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.

Pterins as diagnostic markers of exercise-induced stress: a systematic review.

OBJECTIVES: To evaluate pterins as diagnostic biomarkers of exercise-induced stress. DESIGN: Systematic review of the literature. METHODS: MEDLINE, Scopus and Web of Science were searched in March 2019 for relevant literature. We only considered in vivo studies of healthy humans that reported measurement of a pterin(s) in response to exercise or sport with no underlying prior disease or complication. Relevant articles were independently reviewed and resolved by consensus. RESULTS: We included 29 studies with 644 participants. We classified articles by running/hiking, cycling, rugby, mixed martial arts (MMA) or other. Eighty-six percent of studies measured a significant increase in a pterin in response to exercise. Changes in pterin concentrations were within 24h of the exercise-stimulus in 79% of studies and 17% measured a change from baseline greater than 48h post-exercise (49% did not measure or report beyond 48h). Neopterin or total neopterin (neopterin+7,8-dihydroneopterin) were the primary pterin measured (28 studies) and they were equally sensitive to exercise regardless of whether the stimulus was running, cycling, rugby, MMA or other. CONCLUSIONS: Neopterin and total neopterin increase in response to exercise-induced stress. Pterins may have limited capacity for monitoring long-term stress beyond 48h but further research is required.

Phase I clinical evaluation of CNSA-001 (sepiapterin), a novel pharmacological treatment for phenylketonuria and tetrahydrobiopterin deficiencies, in healthy volunteers.

Tetrahydrobiopterin (BH4) is the natural cofactor of aromatic amino acid hydroxylases and essential for degradation of phenylalanine and synthesis of catecholamines and serotonin. It can be synthesized either de novo from GTP or through the salvage pathway from sepiapterin. Sepiapterin, a natural precursor of BH4, is a more stable molecule and is transported more efficiently across cellular membranes, thus having potentially significant advantage over BH4 as a pharmacological agent for diseases associated with BH4-deficient conditions. We report the results of a first-in-humans, randomized, double-blind, placebo-controlled, dose-ranging, Phase I clinical trial in 83 healthy volunteers of CNSA-001, a novel formulation of sepiapterin. Single oral doses of 2.5-80 mg/kg CNSA-001 caused dose-related increases in plasma sepiapterin (mean Cmax 0.58-2.92 ng/mL) and BH4 (mean Cmax 57-312 ng/mL). Maximum plasma concentrations were achieved in about 1-2 h (sepiapterin) or about 4 h (BH4) after CNSA-001 oral intake. Increases in plasma BH4 were substantially larger in absolute terms and on a dose-for-dose basis following treatment with CNSA-001 vs. sapropterin dihydrochloride, a synthetic form of BH4. The pharmacokinetics of plasma sepiapterin and BH4 were similar before and after seven days of repeat daily dosing with CNSA-001 at 5, 20 or 60 mg/kg indicating little or no drug accumulation. Oral administration of CNSA-001 resulted in higher concentrations of sepiapterin in fasted vs. fed subjects, but overall BH4 plasma exposure following CNSA-001 intake increased by 1.7-1.8-fold in fed subjects. CNSA-001 was well tolerated, with no clear dose-relationship for adverse events (AE), no serious AE and no study discontinuations for AE. These data indicate that CNSA-001 is rapidly and efficiently converted to BH4 in humans supporting further clinical evaluation of CNSA-001 for the management of PKU, primary BH4 deficiencies and other diseases associated with deficient BH4 metabolism.

Plasma levels of soluble VEGF receptor isoforms, circulating pterins and VEGF system SNPs as prognostic biomarkers in patients with acute coronary syndromes.

BACKGROUND: Development of collateral circulation in coronary artery disease is cardio-protective. A key process in forming new blood vessels is attraction to occluded arteries of monocytes with their subsequent activation as macrophages. In patients from a prospectively recruited post-acute coronary syndromes cohort we investigated the prognostic performance of three products of activated macrophages, soluble vascular endothelial growth factor (VEGF) receptors (sFlt-1 and sKDR) and pterins, alongside genetic variants in VEGF receptor genes, VEGFR-1 and VEGFR-2. METHODS: Baseline levels of sFlt-1 (VEGFR1), sKDR (VEGFR2) and pterins were measured in plasma samples from subgroups (n = 513; 211; 144, respectively) of the Coronary Disease Cohort Study (CDCS, n = 2067). DNA samples from the cohort were genotyped for polymorphisms from the VEGFR-1 gene SNPs (rs748252 n = 2027, rs9513070 n = 2048) and VEGFR-2 gene SNPs (rs2071559 n = 2050, rs2305948 n = 2066, rs1870377 n = 2042). RESULTS: At baseline, levels of sFlt-1 were significantly correlated with age, alcohol consumption, NTproBNP, BNP and other covariates relevant to cardiovascular pathophysiology. Total neopterin levels were associated with alcohol consumption at baseline. 7,8 dihydroneopterin was associated with BMI. The A allele of VEGFR-2 variant rs1870377 was associated with higher plasma sFlt-1 and lower levels of sKDR at baseline. Baseline plasma sFlt-1 was univariately associated with all cause mortality with (p < 0.001) and in a Cox's proportional hazards regression model sFlt-1 and pterins were both associated with mortality independent of established predictors (p < 0.027). CONCLUSIONS: sFlt-1 and pterins may have potential as prognostic biomarkers in acute coronary syndromes patients. Genetic markers from VEGF system genes warrant further investigation as markers of levels of VEGF system components in these patients. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry. ACTRN12605000431628 . 16 September 2005, Retrospectively registered.

Use of sapropterin dihydrochloride in maternal phenylketonuria. A European experience of eight cases.

Sapropterin dihydrochloride (SD) is the first drug treatment for phenylketonuria (PKU), but due to the lack of data, its use in maternal PKU must be undertaken with caution as noted in the FDA and EMEA labels. We collected data from eight pregnancies in PKU women treated with SD and we analysed the phenotypes of these patients, their tetrahydrobiopterin (BH4) responsiveness, the indications for SD treatment, the efficacy (metabolic control, phenylalanine (Phe) tolerance and offspring outcome) and the safety data. Results showed that in the seven patients known to be responsive to BH4, the use of SD during pregnancy was efficient in terms of metabolic control and Phe tolerance. The indications for giving SD included the failure of the low-Phe diet (n = 3), the fact that some of these women had never experienced the low Phe diet (n = 2), one unexpected pregnancy in a woman currently on SD and one pregnancy where the foetus was known to have PKU. The offspring of these seven pregnancies were all normal babies with normal birth measurements and outcomes. No side effect related to SD was observed in these seven cases. In the eighth case, SD was prescribed as a rescue treatment without previous knowledge of the BH4 responsiveness to a woman who was already 8 weeks pregnant without diet. The birth occurred at 33 weeks of gestational age with Potter syndrome (probably related to the absence of metabolic control during the first trimester) and the baby died in the first hours of life. In conclusion, the data presented here provides the first evidence that treatment with pharmacological doses of SD appears to be efficient and safe in women with PKU during pregnancy. Its use should, however, be restricted to those women previously identified to be clear responders to BH4.

Changes in selected biochemical indices, leukocyte profile, and pterins as biomarkers of immune system activity due to antipecking measures in pheasants.

The physiological changes in response to beak trimming and spectacle usage as antipecking measures were monitored in 10-mo-old common pheasants (Phasianus colchicus). Short-term analysis conducted before the beginning of the laying period showed immediate increases of plasma corticosterone (P < 0.05) and lactate dehydrogenase (P < 0.001) concentrations and decrease of plasma triglycerides (P < 0.01) levels in response to both beak trimming and the application of spectacles. Beak-trimmed pheasants exhibited higher plasma corticosterone concentrations than pheasants fitted with spectacles (P < 0.001). To assess long-term changes, blood samples for biochemical (neopterin and biopterin determination) and hematological (leukocyte profile determination) examinations were taken from beak-trimmed, spectacles-fitted, and control pheasant hens housed in cages during their laying period. At the end of the laying period, hens fitted with spectacles exhibited lower concentrations of plasma neopterin (P = 0.005) and biopterin (P = 0.005) than beak-trimmed pheasant hens. Our findings suggest that the immune system was suppressed in spectacles-fitted pheasant hens as a result of chronic stress, as also indicated by the higher heterophil-to-lymphocyte ratio (P = 0.001) compared with beak-trimmed hens. Our study found a negative correlation (r = -0.31, P = 0.019) between the heterophil-to-lymphocyte ratio and plasma neopterin concentration. This study demonstrated that both beak trimming and use of spectacles are not only stressful procedures for pheasants, but long-term effects may also include a negative impact on the immune system.

Metabolic effects of sapropterin treatment in autism spectrum disorder: a preliminary study.

Sapropterin, a synthetic form of tetrahydrobiopterin (BH4), has been reported to improve symptoms in children with autism spectrum disorder (ASD). However, as BH4 is involved in multiple metabolic pathway that have been found to be dysregulated in ASD, including redox, pterin, monoamine neurotransmitter, nitric oxide (NO) and immune metabolism, the metabolic pathway by which sapropterin exerts its therapeutic effect in ASD effect remains unclear. This study investigated which metabolic pathways were associated with symptomatic improvement during sapropterin treatment. Ten participants (ages 2-6 years old) with current social and/or language delays, ASD and a central BH4 concentration 30 nM l(-1) were treated with a daily morning 20 mg kg(-1) dose of sapropterin for 16 weeks in an open-label fashion. At baseline, 8 weeks and 16 weeks after starting the treatment, measures of language, social function and behavior and biomarkers of redox, pterin, monoamine neurotransmitter, NO and immune metabolism were obtained. Two participants discontinued the study, one from mild adverse effects and another due to noncompliance. Overall, improvements in subscales of the Preschool Language Scale (PLS), Vineland Adaptive Behavior Scale (VABS), Aberrant Behavior Checklist (ABC) and autism symptoms questionnaire (ASQ) were seen. Significant changes in biomarkers of pterin, redox and NO were found. Improvement on several subscales of the PLS, VABS, ABC and ASQ were moderated by baseline and changes in biomarkers of NO and pterin metabolism, particularly baseline NO metabolism. These data suggest that behavioral improvement associated with daily 20 mg kg(-1) sapropterin treatment may involve NO metabolism, particularly the status of pretreatment NO metabolism.

Pterins as sensors of response to the application of Fe3+ -dextran in piglets.

The aim of the presented study was to assess the effect of a single administration of Fe(3+)-dextran on immune cell counts and pterin biomolecule production as novel sensors of the piglets' immune system activation, and to determine concentrations of cortisol, a traditional hormonal biosensor of the stress response. Pterins (neopterin and biopterin) in the piglets' blood serum were analyzed by separation using reversed-phase HPLC. A single dose of Fe(3+)-dextran produced a special stress situation in the piglets' organism which manifested itself by an increased production of neopterin (p < 0.05) and biopterin (p < 0.01) in the experimental piglets. Changes in cortisol concentrations and leukocyte counts were influenced by handling stress and were not specifically correlated to iron dextran application. Iron concentrations in the internal environment of the experimental piglets' group were higher by an order of magnitude compared with the controls, and the highest serum concentrations of iron (p < 0.01) were reached 24 h following Fe(3+)-dextran administration. The data presented offer a new perspective on the evaluation of stress situations in the animal organism and, not least importantly, extends the rather modest current list of references on the role of pterins in livestock animals.

Quantitation of total biopterin and tetrahydrobiopterin in plasma.

OBJECTIVE: To develop a reliable method for the determination of tetrahydrobiopterin and total biopterin in plasma. METHODS: Modifications include blood collection and handling procedures. RESULTS: Collection of blood in the presence of 0.1% (w/v) dithiothreitol and waiting for 2-3 h before centrifugation are optimal for adequate quantitation of both pterins. CONCLUSIONS: For a reliable determination of tetrahydrobiopterin and total biopterin in plasma, blood tubes must contain a dimercapto-antioxidant and time between blood collection and centrifugation is critical.

Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper.

Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Phenylalanine hydroxylase gene in psychiatric patients: screening and functional assay of mutations.

BACKGROUND: Reports relating phenylalanine kinetics and metabolism to psychiatric disorders led us to undertake the comprehensive screening of the phenylalanine hydroxylase (PAH) coding region and functional testing of discovered mutations in a sample of psychiatric patients and healthy control subjects. METHODS: Genomic DNA from psychiatric patients and control subjects was assayed for sequence variants in all PAH coding regions and splice junctions. In vivo functional analysis of mutations was conducted by assessing the kinetics and conversion to tyrosine of a standardized phenylalanine dose and by measuring fasting pterin levels. RESULTS: A known missense mutation was observed in a schizoaffective subject, and a novel missense mutation was discovered in four subjects with schizophrenia and one normal subject. The schizoaffective patient heterozygous for the known A403V mutation showed the lowest rate of phenylalanine kinetics and lowest conversion to tyrosine in the patient sample. The four schizophrenic patients heterozygous for the novel K274E mutation showed significantly decreased phenylalanine kinetics, reduced conversion to tyrosine, and increased synthesis of the PAH cofactor tetrahydrobiopterin compared with schizophrenic subjects without the mutation. CONCLUSIONS: The study findings suggest that larger scale studies are warranted to test the relationship of the PAH genotype with a psychiatric phenotype.

Novel high-performance liquid chromatographic assay using fluorescence detection for the quantitation of plasma gamma-methylene-10-deazaaminopterin and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin, in patients with solid cancers in a phase I trial.

Gamma-methylene-10-deazaaminopterin (MDAM), a unique dihydrofolate reductase inhibitor, has demonstrated antitumor activity against a broad spectrum of human solid tumors in preclinical studies. A novel reversed-phase, ion-pair high-performance liquid chromatography (HPLC) assay that uses fluorescence detection has been developed to quantitate levels of MDAM and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin (7-OH-MDAM), in human plasma. The recovery of MDAM and 7-OH-MDAM from plasma was >97% by a simple one-step deproteinization process using tetrabutylammonium bromide (TBABr) and methanol. MDAM and 7-OH-MDAM remained stable in plasma over a 28-day test period at ambient temperatures, and neither compound was light-sensitive. The limit of quantitation was 0.005 microM for both MDAM and 7-OH-MDAM. This assay has been found to be simple, sensitive and reproducible in determining plasma concentrations of MDAM and 7-OH-MDAM in patients with solid cancers in a phase I trial.

Studies and comparison of pterin patterns in the plasma of dogs and cats and their alteration in various neoplasias and virus infections.

Physiological pterin levels were evaluated in plasma samples of healthy dogs and cats. Biopterin and 7-xanthopterin were determined quantitatively by HPLC after partial purification by ion exchange chromatography. Neopterin, pterin and 6-hydroxymethylpterin were not detectable in plasma samples. Pterin levels in plasma of dogs with neoplasias and cats with neoplasias or viral infections (FeLV, FIV, FIP) were determined. Dogs with various tumours did not show qualitative but distinct quantitative alterations in the pterin concentration in plasma. Biopterin concentrations in plasma of dogs after tumour exstirpation significantly decreased compared to the levels before surgery. There was only a moderate increase of the biopterin levels in plasma of cats with neoplasias in comparison to healthy animals of this species. Cats suffering from viral infections, however, showed a significant rise in biopterin concentrations. The results underscore the importance of pterins as sensitive indicators for the presence of activation of cell mediated immune response. With emphasis on biopterin the role of pterins as helpful parameters for the preventation and the monitoring of diseases is pointed out.

A yellow component associated with human transthyretin has properties like a pterin derivative, 7,8-dihydropterin-6-carboxaldehyde.

Transthyretin (TTR) in plasma is associated with yellow compounds. Their properties differ, and in the chicken protein a major yellow compound has recently been identified as a carotenoid, lutein, also called xanthophyll. We now show that the major yellow component extracted from human TTR has properties like a pterin derivative, 7,8-dihydropterin-6-carboxyaldehyde (2-amino-4-hydroxy-6-formyl-7,8-dihydropteridine). The human TTR derivative has chromatographic and spectral properties identical to a yellow photochemical degradation product of biopterin and a spectrum like that of the pterin aldehyde.

Effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin on the dopaminergic and cholinergic receptors as evaluated by positron emission tomography in the Rhesus monkey.

The effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP) on the central cholinergic and dopaminergic systems in the Rhesus monkey brain were investigated by positron emission tomography (PET) with the muscarinic cholinergic receptor ligands (N-[11C]methyl-benztropine) and dopaminergic receptor ligands selective for D1, D2, and D3 subtypes ([11C]SCH23390, N-[11C]methyl-spiperone, and (+)[11C]UH232, respectively). None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP used significantly affected the regional cerebral blood flow (rCBF as determined by Raichle's H(2)15O method), and 10 mg/kg of R-THBP had little effect on the regional cerebral metabolic rate of glucose (rCMRglc) in the Rhesus monkey brain, as assessed by the graphical [18F]fluoro-deoxyglucose method. The effect of R-THBP on the muscarinic cholinergic system was dose dependent; while 3 mg/kg of R-THBP did not significantly alter the uptake ratio of N-[11C]methylbenztropine in several brain regions to that in the cerebellum, 10 and 30 mg/kg of R-THBP significantly reduced the uptake ratio in the thalamus, as well as in the frontal and temporal cortices. None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP tested affected [11C]SCH23390 (dopamine D1 receptor) binding. However, the k3 value for N-[11C]methyl-spiperone (dopamine D2 receptor) binding, which represents the association rate X Bmax value, was significantly decreased in the striatum. The uptake ratio of (+)[11C]UH232 (dopamine D3 receptor) in the striatum to that in the cerebellum was also decreased by administration of R-THBP (3 and 30 mg/kg i.v.). These findings suggest that R-THBP acts on dopamine D2 and D3 receptors selectively without markedly affecting dopamine D1 receptor binding. Furthermore, the changes in cholinergic and dopamine D2 and D3 receptors in vivo can not be attributed to a change in rCBF but may depend on the action of R-THBP.

Hyperphenylalaninemia and pterin metabolism in serum and erythrocytes.

The relationship between blood phenylalanine concentrations and serum and erythrocyte biopterin and neopterin concentrations was investigated in 20 phenylketonuric patients with different dietary compliance. At serum phenylalanine concentrations ranging from 43 to 1004 mumol/l, a good correlation was found with serum biopterin (r = 0.76, P < 0.001) and with red blood cell biopterin (r = 0.62, P < 0.001). A similar correlation was found between serum neopterin and phenylalanine (r = 0.60, P < 0.001). The correlation between red blood cell neopterin and serum phenylalanine was less evident, however (r = 0.47, P < 0.005). After oral loading with phenylalanine (100 mg/kg body weight), serum and red blood cell biopterin concentrations increased in patients with classical phenylketonuria as well as in one patient with dihydropteridine reductase deficiency in response to the induced acute hyperphenylalaninemia. One patient suffering from 6-pyruvoyl tetrahydropterin synthase deficiency was loaded orally with tetrahydrobiopterin (20 mg/kg body weight). The kinetics of administered cofactor confirmed its rapid absorption, with early increase of serum concentrations followed by its transport into the red blood cells. The half-life of biopterin was approximately 7 h in serum and 15 h in red blood cells. Because both values are less than the half-life of phenylalanine (20-30 h) in serum, biopterin measurement offers no advantage in monitoring dietary control in hyperphenylalaninemic patients.

Plasma and urinary levels of biopterin, neopterin, and related pterins and plasma levels of folate in infantile autism.

Tetrahydrobiopterin is essential for brain cells to make monoamine neurotransmitters. It has been reported that the concentrations of tetrahydrobiopterin in plasma and urine are low in certain mental disorders and that oral supplements are beneficial. A group of Japanese investigators have been conducting clinical trials of the effect of administration of tetrahydrobiopterin to autistic children and reported that it is beneficial with no significant side effects. We, therefore, initiated a study to assess plasma and urinary levels of tetrahydrobiopterin in infantile autism to see if they are reduced. Besides tetrahydrobiopterin, we also determined plasma and urinary levels of neopterin and monapterin in these individuals in order to evaluate the status of dihydroneopterin triphosphate, a key biosynthetic precursor of tetrahydrobiopterin. Sixteen autistic children and 12 healthy controls were included in this study. Results indicated that the plasma and urinary levels of tetrahydrobiopterin are not statistically different between the two groups and, therefore, no simple explanation for the beneficial effects of administration of tetrahydrobiopterin on autistic children can be offered at the present time. In contrast, plasma and urinary levels of neopterin were depressed (.01 less than p less than .05) and plasma monapterin was also significantly depressed (p less than .01) in autistic subjects compared with controls. Levels of other pterins, including folate, were not statistically different between the two groups. The basis for this depression in neopterin and monapterin is unknown. It does not seem likely that this depression could be attributed to a difference in age or T-lymphocyte/macrophage activity. However, further studies are needed to investigate these possibilities.

7-Substituted pterins. A new class of mammalian pteridines.

Three novel pteridines have been isolated from the urine of patients with a new variant of 6-(L-erythro-1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) deficiency, showing hyperphenylalaninemia. From the results of high performance liquid chromatography, oxidative degradation, and gas chromatography-electron impact mass spectrometry, their structures were identified as 7-(D-erythro-1',2',3'-trihydroxypropyl)-pterin (7-neopterin), 7-(L-erythro-1',2'-dihydroxypropyl)-pterin (7-biopterin), and 6-oxo-7-(L-erythro-1',2'-dihydroxypropyl)-pterin (6-oxo-7-biopterin). The ratio of biopterin to 7-biopterin in the patients' urines was 1:1, and after oral loading with tetrahydrobiopterin, 7-biopterin excretion rose parallel to biopterin. This finding suggests that 7-substituted pterins may be formed endogenously by a yet unknown isomerization reaction. The cause of hyperphenylalaninemia is still unclear. The activities of the enzymes involved in tetrahydrobiopterin biosynthesis and regeneration were found to be normal in the patients, and no effect of 7-biopterin on these enzymes was observed in vitro. However, compared with the normal cofactor, tetrahydrobiopterin, the Km values of tetrahydro-7-biopterin for phenylalanine hydroxylase and dihydropteridine reductase are 20 and 5 times higher, respectively.