Dispel some mist on circulating biopterins: measurement, physiological interval and pathophysiological implication.

BACKGROUND AND AIMS: Biopterins, including tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and biopterin (B), were crucial enzyme cofactors in vivo. Despite their recognized clinical significance, there remain notable research gaps and controversies surrounding experimental outcomes. This study aims to clarify the biopterins-related issues, including analytical art, physiological intervals, and pathophysiological implications. MATERIALS AND METHODS: A novel LC-MS/MS method was developed to comprehensively profile biopterins in plasma, utilizing chemical derivatization and cold-induced phase separation. Subsequently, apparently healthy individuals were enrolled to investigate the physiological ranges. And the relationships between biopterins and biochemical indicators were analyzed to explore the pathophysiological implications. RESULTS: The developed method was validated as reliable for detecting biopterins across the entire physiological range. Timely anti-oxidation was found to be essential for accurate assessment of biopterins. The observed overall mean ± SDs levels were 3.51 ± 0.94, 1.54 ± 0.48, 2.45 ± 0.84 and 5.05 ± 1.14 ng/mL for BH4, BH2, BH4/BH2 and total biopterins. The status of biopterins showed interesting correlations with age, gender, hyperuricemia and overweight. CONCLUSION: In conjunction with proper anti-oxidation, the newly developed method enables accurate determination of biopterins status in plasma. The observed physiological intervals and pathophysiological implications provide fundamental yet inspiring support for further clinical researches.

Children and Adolescents with Early Treated Phenylketonuria: Cognitive Development and Fluctuations of Blood Phenylalanine Levels.

BACKGROUND: We assessed the relationship between the cognitive development of children and adolescents with phenylketonuria (PKU) and fluctuations in peripheral phenylalanine (Phe) levels. METHODS: We examined the neurocognitive performance of 33 children and adolescents with early treated PKU, of whom 18 were treated with sapropterin dihydrochloride, and 15 were on a classic diet. For 26 weeks, patients were assessed weekly for their blood phenylalanine (Phe) levels. Phe levels were analyzed for fluctuations indicated by the individual standard deviation. Fluctuations were compared to the standard deviation of 26 Phe level measurements before the study interval. We also assessed the concurrent IQ of the patients. This was repeated at one-, two-, and seven-year intervals. RESULTS: Full-scale IQ in patients treated with a classic diet did not change within the follow-up. In patients treated with Sapropterin dihydrochloride, however, there was a considerable gain in full-scale IQ. This was particularly true if blood Phe fluctuations increased in patients of this treatment group. CONCLUSIONS: Sapropterin dihydrochloride enhances Phe tolerance in patients with PKU. Increasing blood Phe fluctuations following enhanced Phe tolerance may indicate that the treatment not only allows patients to relax their Phe-restricted diet but also may support cognitive development in patients.

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.

Salivary neopterin and related pterins: their comparison to those in plasma and changes in individuals.

Neopterin (NP), biopterin (BP) and monapterin (MP) exist in saliva. The physiological role of salivary NP as well as the pathophysiological role of increased NP in the immune-activated state has been unclear. Saliva is a characteristic specimen different from other body fluids. In this study, we analysed salivary NP and related pterin compounds, BP and MP and revealed some of its feature. High-performance liquid chromatography (HPLC) analysis of saliva and plasma obtained from 26 volunteers revealed that salivary NP existed mostly in its fully oxidized form. The results suggested that salivary NP as well as BP would mostly originate from the oral cavity, perhaps the salivary glands, and that salivary NP levels might not reflect those in the plasma. We also found that a gender difference existed in correlations between concentrations of salivary total concentrations of NP (tNP) and BP (tBP). HPLC analysis of saliva obtained from 5 volunteers revealed that the concentrations of salivary tNP as well as tBP fluctuated in an irregular fashion in various individuals. MP, a diastereomer of NP, might have come from oral cavity NP itself or its precursor. These results indicated that the nature of salivary NP might be different from that of NP in the blood or urine.

Sapropterin (BH4) Aggravates Autoimmune Encephalomyelitis in Mice.

Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood-brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.

Retrospective analysis of 19 patients with 6-Pyruvoyl Tetrahydropterin Synthase Deficiency: Prolactin levels inversely correlate with growth.

BACKGROUND: Pyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency is the most common form of BH4 deficiency resulting in hyperphenylalaninemia. It can have variable clinical severity and there is limited information on the clinical presentation, natural history and effectiveness of newborn screening for this condition. METHODS: Retrospective data (growth and clinical parameters, biochemical and genetic testing results, treatment) were collected from 19 patients with PTPS deficiency in different centers, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate quantitative variables. RESULTS: Patients with PTPS deficiency had an increased incidence of prematurity (4/18) with an average gestational age only mildly reduced (37.8 ± 2.4 weeks) and low birth weight (-1.14 ± 0.97 SD below that predicted for gestational age). With time, weight and height approached normal. VALUES: All patients were identified by newborn screening for an elevated phenylalanine level. However, phenylalanine levels were normal in two whose testing was performed at or before 24 h of age. Sapropterin dihydrochloride treatment normalized phenylalanine levels. Molecular testing identified novel variants in the PTS gene, some of which present in more than one affected family. The neurotransmitter derivatives 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in the CSF were decreased in most cases except in 2 families with the peripheral form of PTPS deficiency. With time, HVA and 5HIAA became abnormally low in two of these patients requiring therapy. Prolactin (whose secretion is inhibited by dopamine) levels were elevated in several patients with PTPS deficiency and inversely correlated with the z-scores for height (p < 0.01) and weight (p < 0.05). Most patients with PTPS deficiency had delayed development early in life, improving around school age with IQs mostly in the normal range, with a small decline in older individuals. From a neurological standpoint, most patients had normal brain MRI and minor EEG anomalies, although some had persistent neurological symptoms. DISCUSSION: Patients with PTPS deficiency have not only an increased incidence of prematurity, but also decreased birth weight when corrected for gestational age. Hyperphenylalaninemia can be absent in the first day of life. Therapy with sapropterin dihydrochloride normalizes phenylalanine levels and neurotransmitter precursors can improve CSF neurotransmitter metabolites levels. Insufficient dopaminergic stimulation (as seen from elevated prolactin) might result in decreased height in patients with PTPS deficiency. Despite early delays in development, many patients can achieve independence in adult life, with usually normal neuroimaging and EEG.

Neopterin and biopterin levels and tryptophan degradation in patients with diabetes.

This study aimed to evaluate the possible changes of neopterin, biopterin levels and tryptophan degradation in diabetes and to compare the results within diabetes groups and with healthy subjects. Diabetes mellitus patients and healthy controls were recruited the study. Patients were further subgrouped according to their drug therapy. Serum neopterin concentrations were detected by ELISA. Urinary neopterin, biopterin, serum tryptophan (Trp) and kynurenine (Kyn) levels were detected by HPLC. There was no difference between controls and diabetes patients in serum neopterin, urinary neopterin and biopterin levels (p > 0.05, all). Serum Trp and Kyn levels were significantly different in type 1 diabetes (T1DM) patients compared to controls (p < 0.05, both). Serum neopterin levels were significantly higher in type 2 diabetes patients (T2DM) compared to T1DM (p < 0.05). Urinary biopterin levels of T2DM patients using both metformin and vildagliptin were significantly higher than T1DM patients (p < 0.05). The correlations between serum neopterin and urinary neopterin, Kyn and Kyn/Trp were statistically significant in control and patient groups (p < 0.05, all). The study showed that Kyn/Trp was altered in diabetes patients due to immune modulation. On the other hand, although xenobiotic exposure may change pteridine levels, metformin and/or vildagliptin use in T2DM patients did not have any effect on the measured parameters.

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.

Rare variants and biological pathways identified in treatment-refractory depression.

Individuals diagnosed with major depressive disorder not responding to at least two adequate treatments are defined as treatment-refractory major depressive disorder (TR-MDD). Some TR-MDD patients have altered metabolic phenotypes that may be pharmacologically reversed. The characterization of these phenotypes and their underlying etiologies is paramount, particularly their genetic components. In this study, TR-MDD patients (n = 124) were recruited and metabolites were quantified in their cerebrospinal fluid (CSF) and peripheral blood. Three sub-categories of deficiencies were examined, namely 5-methyltetrahydrofolte (in CSF; n = 13), tetrahydrobiopterin (in CSF; n = 11), and abnormal acylcarnitine profiles (in peripheral blood; n = 8). Whole exome sequencing was performed on genomic DNA from the entire TR-MDD cohort and exonic variant allele frequencies for cases were compared to a control cohort (1:5 matching on ancestry). Low frequency, damaging alleles were identified and used for in silico pathway analyses. Three association signals for TR-MDD approached genome-wide significance on chromosomes 22, 7, and 3. Three risk-associated variants from a prior depression study were replicated. Relevant biological pathways were identified that contained an enrichment of rare, damaging variants in central nervous system (CNS)-specific pathways, including neurotransmitter receptors, potassium channels, and synapse transmission. Some TR-MDD patients had rare variants in genes that were previously associated with other psychiatric disorders, psychiatric endophenotypes, CNS structural defects, and CNS-related cellular and molecular functions. Exome analysis of metabolically phenotyped TR-MDD patients has identified potentially functional gene pathways and low frequency, deleterious gene variants for further investigation. Further studies in larger cohorts of biochemically phenotyped TR-MDD patients are desirable to extend and confirm these findings.

Utility of Tetrahydrobiopterin Pathway in the Assessment of Diabetic Foot Ulcer: Significant and Complex Interrelations.

OBJECTIVES: Tetrahydrobiopterin (BH4) pathway that included generation of neopterin (Neop), biopterin (Biop), and nitric oxide (NO) is altered in type 2 diabetes (T2D). The aim of this study was to assess the biomarkers of BH4 pathway in noninfected DFUs and to relate these levels to the variables of diabetes as well as to the hematological indices. METHODS: We performed a cross-sectional investigating study in a Kurdish people including 30 healthy subjects (group I), 66 T2D patients (group II), and 57 DFUs patients (group III). Hematological indices including red cell distribution width (RDW), mean platelet volume (MPV), and platelet distribution width (PDW) were determined by Coulter hematological analysis. Serum BH4 markers including NO, Neop, and Biop were determined by using an enzyme-linked immunosorbent assay (ELISA) technology. The relationship between BH4 markers with glycemic and hematological indices was assessed by Spearman's correlation and multivariable regression analysis. RESULTS: Neop was significantly increased while PDW was significantly decreased in group III compared with group II patients. Nitric oxide was found to be inversely correlated with age (r = -0.382), duration of diabetes (r = -0.264), mean arterial blood pressure (r = -0.532), body mass index (r = -0.321), RDW (r = -0.322), and PDW (r = -0.284) in group III patients. Circulating Neop and Biop significantly correlated with RDW and erythrocyte sedimentation rate. Multivariable regression analysis revealed that serum Neop predicted the DFUs in 92.5% of group III patients. CONCLUSION: Tetrahydrobiopterin biomarkers are predictors of DFUs and the significant correlation of neopterin with red distribution width and erythrocyte sedimentation rate indicating the role of neopterin in the vascular and inflammation concerns of noninfected DFUs.

Usefulness of serum biopterin as a predictive biomarker for childhood asthma control: A prospective cohort study.

BACKGROUND: Pteridines are metabolites of tetrahydrobiopterin, which serves as co-enzyme of nitric oxide synthase. We sought to investigate the usefulness of pteridines as biomarkers for childhood asthma control. METHODS: We conducted a single-center prospective cohort study involving 168 asthmatic children aged 4-17 years who visited the periodical asthma checkup program. Serum neopterin and biopterin levels were measured as pteridines at each visit along with measurement of FeNO, respiratory function tests, nasal eosinophil test, blood eosinophil count, and IgE level. We calculated coefficients for relation between pteridines and asthma control, which was assessed by questionnaires (JPAC: Japanese Pediatric Asthma Control Program). RESULTS: A total of 168 participants aged 10.3 ± 3.39 years (mean ± SD) with asthma were recruited. The participants in this study contained 58 patients (34.5%) of complete-controlled based on JPAC, 132 patients (76.0%) of well-controlled group based on GINA. FeNO and serum neopterin level did not correlate with following period's JPAC scores. In contrast, serum biopterin level significantly correlated with following period's JPAC total score (Coefficients 0.398; 95% CI 0.164 to 0.632; p value 0.001) and frequency of wheezing during exercise (Coefficients 0.272; 95% CI 0.217 to 0.328; p value < 0.001). CONCLUSIONS: We found serum biopterin effected the following period's control status of asthmatic children, thus monitoring biopterin level will be a useful for management of asthma to adjust treatment.

A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with liquid chromatography-tandem mass spectrometry analysis.

Tetrahydrobiopterin (BH4) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH4 has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH4 by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH4-BC), ultra-sensitive and reliable quantification of endogenous BH4 in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH4-BC-d5 was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL-1. In the end, after investigation of plasma BH4 in healthy volunteers (n = 38), we found that the levels of BH4 were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH4-related fields.

13C-phenylalanine breath test and serum biopterin in schizophrenia, bipolar disorder and major depressive disorder.

Phenylalanine is required for the synthesis of the neurotransmitters dopamine, noradrenaline, and adrenaline. The rate-limiting step for phenylalanine metabolism is catalyzed by phenylalanine hydroxylase (PAH) and its cofactor tetrahydrobiopterin. We aimed to detect altered phenylalanine metabolism in major psychiatric disorders using the l-[1-13C]phenylalanine breath test (13C-PBT) and serum biopterin levels. We also investigated association of PAH mutations with schizophrenia and phenylalanine metabolism. 13C-phenylalanine (100 mg) was orally administered, and the breath 13CO2/12CO2 ratio was monitored for 120 min in four groups: 103 patients with schizophrenia (DSM-IV), 39 with bipolar disorder, 116 with major depressive disorder (MDD), and 241 healthy controls. Serum biopterin levels were measured by high performance liquid chromatography. Mutation screening of PAH exons was performed by direct sequencing in 46 schizophrenia patients. Association analysis was performed using six tag single nucleotide polymorphisms and the PAH Arg53His mutation by TaqMan assays in 616 schizophrenia patients and 1194 healthy controls. Analyses of covariance controlling for age, sex, and body weight showed that the index for the amount of exhaled 13CO2 was significantly lower in the schizophrenia group than in the other three groups (all p < 0.05). Biopterin levels in schizophrenia and MDD were significantly lower than those in controls. Biopterin levels correlated with 13C-PBT indices in controls. PAH polymorphisms were not associated with schizophrenia or 13C-PBT indices. 13C-PBT revealed reduced phenylalanine metabolism in schizophrenia, though we obtained no evidence of involvement of PAH polymorphism. Serum biopterin levels were lower in schizophrenia and MDD, warranting further investigation.

Regulation of cortical and peripheral GCH1 expression and biopterin levels in schizophrenia-spectrum disorders.

Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine, serotonin and nitric oxide synthesis. Deficits of plasma total biopterin (a measure of BH4) have been described in schizophrenia and schizoaffective disorder. GCH1 encodes the first and rate-limiting enzyme in BH4 synthesis. Peripheral GCH1 expression is lower in first episode psychosis patients versus controls, and we hypothesized that a GCH1 promoter polymorphism associated with psychiatric illness, contributes to regulation of both GCH1 expression and BH4 levels. We tested this hypothesis in 120 subjects (85 patients with schizophrenia or schizoaffective disorder and 35 controls): Patients with the rs10137071 A allele had significantly lower plasma biopterin than GG patients and controls. In additional samples we assessed the relationship between genotype and diagnosis (schizophrenia or control) on GCH1 expression in the prefrontal cortex (n = 67) and peripheral leukocytes (n = 53). We found a significant linear relationship between GCH1 and study group in the CNS and periphery, with A allele patients having lower expression. Finally, in antipsychotic naïve patients (n = 13) we tested for an effect of medication on GCH1: Expression rose significantly after the onset of medication, primarily in A allele patients. These data suggest the potential for personalized genetic approaches to ameliorating BH4 deficits in schizophrenia-spectrum disorders.

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.

Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs.

Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10-12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP), U46619, sarafotoxin 6c (S6c) and endothelin-1 (ET-1) were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001). Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001). Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling.

[Neopterin levels and systemic inflammatory response syndrome in pediatric critically ill patients]. / Niveles de neopterina y síndrome de respuesta inflamatoria sistémica en pacientes críticos pediátricos.

INTRODUCTION: Neopterin and biopterin are sub-products of redox reactions, which act as cofactors of enzymes responsible for nitric oxide production. The hypothesis is presented that plasma neopterin and biopterin evolve differently during the first days in a critically ill child. METHODS: A single-centre prospective observational study was conducted on patients 7 days to 14 years admitted to our Paediatric Intensive Care Unit (PICU) and that met Systemic inflammatory response syndrome (SIRS) criteria. Neopterin and biopterin levels, as well as other acute phase reactants, were collected at admission and at 24 h. RESULTS: A total of 28 patients were included, of which 78.9% were male, The median age was 5.04 years (interquartile range [IQR] 1.47-10.26), and PRISM II 2.0% (IQR 1.1-5.0). Mechanical ventilation (MV) was used in 90% of patients, with a median duration of 6.0 hrs (IQR 3.7-102.0). The median length of stay in PICU was 5.0 days (IQR 2.7-18.7), maximum VIS mean of 0 (IQR 0-14). Baseline neopterin level was 2.3±1.2 nmol/l and at 24 h it was 2.3±1.4 nmol/l. Baseline biopterin was 1.3±0.5 nmol/l and 1.4±0.4 nmol/l at 24 h. Neopterin levels were significantly higher in patients with PICU length of stay > 6 days (P=.02), patients who needed MV >24 h (P=.023), and those who developed complications (P=.05). Neopterin correlates directly and is statistically significant with the duration of MV (rho=.6, P=.011), PICU length of stay (rho=.75, P<.0001), and VIS (rho=.73, P=.001). Additionally, biopterin directly correlates with the PRISM (rho=.61, P=.008). DISCUSSION: There is a higher neopterin level when there is a longer PICU stay, higher VIS score, longer time on MV, and occurrence of complications, indicating the involvement of an activation of the cellular immune system.

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). METHODS: After DBS extraction, samples were filtered and injected into the UPLC column coupled with a tandem mass spectrometer working in positive electrospray ionization. RESULTS: The 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. CONCLUSIONS: The 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.

Tetrahydrobiopterin Supplementation: Elevation of Tissue Biopterin Levels Accompanied by a Relative Increase in Dihydrobiopterin in the Blood and the Role of Probenecid-Sensitive Uptake in Scavenging Dihydrobiopterin in the Liver and Kidney of Rats.

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 Supplementation Improves Phenylalanine Metabolism in a Murine Model of Severe Malaria.

Tetrahydrobiopterin (BH4) is an essential cofactor for both phenylalanine hydroxylase and nitric oxide synthase. Patients with severe malaria have low urinary BH4, elevated plasma phenylalanine, and impaired endothelium-dependent vasodilation, suggesting that BH4 depletion may limit phenylalanine metabolism and nitric oxide synthesis. We infected C57BL/6 mice with Plasmodium berghei ANKA to characterize BH4 availability and to investigate the effects of BH4 supplementation. P. berghei ANKA infection lowered BH4 in plasma, erythrocytes, and brain tissue but raised it in aorta and liver tissue. The ratio of BH4 to 7,8-BH2 (the major product of BH4 oxidation) was decreased in plasma, erythrocytes, and brain tissue, suggesting that oxidation contributes to BH4 depletion. The continuous infusion of sepiapterin (a BH4 precursor) and citrulline (an arginine precursor) raised the concentrations of BH4 and arginine in both blood and tissue compartments. The restoration of systemic BH4 and arginine availability in infected mice produced only a minor improvement in whole blood nitrite concentrations, a biomarker of NO synthesis, and failed to prevent the onset of severe disease symptoms. However, sepiapterin and citrulline infusion reduced the ratio of phenylalanine to tyrosine in plasma, aortic tissue, and brain tissue. In summary, BH4 depletion in P. berghei infection may compromise both nitric oxide synthesis and phenylalanine metabolism; however, these findings require further investigation in human patients with severe malaria.