Tetrahydrobiopterin Deficiency Research Articles

Research progress on phenotype and genotype of hyperphenylalaninemia

Hyperphenylalaninemia(HPA), an autosomal recessive disease, is the most common inborn error of amino acid metabolism, caused by the deficiency of phenylalanine hydroxylase(PAH) or tetrahydrobiopterin(BH4) which induced by mutations of genes. The accumulation of the clinical database and genetic information will enhance the development of novel personalized medicine and to provide more accurate and timely diagnostic and therapeutic approaches for HPA. This paper summarizes the correlations between HPA metabolism and PAH, BH4, pathogenic genes and their distributions in HPA, as well as the phenotypes and genotypes of HPA, so as to provide reference for personalized medicine for HPA.

Analysis of types and metabolic profiles of hyperphenylalaninemia

Objective To study the characteristics of hyperphenylalaninemia (HPA) and the differences in blood and urine metabolic index and their correlation. Methods A total of 137 patients with HPA diagnosed by the Pediatric Inherit Metabolism and Endocrine Department, Guangdong Women and Children′s Hospital, Guangzhou Medical University from January 2014 to June 2017, were enrolled.Tandem mass spectrometry (MS/MS), gas chromatography/mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) were used to analyze the concentration of blood and urine metabolites in children, and the patients were divided into different groups according to the drug load test of tetrahydrobiopterin (BH4) and dihydrobiopterindine reductase (DHPR) deficiency.The HPA metabolite analysis of horizontal concentration by statistical differences and correlation analysis were performed. Results Among the 137 cases of HPA, there were 101 cases (73.7%) of phenylalanine hydroxylase deficiency (PAH), and among them 21 cases (15.3%) were classic phenylketonuria (PKU), 37 cases were mild PKU (27.0%), 43 cases (31.4%) were mild HPA.There were 22 cases (16.1%) with BH4 reaction, and 79 cases (57.7%) of non-reactive type.Besides, there were 36 cases(26.3%) of tetrahydrobiopterin deficiency (BH4D), of which 6-pyruvoyl tetrahydropterin synthase deficiency(PTPS) in 34 cases(24.8%) and dihydrobiopterindine reductase deficiency (DHPR) in 2 cases (1.5%). Urinary phenylacetic acid (r=0.673, P 0.05). Conclusions Through the analysis of the different types of HPA metabolic profiles, it can help to master the incidence and characteristics in the region, within a certain concentration range of blood Phe, the phenylacetic acid, phenyllactic acid, phenylpyruvic acid should not be tested by GC-MS alone.Uterine erythropoietin analysis of BH4D classification and identification of BH4 reaction, non-reactive PKU have a supporting role, so master the metabolic index of various types of concentration and relevance of HPA, it can provide basis for early diagnosis, accurate treatment and follow-up. Key words: Hyperphenylalaninemia; Metabolic profiling; Phenylalanine hydroxylase deficiency; Tetrahydrobiopterin deficiency

Pregnancy management and outcome in patients with four different tetrahydrobiopterin disorders.

Inborn errors of tetrahydrobiopterin (BH4) biosynthesis or recycling are a group of very rare neurometabolic diseases. Following growing awareness and improved availability of drug treatment the number of patients with BH4 disorders reaching adulthood is constantly increasing. Pregnancy care of patients with these disorders is therefore a new challenge for clinicians. This retrospective study summarises for the first time clinical and biochemical monitoring data of 16 pregnancies in seven women with different disorders of BH4 metabolism and evaluates treatment regimens before and during pregnancy in relation to the obstetrical outcome and paediatric follow-up. Worsening of pre-existing neurological symptoms or occurrence of new symptoms during pregnancy was not observed in most of the cases. Treatment regimens remained mostly unchanged. Pregnancies were not complicated by disease-specific features. Organ abnormalities, miscarriage, prematurity, IUGR and chromosomal changes were occasionally reported, without showing any association with the standard drug treatment for BH4 deficiencies. Although our data on 16 pregnancies in seven patients did notpresent any association of standard drug treatment with an increased rate of pregnancy complications, abnormal obstetrical or paediatric outcome, an intensive clinical and biochemical supervision by a multidisciplinary team before, during and after the pregnancy in any BH4 deficiency is essential since available data on pregnancies in patients with BH4 deficiencies is limited.

Roles for endothelial cell and macrophage Gch1 and tetrahydrobiopterin in atherosclerosis progression.

AimsGTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown.Methods and resultsHyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE–/– mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE–/– mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE–/– mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis.ConclusionBoth endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.

Secondary BH4 deficiency links protein homeostasis to regulation of phenylalanine metabolism.

Metabolic control of phenylalanine concentrations in body fluids is essential for cognitive development and executive function. The hepatic phenylalanine hydroxylating system is regulated by the ratio of l-phenylalanine, which is substrate of phenylalanine hydroxylase (PAH), to the PAH cofactor tetrahydrobiopterin (BH4). Physiologically, phenylalanine availability is governed by nutrient intake, whereas liver BH4 is kept at constant level. In phenylketonuria, PAH deficiency leads to elevated blood phenylalanine and is often caused by PAH protein misfolding with loss of function. Here, we report secondary hepatic BH4 deficiency in Pah-deficient mice. Alterations in de novo synthesis and turnover of BH4 were ruled out as molecular causes. We demonstrate that kinetically instable and aggregation-prone variant Pah proteins trap BH4, shifting the pool of free BH4 towards bound BH4. Interference of PAH protein misfolding with metabolite-based control of l-phenylalanine turnover suggests a mechanistic link between perturbation of protein homeostasis and disturbed regulation of metabolic pathways.

BH4 deficiency identified in a neonatal screening program for hyperphenylalaninemia

ObjectivesTo show the general prevalence and to characterize tetrahydrobiopterin (BH4) deficiencies with hyperphenylalaninemia, identified by the Neonatal Screening Program of the State of Minas Gerais. MethodsDescriptive study of patients with BH4 deficiency identified by the Neonatal Screening Program of the State of Minas Gerais. ResultsThe prevalence found was 2.1 for 1,000,000 live births, with a frequency of 1.71% among hyperphenylalaninemias. There were four cases (40%) with 6-pyruvoyl-tetrahydropterin synthase deficiency, three with GTP cyclohydrolase I – autosomal recessive form deficiency, and three with dihydropteridine reductase deficiency (30% each). Six patients were diagnosed due to clinical suspicion and four cases due to systematic screening in neonatal screening. After the start of the treatment, patients identified by neonatal screening had rapid improvement and improved neuropsychomotor development compared to those diagnosed by the medical history. ConclusionsThe prevalence of BH4 deficiencies in Minas Gerais was slightly higher than that found in the literature, but the frequency among hyperphenylalaninemias was similar. Although rare, they are severe diseases and, if left untreated, lead to developmental delays, abnormal movements, seizures, and premature death. Early treatment onset (starting before 5 months of age) showed good results in preventing intellectual disability, justifying the screening of these deficiencies in newborns with hyperphenylalaninemia identified at the neonatal screening programs for phenylketonuria.

Mutation spectrum of hyperphenylalaninemia candidate genes and the genotype-phenotype correlation in the Chinese population

BackgroundHyperphenylalaninemia (HPA) is an inherited metabolic disorder that is caused by a deficiency of phenylalanine hydroxylase (PAH) or tetrahydrobiopterin. The prevalence of HPA varies widely around the world. MethodsA spectrum of HPA candidate genes in 1020 Chinese HPA patients was reported. Sanger sequencing, next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and quantitative real-time PCR (qRT-PCR) were applied to precisely molecular diagnose HPA patients. The allelic phenotype values (APV) and genotypic phenotype values (GPV) were calculated in PAH-deficient patients based on a recently developed formula. ResultsApart from genetic diagnoses confirmed in 915 HPA patients (89.7%) by Sanger sequencing, pathogenic variants were discovered in another 57 patients (5.6%) through deep detections (NGS, MLPA and qRT-PCR). We identified 196, 42, 10 and 2 variants in PAH, PTS, QDPR and GCH1, respectively. And a total of 47 novel variants were found in these genes. Through the APV and GPV calculations, it was found that the new GPV system was well correlated with metabolic phenotypes in most PAH-deficient patients. ConclusionsMore HPA candidate variants were identified using new molecular diagnostic methods. The new APV and GPV system is likely to be highly beneficial for predicting clinical phenotypes for PAH-deficient patients.

An Overview of Traditional and Novel Therapeutic Options for the Management of Phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by the deficiency of phenylalanine hydroxylase enzyme that catalyzes the conversion of L-phenylalanine to L-tyrosine using tetrahydrobiopterin (BH4) as a cofactor. Among aminoacidopathies, PKU is one of the most prevalent disorders in different populations. It may be caused by deficiency of BH4 or mutations in PAH. About 98% of PKU patients have mutations in the PAH, while the remaining have BH4 deficiency. If PKU is diagnosed earlier in life using advance analytical techniques (e.g., high performance liquid chromatography, mass spectrometry, and polymerase chain reaction), then it is potentially treatable by special diets (L-phenylalanine-free medical formula). However, some complications such as vitamin B12 deficiency, cardiovascular problems, and neurodevelopmental problems have been reported in PKU patients when they ate special diets for a long period. Hence, special diet alone is not a good option for proper treatment. Next generation therapies require structure-function based development. For therapies which target PAH gene (e.g., gene therapy, RNAi, gene editing), a lot of research has yet to be done. Treatment with BH4 therapy is safe and effective but only in BH4-responsive PKU patients. Therefore, research efforts should be focused on the development of more targeted pharmacological and genetic therapies especially PAH gene therapy, which can reduce the burden or deleterious effects of this disease in affected patients.

Tetrahydrobiopterin (BH4) deficiency - diagnosis and treatment

Since the initial breaking discovery of Følling that the severe neurological consequences of phenylketonuria could be prevented by use of low phenylalanine (Phe) diet, it has been shortly recognised that defective phenylalanine metabolism may also arise from the deficiency of tetrahydrobiopterin (BH4) cofactor, required for phenylalanine-hydroxylase activity. Furthermore, as BH4 is in Phe metabolism, it is also a cofactor for the activities of tyrosine hydroxylase and tryptophane hydroxylase, enzymes required for the synthesis of catecholamines and serotonin neurotransmitters. Besides hyperphenylalaninemia in patients with tetrahydrobiopterin deficiencies, dopamine and serotonin deficiencies, with different disorders of the central nervous system also develop. Mild form of tetrahydrobiopterin deficiency is rare, most of the patients have severe neurological abnormalities including progressive mental retardation if not treated properly. Early diagnosis and treatment are essential and can improve the clinical course and prognosis. Orv Hetil. 2017; 158(48): 1897-1902.

Massive parallel sequencing as a new diagnostic approach for phenylketonuria and tetrahydrobiopterin-deficiency in Thailand

BackgroundHyperphenylalaninemia (HPA) can be classified into phenylketonuria (PKU) which is caused by mutations in the phenylalanine hydroxylase (PAH) gene, and BH4 deficiency caused by alterations in genes involved in tetrahydrobiopterin (BH4) biosynthesis pathway. Dietary restriction of phenylalanine is considered to be the main treatment of PKU to prevent irreversible intellectual disability. However, the same dietary intervention in BH4 deficiency patients is not as effective, as BH4 is also a cofactor in many neurotransmitter syntheses.MethodWe utilized next generation sequencing (NGS) technique to investigate four unrelated Thai patients with hyperphenylalaninemia.ResultWe successfully identified all eight mutant alleles in PKU or BH4-deficiency associated genes including three novel mutations, one in PAH and two in PTS, thus giving a definite diagnosis to these patients. Appropriate management can then be provided.ConclusionThis study identified three novel mutations in either the PAH or PTS gene and supported the use of NGS as an alternative molecular genetic approach for definite diagnosis of hyperphenylalaninemia, thus leading to proper management of these patients in Thailand.

Tetrahydrobiopterin (BH4) deficiency is associated with augmented inflammation and microvascular degeneration in the retina

BackgroundTetrahydrobiopterin (BH4) is an essential cofactor in multiple metabolic processes and plays an essential role in maintaining the inflammatory and neurovascular homeostasis. In this study, we have investigated the deleterious effects of BH4 deficiency on retinal vasculature during development.Methodshph-1 mice, which display deficiency in BH4 synthesis, were used to characterize the inflammatory effects and the integrity of retinal microvasculature. BH4 levels in retinas from hph-1 and wild type (WT) mice were measured by LC-MS/MS. Retinal microvascular area and microglial cells number were quantified in hph-1 and WT mice at different ages. Retinal expression of pro-inflammatory, anti-angiogenic, and neuronal-derived factors was analyzed by qPCR. BH4 supplementation was evaluated in vitro, ex-vivo, and in vivo models.ResultsOur findings demonstrated that BH4 levels in the retina from hph-1 mice were significantly lower by ~ 90% at all ages analyzed compared to WT mice. Juvenile hph-1 mice showed iris atrophy, persistent fetal vasculature, significant increase in the number of microglial cells (p < 0.01), as well as a marked degeneration of the retinal microvasculature. Retinal microvascular alterations in juvenile hph-1 mice were associated with a decreased expression in Norrin (0.2-fold) and its receptor Frizzled-4 (FZD4; 0.51-fold), as well as with an augmented expression of pro-inflammatory factors such as IL-6 (3.2-fold), NRLP-3 (4.4-fold), IL-1β (8.6-fold), and the anti-angiogenic factor thrombospondin-1 (TSP-1; 17.5-fold). We found that TSP-1 derived from activated microglial cells is a factor responsible of inducing microvascular degeneration, but BH4 supplementation markedly prevented hyperoxia-induced microglial activation in vitro and microvascular injury in an ex-vivo model of microvascular angiogenesis and an in vivo model of oxygen-induced retinopathy (OIR).ConclusionOur findings reveal that BH4 is a key cofactor in regulating the expression of inflammatory and anti-angiogenic factors that play an important function in the maintenance of retinal microvasculature.

BH4 deficiency identified in a neonatal screening program for hyperphenylalaninemia

ObjectivesTo show the general prevalence and to characterize tetrahydrobiopterin (BH4) deficiencies with hyperphenylalaninemia, identified by the Neonatal Screening Program of the State of Minas Gerais. MethodsDescriptive study of patients with BH4 deficiency identified by the Neonatal Screening Program of the State of Minas Gerais. ResultsThe prevalence found was 2.1 for 1,000,000 live births, with a frequency of 1.71% among hyperphenylalaninemias. There were four cases (40%) with 6-pyruvoyl-tetrahydropterin synthase deficiency, three with GTP cyclohydrolase I – autosomal recessive form deficiency, and three with dihydropteridine reductase deficiency (30% each). Six patients were diagnosed due to clinical suspicion and four cases due to systematic screening in neonatal screening. After the start of the treatment, patients identified by neonatal screening had rapid improvement and improved neuropsychomotor development compared to those diagnosed by the medical history. ConclusionsThe prevalence of BH4 deficiencies in Minas Gerais was slightly higher than that found in the literature, but the frequency among hyperphenylalaninemias was similar. Although rare, they are severe diseases and, if left untreated, lead to developmental delays, abnormal movements, seizures, and premature death. Early treatment onset (starting before 5 months of age) showed good results in preventing intellectual disability, justifying the screening of these deficiencies in newborns with hyperphenylalaninemia identified at the neonatal screening programs for phenylketonuria.

Organic anion transporters, OAT1 and OAT3, are crucial biopterin transporters involved in bodily distribution of tetrahydrobiopterin and exclusion of its excess

Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

Tetrahydrobiopterin activates brown adipose tissue and regulates systemic energy metabolism.

Brown adipose tissue (BAT) is a central organ that acts to increase energy expenditure; its regulatory factors could be clinically useful in the treatment of obesity. Tetrahydrobiopterin (BH4) is an essential cofactor of tyrosine hydroxylase and nitric oxide synthase (NOS). Although BH4 regulates the known regulatory factors of BAT, such as noradrenaline (NA) and NO, participation of BH4 in BAT function remains unclear. In the present study, we investigate the role of BH4 in the regulation of BAT. Hph-1 mice, a mouse model of BH4 deficiency, exhibit obesity, adiposity, glucose intolerance, insulin resistance, and impaired BAT function. Impaired BAT function was ameliorated together with systemic metabolic disturbances by BAT transplantation from BH4-sufficient mice (control mice) into BH4-deficient mice, strongly suggesting that BH4-induced BAT has a critical role in the regulation of systemic energy metabolism. Both NA derived from the sympathetic nerve and NO derived from endothelial NOS in the blood vessels participate in the regulation of BH4. In addition, a direct effect of BH4 in the stimulation of brown adipocytes via NO is implicated. Taken together, BH4 activates BAT and regulates systemic energy metabolism; this suggests an approach for metabolic disorders, such as obesity and diabetes.

A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila.

Here, we show that the enzymatic cofactor tetrahydrobiopterin (BH4) inhibits feeding in Drosophila. BH4 biosynthesis requires the sequential action of the conserved enzymes Punch, Purple, and Sepiapterin Reductase (Sptr). Although we observe increased feeding upon loss of Punch and Purple in the adult fat body, loss of Sptr must occur in the brain. We found Sptr expression is required in four adult neurons that express neuropeptide F (NPF), the fly homologue of the vertebrate appetite regulator neuropeptide Y (NPY). As expected, feeding flies BH4 rescues the loss of Punch and Purple in the fat body and the loss of Sptr in NPF neurons. Mechanistically, we found BH4 deficiency reduces NPF staining, likely by promoting its release, while excess BH4 increases NPF accumulation without altering its expression. We thus show that, because of its physically distributed biosynthesis, BH4 acts as a fat-derived signal that induces satiety by inhibiting the activity of the NPF neurons.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Development of a new UPLC-ESI-MS/MS method for the determination of biopterin and neopterin in dried blood spot

Background(6R)-5,6,7,8-tetrahydrobiopterin (BH4) deficiencies are rare inherited defects of synthesis or regeneration of BH4. Due to the resulting hyperphenylalaninemia (HPA), some of them are detected by newborn screening and require the assessment of the pattern of neopterin (Neo) and biopterin (Bio) excretion in urine to be confirmed. Aim of present study was to develop a method for the measurement of these diagnostic biomarkers in dried blood spot (DBS). MethodsAfter DBS extraction, samples were filtered and injected into the UPLC column coupled with a tandem mass spectrometer working in positive electrospray ionization. ResultsThe chromatographic separation was accomplished in 6min. The LoQ was 0.57 and 1.45nmol/l of blood for Neo and Bio respectively and the response was linear over the range 0–100nmol/l of blood. The within- and between-day imprecision was <6.4 and 10.8% respectively. Reference ranges for newborns, infants and children/adult were established. The method was tested in 11 patients affected by BH4 defects. ConclusionsThe assessment of Neo and Bio in DBS is reliable and sensitive and may be proposed as a second tier test for the newborns with hyperphenylalaninemia (HPA) as well as a new potential diagnostic tool for symptomatic subjects with BH4 deficiencies.

A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance

The redox co-factor tetrahydrobiopterin (BH4) regulates nitric oxide (NO) and reactive oxygen species (ROS) production by endothelial NOS (eNOS) and is an important redox-dependent signalling molecule in the endothelium. Loss of endothelial BH4 is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O2-) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH4 to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH4 deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH4 and a striking induction of O2- generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Moreover, this BH4-dependent, mitochondria-derived ROS further oxidised mitochondrial BH4, concomitant with changes in the thioredoxin and glutathione antioxidant pathways. These changes were accompanied by a modest increase in mitochondrial size, mildly attenuated basal respiratory function, and marked changes in the mitochondrial proteome and cellular metabolome, including the accumulation of the TCA intermediate succinate. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and bioenergetic metabolism.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease