Aging increases circulating BH2 without modifying BH4 levels and impairs peripheral vascular function in healthy adults.

Little is known about the mechanisms of aging on vascular beds and its relationship with tetra and di-hydrobiopterin (BH4 and BH2) levels. This observational clinical study analyzed the impact of aging on plasma and platelet biopterins, cutaneous blood flow (CBF), and coronary flow reserve (CFR) in healthy adults. The study enrolled healthy adults in 3 age groups: 18-30, 50-59, and 60-70 years (n = 25/group). Biopterins were assessed by LC-MS/MS using newly defined pre-analytical conditions limiting BH4 oxidation and improving long-term stability. CBF was measured by Laser Speckle Contrast Imaging coupled with acetylcholine-iontophoresis and CFR by adenosine stress cardiac magnetic resonance. In healthy adults, aging (60-70 years vs 18-30 years) significantly increased platelet BH2 (+75%, P = 0.033) and BH2 + BH4 (+31%, P = 0.033), and to a lesser extent plasma BH2 (+29%, P = 0.009) without affecting BH4 and BH4/BH2. Simultaneously, CBF was decreased (-23%, P = 0.004) but not CFR, CBF being inversely correlated with platelet BH2 (r = -0.42, P = 0.001) and BH2 + BH4 (r = -0.41, P = 0.002). The proportion of adults with abnormal platelet BH2 increased with age (+28% in 60-70y). These abnormal BH2 levels were significantly associated with reduced CBF and CFR (-16%, P = 0.03 and -26%, P = 0.02). In conclusion, our study showed that age-related peripheral endothelial dysfunction was associated with an increase in circulating BH2 without decreasing BH4, the effect being more marked in platelets, the most relevant blood compartment to assess biopterin bioavailability. Peripheral but not coronary vascular function is progressively impaired with aging in healthy adults. All these findings support biopterins as therapeutic targets to improve vascular function.

Nitric oxide in exhaled gas and tetrahydrobiopterin in plasma after exposure to hyperoxia.

The fraction of nitric oxide in exhaled gas (FENO) is decreased after exposure to hyperoxia in vivo, although the mechanisms for this decrease is not clear. A key co-factor for nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), has been shown to be oxidized in vitro when exposed to hyperoxia. We hypothesized that the decrease of FENO is due to decreased enzymatic generation of NO due to oxidation of BH4. The present study was performed to investigate the relationship between levels of FENO and plasma BH4 following hyperoxic exposure in humans. Two groups of healthy subjects were exposed to 100% oxygen for 90 minutes. FENO was measured before and 10 minutes (n = 13) or 60 minutes (n = 14) after the exposure. Blood samples were collected at the same time points for quantification of biopterin levels (BH4, BH2 and B) using LC-MS/MS. Each subject was his or her own control, breathing air for 90 minutes on a separate day. Hyperoxia resulted in a 28.6 % decrease in FENO 10 minutes after exposure (p < 0.001), confirming previous findings. Moreover, hyperoxia also caused a 14.2% decrease in plasma BH4 (p = 0.012). No significant differences were observed in the group measured 60 minutes after exposure. No significant correlation was found between the changes in FENO and BH4 after the hyperoxic exposure (r = 0.052, p = 0.795), this might be due to the recovery of BH4 being faster than the recovery of FENO.

Early Screening for Tetrahydrobiopterin Responsiveness in Phenylketonuria.

Since 2007, synthetic tetrahydrobiopterin (BH4) has been approved as a therapeutic option in BH4-responsive phenylketonuria (PKU) and since 2015 extended to infants younger than 4 years in Europe. The current definition of BH4 responsiveness relies on the observation of a 20% to 30% blood phenylalanine (Phe) decrease after BH4 administration, under nonstandardized conditions. By this definition, however, patients with the same genotype or even the same patients were alternatively reported as responsive or nonresponsive to the cofactor. These inconsistencies are troubling, as frustrating patient expectations and impairing cost-effectiveness of BH4-therapy. Here we tried a quantitative procedure through the comparison of the outcome of a simple Phe and a combined Phe plus BH4 loading in a series of infants with PKU, most of them harboring genotypes already reported as BH4 responsive. Under these ideal conditions, blood Phe clearance did not significantly differ after the 2 types of loading, and a 20% to 30% decrease of blood Phe occurred irrespective of BH4 administration in milder forms of PKU. Such early screening for BH4 responsiveness, based on a quantitative assay, is essential for warranting an evidence-based and cost-effective therapy in those patients with PKU eventually but definitely diagnosed as responsive to the cofactor.

Biopterin status in dogs with myxomatous mitral valve disease is associated with disease severity and cardiovascular risk factors.

BACKGROUND: Endothelial dysfunction (ED) has been suggested to be associated with myxomatous mitral valve disease (MMVD) in dogs. Tetrahydrobiopterin (BH4) is an important cofactor for production of the endothelium-derived vasodilator nitric oxide (NO). Under conditions of oxidative stress, BH4 is oxidized to the biologically inactive form dihydrobiopterin (BH2). Thus, plasma concentrations of BH2 and BH4 may reflect ED and oxidative stress. OBJECTIVE: To determine plasma concentrations of BH2 and BH4 in dogs with different degrees of MMVD. ANIMALS: Eighty-four privately owned dogs grouped according to ACVIM guidelines (37 healthy control dogs including 13 Beagles and 24 Cavalier King Charles Spaniels [CKCSs], 33 CKCSs with MMVD of differing severity including 18 CKCSs [group B1] and 15 CKCSs [group B2], and 14 dogs of different breeds with clinical signs of congestive heart failure [CHF] because of MMVD [group C]). METHODS: Dogs underwent clinical examination including echocardiography. Plasma concentrations of BH2 and BH4 were measured using high-performance liquid chromatography with fluorescence detection. RESULTS: Higher plasma BH4 and BH2 concentrations were found with dogs in CHF compared with all other groups (control, B1 and B2; P ≤ .001). Females had higher concentrations of BH4 and BH4/BH2 (P ≤ .0003). BH4/BH2 was found to decrease with age (P < .0001). Cardiovascular risk factors in humans such as passive smoking (P ≤ .01) and increased body weight (P ≤ .009) were associated with lower BH4 concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: Age, sex, body weight, passive smoking, and cardiac status are associated with plasma biopterin concentration in dogs. Additional studies should clarify the clinical implications of the findings.

Circulating tetrahydrobiopterin as a novel biomarker for abdominal aortic aneurysm.

Rupture of abdominal aortic aneurysm (AAA) is unpredictable and lethal. A clinically valid biomarker to monitor the disease has not been available. Based on our recent discoveries that uncoupled endothelial nitric oxide synthase (eNOS)/tetrahydrobiopterin deficiency plays a causal role in various models of AAA, the present study examined the relationship between circulating and tissue levels of tetrahydrobiopterin (H4B) in angiotensin II-infused hyperphenylalaninemia (hph-1) and apoE null mice. For apoE null mice, tissue and plasma H4B levels decreased time dependently, to 2.69 ± 0.15 and 1.99 ± 0.06 pmol/mg, respectively (from 4.86 ± 0.32 and 3.31 ± 0.13 pmol/mg at baseline) by week 3, when aneurysms developed. For hph-1 mice, tissue and plasma H4B levels decreased significantly to 1.02 ± 0.10 and 0.98 ± 0.09 pmol/mg, respectively (from 1.84 ± 0.18 and 1.48 ± 0.12 pmol/mg at baseline), by week 1, when aneurysms developed. Oral folic acid administration, which has been shown to improve aortic H4B levels to completely prevent or markedly decrease the incidence of AAA, significantly increased tissue and plasma H4B levels in both animal models starting at week 1. The two H4B measurements at all conditions showed significant linear correlation, suggesting that plasma H4B accurately predicts its tissue levels when H4B is either reduced or enhanced. Together, these data demonstrate that H4B levels decrease with AAA development and increase with folic acid treatment in two different murine models of AAA and that plasma H4B levels accurately reflect H4B levels in the tissue, suggesting that circulating H4B levels may be used clinically as a novel and powerful biomarker for the development and response to treatment of AAA.

Early stage of obesity potentiates nitric oxide reduction during the development of renal failure.

BACKGROUND: Obesity is a serious health problem associated with the pathogenesis of various metabolic diseases. Nitric Oxide (NO) plays an important role in kidney function and altered NO levels have been associated with the pathogenesis of obesity. Therefore, we aimed to study whether an early stage of obesity contributes with progression of renal failure through further NO impairment. METHODS: Male C57BL/6 mice were fed with a high-fat diet (HFD) or a normal diet (ND) during 2 weeks. All mice underwent either sham surgery (sham) or 5/6 nephrectomy (Np). One group of HFD Np mice was treated with antioxidants plus L-arginine. Kidney damage parameters were assessed and eNOS metabolism was evaluated. RESULTS: Mice on a HFD increased body weight, eNOS protein and mRNA expression, and radical oxygen species (ROS). Urine nitrites excretion, urine volume, and plasma BH4 were decreased. In HFD mice, 5/6 Np further increased BH2 and urine protein concentration, ROS levels, and eNOS mRNA expression. The decrease in BH4 plasma levels and urine nitrites excretion was accentuated. NO synthesis stimulation with the antioxidants + L-arginine treatment prevented all these changes. CONCLUSIONS: The early changes in NO metabolism are associated with an early stage of obesity. This effect on NO potentiates kidney damage development.

Resveratrol improves vascular function in patients with hypertension and dyslipidemia by modulating NO metabolism.

Epidemiological studies have demonstrated that the Mediterranean diet, which is rich in resveratrol, is associated with a significantly reduced risk of cardiovascular disease. However, the molecular mechanisms that underlie the beneficial effects of resveratrol on cardiovascular function remain incompletely understood. Therefore, we set out to identify the molecular target(s) mediating the protective action of resveratrol on vascular function. To this end, we performed vascular reactivity studies to evaluate the effects of resveratrol on superior thyroid artery obtained from 59 patients with hypertension and dyslipidemia. We found that resveratrol evoked vasorelaxation and reduced endothelial dysfunction through the modulation of NO metabolism via (1) an 5' adenosine monophosphate-activated protein kinase-mediated increase in endothelial NO synthase activity; (2) a rise in tetrahydrobiopterin levels, which also increases endothelial NO synthase activity; and (3) attenuation of vascular oxidative stress, brought about by overexpression of manganese superoxide dismutase via an nuclear factor erythroid-derived 2-like 2-dependent mechanism. The effects of resveratrol on acetylcholine vasorelaxation were also tested in vessels from patients with nonhypertensive nondyslipidemia undergoing thyroid surgery. In this setting, resveratrol failed to exert any effect. Thus, our finding that resveratrol reduces endothelial dysfunction, an early pathophysiological feature and independent predictor of poor prognosis in most forms of cardiovascular disease, supports the concept that the risk of vascular events could be further reduced by adherence to a set of dietary and behavioral guidelines.

A GCH1 haplotype and risk of neural tube defects in the National Birth Defects Prevention Study.

Tetrahydrobiopterin (BH(4)) is an essential cofactor and an important cellular antioxidant. BH(4) deficiency has been associated with diseases whose etiologies stem from excessive oxidative stress. GTP cyclohydrolase I (GCH1) catalyzes the first and rate-limiting step of de novo BH(4) synthesis. A 3-SNP haplotype in GCH1 (rs8007267, rs3783641, and rs10483639) is known to modulate GCH1 gene expression levels and has been suggested as a major determinant of plasma BH(4) bioavailability. As plasma BH(4) bioavailability has been suggested as a mechanism of neural tube defect (NTD) teratogenesis, we evaluated the association between this GCH1 haplotype and the risk of NTDs. Samples were obtained from 760 NTD case-parent triads included in the National Birth Defects Prevention Study (NBDPS). The three SNPs were genotyped using TaqMan® SNP assays. An extension of the log-linear model was used to assess the association between NTDs and both offspring and maternal haplotypes. Offspring carrying two copies of haplotype C-T-C had a significantly increased NTD risk (risk ratio [RR]=3.40, 95% confidence interval [CI]: 1.02-11.50), after adjusting for the effect of the maternal haplotype. Additionally, mothers carrying two copies of haplotype C-T-C had a significantly increased risk of having an NTD-affected offspring (RR=3.46, 95% CI: 1.05-11.00), after adjusting for the effect of the offspring haplotype. These results suggest offspring and maternal variation in the GCH1 gene and altered BH(4) biosynthesis may contribute to NTD risk.

Clinical efficacy of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine (PMPDAP) in the treatment of feline immunodeficiency virus-infected cats.

In in vitro studies, the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine (PMPDAP) inhibited the replication of feline immunodeficiency virus (FIV). No information about its clinical efficacy is available so far. The aim of this prospective placebo-controlled, double-blinded study was to evaluate the antiviral efficacy of PMPDAP in cats naturally infected with FIV. Twenty cats were randomly assigned to two treatment groups receiving either PMPDAP (25 mg/kg) or placebo twice per week subcutaneously for 6 weeks. The general health status (Karnofsky's score), clinical signs, laboratory, immunological, and surrogate parameters were evaluated. No significant differences were found between PMPDAP- and placebo-treated cats, although cats treated with PMPDAP showed a tendency for improvement in their Karnofsky's score and clinical signs. Haematological side effects were noted in the PMPDAP-treated cats. Thus, PMPDAP may be an option in treating cats if it becomes available for veterinarians, but side effects have been monitored.

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

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.

Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis.

BACKGROUND: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. METHODS AND RESULTS: In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. CONCLUSIONS: The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Neopterin and biopterin as biomarkers of immune system activation associated with castration in piglets.

Recent reports have shown that stressful situations may affect the production of unconjugated pterins (neopterin and biopterin). The aim of the study was to investigate the effect of castration on neopterin and biopterin plasma concentrations in piglets, using 2 groups of 12 piglets allocated to castrated and uncastrated (control) groups. Pterin concentrations were determined by HPLC with fluorescence detection. Blood samples were also analzyed for leukocyte profiles and plasma cortisol concentrations. A time × treatment interaction (P < 0.05) was detected for neopterin concentrations, such that neopterin was greater (P < 0.01) at 1 h after surgery in castrated piglets compared with precastration concentrations, and neopterin was greater (P = 0.05) in castrated than in control piglets at 1 h. Castration had no effect on biopterin concentration (P > 0.1). Time effects (P < 0.05) for neutrophil and lymphocyte concentrations and neutrophil-to-lymphocyte ratios were found. A time × treatment interaction (P < 0.01) was detected for plasma cortisol concentrations, such that cortisol was greater (P < 0.01) at 1 and 24 h after surgery in castrated piglets compared with precastration concentrations and was greater (P < 0.01) in castrated than in control piglets at 1 and 24 h. This study showed that castration activated the immune system of piglets as demonstrated by an increase in plasma neopterin concentrations.

L-arginine and antioxidant diet supplementation partially restores nitric oxide-dependent regulation of phenylephrine renal vasoconstriction in diabetics rats.

OBJECTIVE: The increase of reactive oxygen species (ROS) in diabetes potentiates the vascular effects of phenylephrine through nitric oxide (NO) impairment, facilitating the development of diabetic nephropathy. We propose that the combination of an antioxidant and L-arginine as diet supplements could prevent the increased vascular response to phenylephrine in diabetic animals. DESIGN: Changes in the adrenergic system play an important role in the development of vascular complications in the prediabetic condition. The vasoconstrictor effects of phenylephrine are regulated by NO, and the impairment of endothelium-dependent vasodilation in diabetes is associated with ROS. SETTING: Diabetes was induced with a low dose (55 mg/kg body weight) of streptozotocin in 7-week-old rats. Diabetic rats were fed with a diet supplement containing a combination of vitamin E, vitamin C, eicosapentaenoic acid, docosahexaenoic acid, and L-arginine, and the effects on phenylephrine-induced renal vascular responses were evaluated. RESULTS: Phenylephrine increased the renal perfusion pressure of isolated perfused kidneys from diabetic rats compared with nondiabetic rats. This effect was associated with reduced nitrite release as well as reduced plasma tetrahydrobiopterin and increased superoxide anions in the renal tissue. Diet supplementation with a combination of L-arginine and vitamins in diabetic rats partially prevented the generation of superoxide associated with recovery of the renal release of NO and decreased phenylephrine-induced vasoconstrictor effects, compared with untreated diabetic rats. However, the administration of L-arginine or vitamins alone did not affect phenylephrine-induced vasoconstriction. Vitamin treatment alone did decrease superoxide generation. CONCLUSION: The protective mechanism of NO on the vasoconstrictor effects of phenylephrine in the kidney is lost during the development of diabetes, probably via the actions of ROS through a decrease in tetrahydrobiopterin, thus contributing to the pathogenesis of diabetic nephropathy. Restoration of this protective NO mechanism can be achieved by simultaneously stimulating NO synthesis and preventing the effects of ROS through the use of L-arginine and a combination of vitamins E and C as diet supplementation.

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP.

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

Systematic evaluation of nitric oxide, tetrahydrobiopterin, and anandamide levels in a porcine model of endotoxemia.

PURPOSE: Using a lipopolysaccharide (LPS)-treated porcine model, we examined: (1) whether nitric oxide (NO), anandamide, and tetrahydrobiopterin (BH4) increased or not in early endotoxic shock; and (2) the location of the major site of production of these molecules, by comparing their concentrations in arteries and the portal and hepatic veins. METHODS: Ten pigs received an infusion of LPS at 1.7 microg x kg(-1)x h(-1) via the portal vein for 240 min. Consecutive changes in systemic hemodynamics, hepatosplanchnic circulation, and oxygen delivery were measured. Furthermore, the variable changes in the concentrations of nitrite and nitrate (NOx), anandamide, and BH4 were measured. To access the effects of surgery, anesthesia, and fluid management on BH4, an experiment without LPS infusion was performed in two other animals. RESULTS: Mean arterial pressure and cardiac index started to decrease at 60 min after LPS infusion. However, systemic vascular resistance remained unchanged. Total hepatic blood flow and hepatic oxygen delivery also decreased significantly. NOx and anandamide did not change during LPS infusion. BH4 values did not change without LPS infusion. However, BH4 values increased significantly in the arterial, portal, and hepatic circulation during LPS infusion, especially in the hepatic vein (from 136.8 +/- 27.5 to 281.3 +/- 123.2 mol/ml; P < 0.01). CONCLUSION: Our data suggest that the BH4 values were significantly increased in several organs, especially in the liver during endotoxic shock. Impaired cardiac output and decreased blood pressure appeared in the early phase of porcine endotoxemia. Longer-term observation of these parameters after LPS treatment should be performed as the next step in future studies.

Biopterin in the acute phase of hypoxia-ischemia in a neonatal pig model.

To clarify the participation of inducible NOS (iNOS) in the hypoxia-ischemia, we examined iNOS and its tetrahydrobiopterin co-factor in the cerebral cortex and plasma in a newborn-piglet model. We also investigated the role of hypothermia in iNOS expression and biopterin production. Male newborn piglets were ventilated 6% oxygen for 45 min. Their common carotid arteries were clamped during hypoxia. Then they were resuscitated with 30% oxygen (HI group). Piglets of the hypothermia group were treated as the HI group and their body was cooled to 35.5 degrees C after hypoxic-ischemic insults. Sham-treated piglets were also reserved. In the HI group, iNOS was present in neurons and macrophages of the cerebral cortex 12h after the insult. The concentrations of nitrite and nitrate were elevated in the cerebral cortex 12h after hypoxic-ischemic insults but the biopterin level was unchanged. The plasma biopterin concentration after the insult (377.9+/-78.7 nM) was five times higher than before the insult (80.1+/-4.3 nM); this level peaked 4h after the insult (604.8+/-200.9 nM) and only slightly decreased after 12h (445.9+/-57.8 nM). In the hypothermia group, no iNOS expression was observed 12h after the insult. The plasma biopterin concentration after the insult (464.2+/-92.3 nM) was similar to that in the HI group, but was suppressed by 4h of hypothermia (229.3+/-106.8 nM). In this study, neuronal iNOS expression and increase of NO production were found in the acute phase of hypoxia-ischemia. Brain biopterin did not increase in hypoxia-ischemia although plasma biopterin was five-fold elevated. The discrepancy may also affect hypoxic-ischemic organ damage.

Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation.

BACKGROUND: Tetrahydrobiopterin (BH4) is a key regulator of endothelial nitric oxide synthase (eNOS) activity and coupling. However, the extent to which vascular and/or systemic BH4 levels are altered in human atherosclerosis and the importance of BH4 bioavailability in determining endothelial function and oxidative stress remain unclear. We sought to define the relationships between plasma and vascular biopterin levels in patients with coronary artery disease and to determine how BH4 levels affect endothelial function, eNOS coupling, and vascular superoxide production. METHODS AND RESULTS: Samples of saphenous veins and internal mammary arteries were collected from 219 patients with coronary artery disease undergoing coronary artery bypass grafting. We determined plasma and vascular levels of biopterins, vasomotor responses to acetylcholine, and vascular superoxide production in the presence and absence of the eNOS inhibitor N(G)-nitro-L-arginine methyl ester. High vascular BH4 was associated with greater vasorelaxations to acetylcholine (P<0.05), whereas high plasma BH4 was associated with lower vasorelaxations in response to acetylcholine (P<0.05). Furthermore, an inverse association was observed between plasma and vascular biopterins (P<0.05 for both saphenous veins and internal mammary arteries). High vascular (but not plasma) BH4 was associated with reduced total and N(G)-nitro-L-arginine methyl ester-inhibitable superoxide, suggesting improved eNOS coupling. Finally, plasma but not vascular biopterin levels were correlated with plasma C-reactive protein levels (P<0.001). CONCLUSIONS: An inverse association exists between plasma and vascular biopterins in patients with coronary artery disease. Vascular but not plasma BH4 is an important determinant of eNOS coupling, endothelium-dependent vasodilation, and superoxide production in human vessels, whereas plasma biopterins are a marker of systemic inflammation.

17beta-estradiol antagonizes the down-regulation of endothelial nitric-oxide synthase and GTP cyclohydrolase I by high glucose: relevance to postmenopausal diabetic cardiovascular disease.

In postmenopausal women, the risk of diabetic cardiovascular disease drastically increases compared with that of men or premenopausal women. However, the mechanism of this phenomenon has not yet been clarified. We hypothesized that the beneficial effects of estrogen on endothelial function may be relevant to protection against hyperglycemia-induced vascular derangement. Bovine aortic endothelial cells were incubated for 72 h in the presence and absence of the physiological concentration of 17beta-estradiol (17beta-E2) under normal and high-glucose conditions. The presence of 17beta-E2 significantly counteracted the reduction in basal nitric oxide production under high-glucose conditions. This finding was associated with the recovery of endothelial nitric-oxide synthase (eNOS) protein expression, tetrahydrobiopterin (BH4) levels, and the activity and gene expression of GTP cyclohydrolase I (GTPCH-I), a rate-limiting enzyme for BH4 synthesis. Both the gene transfer of estrogen receptor alpha using adenovirus and treatment with the protein kinase C inhibitor bisindolylmaleimide I significantly enhanced the effects of 17beta-E2 treatment under high-glucose conditions, whereas these effects were abolished by the estrogen receptor antagonist ICI 182,780 (faslodex). Transfection of small-interfering RNA targeting eNOS resulted in a marked reduction in GTPCH-I mRNA under both normal and high-glucose conditions, but this reduction was strongly reversed by 17beta-E2. These results suggest that the activation of ERalpha with 17beta-E2 can counteract high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen deficiency may result in an exaggeration of hyperglycemia-induced endothelial dysfunction, leading to the development of cardiovascular disease in postmenopausal diabetic women.

Estrogen therapy replenishes vascular tetrahydrobiopterin and reduces oxidative stress in ovariectomized rats.

OBJECTIVE: We investigated whether the effect of estrogen therapy on vascular endothelial function is mediated through increasing the bioavailability of tetrahydrobiopterin (BH4) and associated antioxidant capacity in ovariectomized (Ovx) rats. DESIGN: Aortas of sham-operated, Ovx, and Ovx plus estrogen therapy (Ovx + ET) female Sprague-Dawley rats were used to measure vascular reactivity. Plasma levels of nitric oxide (NO) metabolites, total antioxidant capacity, aortic superoxide anion (O2.), and BH4 contents were determined. RESULTS: Vascular reactivity, assessed on isolated aortic segments, indicated that phenylephrine-induced contraction in the Ovx group was significantly greater than that in the sham and Ovx + ET groups. The vasodilator responses to acetylcholine (10 to 10 M) and L-arginine (L-Arg; 10 M) in the sham and Ovx + ET groups were significantly greater than those in the Ovx group. Pretreatment with BH4 (10 M) enhanced the vasodilator responses to L-Arg in the Ovx group compared with the untreated Ovx group. An inhibitor of BH4 synthesis, 2,4-diamino-6-hydroxypyrimidine (2 mM), significantly attenuated the vasodilator response to L-Arg in the sham and Ovx + ET groups. In addition, Ovx significantly increased O2. production in aortic tissues and decreased plasma NO metabolites levels, whereas ET significantly prevented these effects. Pretreatment with BH4 also significantly decreased aortic O2. production in the Ovx group; both plasma total antioxidant capacity and aortic BH4 contents in the Ovx group decreased significantly compared with those in the sham group, which were also improved by ET. There were no significant differences in the protein expression of endothelial NO synthase in aortas in these groups. CONCLUSIONS: ET increases the availability of vascular BH4 to attenuate O2. production and restores total antioxidant capacity, leading to improved NO-mediated vasodilation in Ovx rats.

[Tetrahydrobiopterin loading test in differential diagnosis among hyperphenylalaninemia patients].

OBJECTIVE: To perform tetrahydrobiopterin (BH(4)) loading test and to further understand its usefulness in differential diagnosis among hyperphenylalaninemia(HPA) patients. METHODS: BH(4) loading test was carried out in 73 HPA patients, including the positive cases unveiled by neonatal screening and the clinically suspected cases. These patients, 47 males and 26 females, were at a mean age of 1.93 months. BH(4) (20 mg/kg) loading test was performed in all patients, and a combined phenylalanine (Phe)(100 mg/kg) and BH(4) loading test was performed among the patient who had a basic blood Phe concentration less than 600 micro mol/L. The urine pterine profile analysis and the dihydropteridine reductase activity in dry blood filter spot were tested simultaneously. RESULTS: During BH(4) loading test or combined Phe and BH(4) loading test, the patients with classic phenylketonuria showed no response to BH(4), the patients with moderate HPA caused by Phe hydroxylase deficiency decreased 32.8% of blood Phe level and the patients with BH(4) deficiency showed a prompt reduction in blood Phe level and it decreased to normal level at 4 h and lasted until 24 h. Twenty-two cases were diagnosed as classic phenylketonuria, 39 were moderate phenylketonuria and 12 were BH(4) deficiency. CONCLUSION: Hyperphenylalaninemia may be caused by deficiency of Phe hydroxylase or by deficiency of co-factor BH(4). Early diagnosis is important. BH(4) loading test is a safe and fast test in vivo. It is sensitive, easy-to-do, and is highly useful in differential diagnosis for suspected cases of HPA.