, 1000, or 1500 ppm vitamin C until euthanasia at age 60-64 days. Blood samples were drawn from the heart and analyzed for ascorbate, dehydroascorbic acid (DHA), BH₄ and BH₂ by high-performance liquid chromatography. Plasma BH₄ levels were found to be significantly lower in animals fed 100 ppm vitamin C compared to all other groups (P < .05 or less). BH₂ levels were not significantly different between groups but the BH₂-to-BH₄ ratio was higher in the group fed 100 ppm vitamin C (P < .001 all cases). Significant positive correlations between BH4 and ascorbate and between BH₂-to-BH₄ ratio and DHA were observed (P < .0001 both cases). Likewise, BH₂-to-BH₄ ratio was negatively correlated with ascorbate (P < .0001) as was BH₄ and DHA (P < .005). In conclusion, the redox status of plasma biopterins, essentially involved in vasodilation, depends on the vitamin C status in vivo. Thus, ingestion of insufficient quantities of vitamin C not only leads to vitamin C deficiency but also to increased BH₄ oxidation which may promote endothelial dysfunction.

Topics: Animals; Antioxidants; Ascorbic Acid; Ascorbic Acid Deficiency; Biopterins; Dehydroascorbic Acid; Endothelium, Vascular; Guinea Pigs; Oxidation-Reduction; Oxidative Stress

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Topics: Antioxidants; Arginine; Ascorbic Acid; Biopterins; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Ethidium; Fluorescent Dyes; Homocysteine; Humans; L-Lactate Dehydrogenase; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; Pterins; Reactive Nitrogen Species; Superoxides; Thrombin; Time Factors

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Topics: Animals; Aorta; Apolipoproteins E; Arteriosclerosis; Ascorbic Acid; Biopterins; Cyclic AMP; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; In Vitro Techniques; Lipids; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Superoxides; Time; Tyrosine; Vasomotor System; Vitamin E

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

Topics: Ascorbic Acid; Biopterins; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Kinetics; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Pterins; Superoxides

To investigate underlying mechanisms responsible for the impaired nitric oxide (NO)-dependent vascular relaxation in the insulin-resistant state, we examined production of both NO and superoxide anion radical (O2-) and those modulating factors in aortas obtained from normal (CTR), insulin-treated (INS), or high fructose-fed (FR) rats. FR rats showed insulin resistance with endogenous hyperinsulinemia, whereas INS rats showed normal insulin sensitivity. Only FR aortic strips with endothelium elicited impaired relaxation in response to either acetylcholine or calcium ionophore A23187. Endothelial NO synthase (eNOS) activity and its mRNA levels were increased only in vessels from INS rats (P < 0.001), whereas eNOS activity in FR rats was decreased by 58% (P < 0.05) when compared with CTR rats. NO production from aortic strips stimulated with A23187 was significantly lower in FR than CTR rats. In contrast, A23187-stimulated O2- production was higher (P < 0.01) in FR than CTR rats. These differences were abolished when aortic strips were preincubated in the media including (6R)-5,6,7,8-tetrahydrobiopterin (BH4), an active cofactor for eNOS. Furthermore, as compared with CTR rats, aortic BH4 contents in FR rats were decreased (P < 0.001), whereas the levels of 7,8-dihydrobiopterin, the oxidized form of BH4, were increased, with opposite results in INS rats. These results indicate that insulin resistance rather than hyperinsulinemia itself may be a pathogenic factor for decreased vascular relaxation through impaired eNOS activity and increased oxidative breakdown of NO due to enhanced formation of O2- (NO/O2- imbalance), which are caused by relative deficiency of BH4 in vascular endothelial cells.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Ascorbic Acid; Biopterins; Blood Glucose; Blood Pressure; Calcimycin; Endothelium, Vascular; Fructose; Hyperinsulinism; In Vitro Techniques; Insulin; Insulin Resistance; Isometric Contraction; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Rats; Rats, Sprague-Dawley; RNA, Messenger; Superoxides; Transcription, Genetic; Vasodilation

Luminol-dependent chemiluminescence of normal human monocytes activated by zymosan is demonstrated to be inhibited by tetrahydrobiopterin in a concentration-dependent manner. The reduced pterins tetrahydrobiopterin, dihydrobiopterin, and dihydroneopterin are all shown to be readily oxidized by the hydroxyl radical. The susceptibility of reduced pterins to free radical attack may explain the inhibition of chemiluminescence observed and an additional role of reduced pterins as free radical scavengers in tissues is considered.

Topics: Ascorbic Acid; Biopterins; Dithioerythritol; Free Radicals; Humans; Hydrogen Peroxide; In Vitro Techniques; Luminescent Measurements; Macrophages; Monocytes; Neopterin; Oxidation-Reduction; Pteridines