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Background:Reduced availability of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide (NO) synthase (NOS), decreases NO production and increases reactive oxygen species. Both mechanisms contribute to atherosclerotic vascular disease. Although acute supplementation of BH4 improves endothelial dysfunction, the effect of chronic BH4 in humans is unknown.Objective:To investigate the effect of chronic BH4 supplementation on endothelial function and oxidative stress in hypercholesterolaemia.Design:Randomised double-blind, placebo-controlled trial.Setting:University Hospital.Patients:22 hypercholesterolaemic patients (low-density lipoprotein (LDL) >4.5 mmol/l) were randomised to 4 weeks of oral BH4 (400 mg twice daily) or placebo. Age-matched healthy volunteers served as controls.Main outcome measures:Endothelium-dependent and -independent vasodilatation was assessed by venous occlusion plethysmography. To elucidate the mechanisms of BH4 effect, NO release and superoxide anion (O2−) production were measured in human aortic endothelial cells exposed to native LDL (2.6 mmol cholesterol/l).Results:BH4 plasma levels were significantly increased by oral supplementation. NO-mediated vasodilatation to acetylcholine was reduced in patients compared with controls and restored by BH4. No effect of BH4 on endothelium-independent vasodilatation was seen. Furthermore, 8-F2 isoprostane plasma levels, a marker of vascular oxidative stress, were reduced by BH4. In LDL-treated endothelial cells, BH4 levels and NO release were reduced and O2− production increased compared with control cells. Exogenous BH4 normalised NO and O2− production.Conclusions:In hypercholesterolaemia, endothelial dysfunction and oxidative stress can be reversed by chronic oral treatment with BH4. Thus, BH4 availability is essential for maintaining NO synthesis and low O2− production by endothelial NOS in vivo, and may provide a rational therapeutic approach to prevent cardiovascular disease.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation. Genetic screens reveal a compendium of metabolic modifiers of lipid peroxidation. Tetrahydrobiopterin is essential under GPX4 inhibition, acting as a radical-trapping antioxidant that inhibits lipid peroxidation and is regenerated by DHFR.

We performed a pilot study to test the hypothesis that acute oral ingestion of tetrahydrobiopterin (BH(4)), a key cofactor modulating vascular nitric oxide (NO) synthase activity, improves large elastic artery stiffness with aging in men. Healthy older (63 ± 2 years; n = 8) and young (age 25 ± 1 years; n = 6) men were studied 3 h after ingestion of BH(4) (10 mg·kg(-1) body weight) or placebo on separate days in a randomized, placebo-controlled, double-blind study. Baseline carotid artery compliance was 37% lower (0.17 ± 0.02 vs. 0.22 ± 0.02 mm/mm Hg·10(-1)) and β-stiffness was 42% higher (7.3 ± 1.1 vs. 4.2 ± 0.5 AU) in the older men (both P < 0.05). BH(4) ingestion markedly increased circulating BH(4) concentrations in both groups (17-19-fold, P < 0.05), but increased compliance (+39% to 0.23 ± 0.02 mm/mm Hg·10(-1), P < 0.01) and decreased β-stiffness index (-27% to 5.3 ± 0.7 AU, P < 0.01) only in the older men. BH(4) also reduced carotid systolic blood pressure (SBP) in the older men (P < 0.05). These preliminary results support the possibility that limited BH(4) bioavailability contributes to impaired carotid artery compliance in healthy older men. Further studies are needed to determine if increasing BH(4) bioavailability though oral BH(4) supplementation may have therapeutic efficacy for improving large elastic artery compliance and reducing central SBP with aging.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain 1 , 2 . Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine—a tryptophan metabolite that blocks antitumour immunity—inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity. Tetrahydrobiopterin (BH4) is an enzyme co-factor that is involved in the nervous system; it is shown here to also function in T cell activation and proliferation, with roles in autoimmunity, allergic inflammation and cancer.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS) and aromatic amino acid hydroxylases. BH4 and 7,8-dihydrobiopterin (BH2) are metabolically interchangeable at the expense of NADPH. Exogenously administered BH4 can be metabolized by the body, similar to vitamins. At present, synthetic BH4 is used as an orphan drug for patients with inherited diseases requiring BH4 supplementation. BH4 supplementation has also drawn attention as a means of treating certain cardiovascular symptoms, however, its application in human patients remains limited. Here, we tracked biopterin (BP) distribution in blood, bile, urine, liver, kidney and brain after BH4 administration (5 mg/kg rat, i.v.) with or without prior treatment with probenecid, a potent inhibitor of uptake transporters particularly including organic anion transporter families such as OTA1 and OAT3. The rapid excretion of BP in urine was driven by elevated blood concentrations and its elimination reached about 90% within 120 min. In the very early period, BP was taken up by the liver and kidney and gradually released back to the blood. BH4 administration caused a considerable decrease in the BH4% in blood BP as an inevitable compensatory process. Probenecid treatment slowed down the decrease in blood BP and simultaneously inhibited its initial rapid excretion in the kidney. At the same time, the BH4% was further lowered, suggesting that the probenecid-sensitive BP uptake played a crucial role in BH2 scavenging in vivo. This suggested that the overproduced BH2 was taken up by organs by means of the probenecid-sensitive process, and was then scavenged by counter-conversion to BH4 via the BH4 salvage pathway. Taken together, BH4 administration was effective at raising BP levels in organs over the course of hours but with extremely low efficiency. Since a high BH2 relative to BH4 causes NOS dysfunction, the lowering of the BH4% must be avoided in practice, otherwise the desired effect of the supplementation in ameliorating NOS dysfunction would be spoiled.

Tetrahydrobiopterin is a critical cofactor for the NO synthases, and in its absence these enzymes become \"uncoupled,\" producing reactive oxygen species (ROSs) rather than NO. In aortas of mice with deoxycorticosterone acetate-salt (DOCA-salt) hypertension, ROS production from NO synthase is markedly increased, and tetrahydrobiopterin oxidation is evident. Using mice deficient in the NADPH oxidase subunit p47(phox) and mice lacking either the endothelial or neuronal NO synthase, we obtained evidence that hypertension produces a cascade involving production of ROSs from the NADPH oxidase leading to oxidation of tetrahydrobiopterin and uncoupling of endothelial NO synthase (eNOS). This decreases NO production and increases ROS production from eNOS. Treatment of mice with oral tetrahydrobiopterin reduces vascular ROS production, increases NO production as determined by electron spin resonance measurements of nitrosyl hemoglobin, and blunts the increase in blood pressure due to DOCA-salt hypertension. Endothelium-dependent vasodilation is only minimally altered in vessels of mice with DOCA-salt hypertension but seems to be mediated by hydrogen peroxide released from uncoupled eNOS, since it is inhibited by catalase. Tetrahydrobiopterin oxidation may represent an important abnormality in hypertension. Treatment strategies that increase tetrahydrobiopterin or prevent its oxidation may prove useful in preventing vascular complications of this common disease.

The enzyme tryptophan hydroxylase 2 (TPH2) hydroxylates L-tryptophan to L-5-hydroxytryptophan (5-HTP) in the presence of tetrahydrobiopterin (BH 4 ), iron ions (Fe 2+ ), and molecular oxygen, the first and rate-limiting step in the synthesis of the neurotransmitter serotonin (5-HT) in the mammalian brain. We compared the effect of L-tryptophan and bi- and trivalent iron ions on the thermal stability of the mutant (mt) and wild type (wt) TPH2 molecules with those of BH 4 . Mt TPH2 and wt TPH2 molecules were extracted from the midbrain of BALB/c and C57BL/6 mice, respectively. Thermal stability was examined using a thermal denaturation curve and assessed by T 50 , the temperature at which half of the enzyme molecules are denatured. L-tryptophan at a concentration of 0.2 mM did not stabilize TPH2. BH 4 increased T 50 of the mt enzyme at concentration of 0.2 mM, but not 0.1 mM. It was shown for the first time that divalent and trivalent iron ions at concentrations of 0.01, 0.05, and 0.2 mM increased T 50 of the mt and wt enzyme significantly higher than BH 4 . The results prompt using iron ions for elucidation of the molecular mechanisms of thermal stability of TPH2 in order to create pharmacological chaperones for correcting mutation-induced disturbances in TPH2 activity and the associated psychopathologies.

Administration of tetrahydrobiopterin (BH4) has been shown to attenuate acute allograft rejection in a murine heart transplantation model in a manner similar to that of cyclosporine A. However, its mechanism of action on immune cells remains largely unknown. A fully MHC-mismatched (C3H/He to C57BL/6) mouse heart transplant model was used in this study. The recipients were treated with BH4 or cyclosporine A six days. The degree of acute rejection was assessed by histopathological analysis, splenocytes were analyzed by flow cytometry, and cytokine production was estimated based on the level of protein and RNA in sera and grafts and in vitro in T cell cultures. Proliferation of regulatory T cells and mast cells, suppressor capacity of Tregs, and MLR of T cells were conducted in vitro. Survival curves confirmed the significant improvement observed in the BH4-treated animals. BH4-treatment resulted in a substantial increase in Tregs and mast cells in the secondary lymphoid organs. In vitro assays showed increased proliferation of BH4-treated Tregs and mast cells. Cytokine production in vivo and in vitro in BH4-treated animals revealed an increase in the expression of IL-10, IL-5 and IL-4. BH4-dependent mast cell-derived tryptophan hydroxylase-1 could be excluded as a treatment target in recipient knockout mice. These data suggest that BH4 modulates the innate and adaptive immune systems, resulting in increased proliferation of regulatory T and mast cells accompanied by a modulation of anti-inflammatory cytokines.

This study aimed to investigate the influence of chronic ischemia on nitric oxide biosynthesis in the bladder and the effect of administering tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase (eNOS), on chronic ischemia-related lower urinary tract dysfunction (LUTD). This study divided male Sprague–Dawley rats into Control, chronic bladder ischemia (CBI) and CBI with oral BH4 supplementation (CBI/BH4) groups. In the CBI group, bladder capacity and bladder muscle strip contractility were significantly lower, and arterial wall was significantly thicker than in Controls. Significant improvements were seen in bladder capacity, muscle strip contractility and arterial wall thickening in the CBI/BH4 group as compared with the CBI group. Western blot analysis of bladder showed expressions of eNOS ( p  = 0.043), HIF-1α ( p  < 0.01) and dihydrofolate reductase (DHFR) ( p  < 0.01), which could regenerate BH4, were significantly higher in the CBI group than in Controls. In the CBI/BH4 group, HIF-1α ( p  = 0.012) and DHFR expressions ( p  = 0.018) were significantly decreased compared with the CBI group. Our results suggest that chronic ischemia increases eNOS and DHFR in the bladder to prevent atherosclerosis progression. However, DHFR could not synthesize sufficient BH4 relative to the increased eNOS, resulting in LUTD. BH4 supplementation protects lower urinary tract function by promoting eNOS activity.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and “alternatively activated” M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γand lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide–induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerolmonooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.