Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression.

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O2•-) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O2•- levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.

The Role of the BH4 Cofactor in Nitric Oxide Synthase Activity and Cancer Progression: Two Sides of the Same Coin.

Cancer development is associated with abnormal proliferation, genetic instability, cell death resistance, metabolic reprogramming, immunity evasion, and metastasis. These alterations are triggered by genetic and epigenetic alterations in genes that control cell homeostasis. Increased reactive oxygen and nitrogen species (ROS, RNS) induced by different enzymes and reactions with distinct molecules contribute to malignant transformation and tumor progression by modifying DNA, proteins, and lipids, altering their activities. Nitric oxide synthase plays a central role in oncogenic signaling modulation and redox landscape. Overexpression of the three NOS isoforms has been found in innumerous types of cancer contributing to tumor growth and development. Although the main function of NOS is the production of nitric oxide (NO), it can be a source of ROS in some pathological conditions. Decreased tetrahydrobiopterin (BH4) cofactor availability is involved in NOS dysfunction, leading to ROS production and reduced levels of NO. The regulation of NOSs by BH4 in cancer is controversial since BH4 has been reported as a pro-tumoral or an antitumoral molecule. Therefore, in this review, the role of BH4 in the control of NOS activity and its involvement in the capabilities acquired along tumor progression of different cancers was described.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2-•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

Role of the Transient Receptor Potential Vanilloid Type 1 (TRPV1) in the Regulation of Nitric Oxide Release in Wistar Rat Aorta.

The potential transient vanilloid receptor type 1 (TRPV1) plays important functional roles in the vascular system. In the present study, we explored the role of the TRPV1 in the production of nitric oxide (NO), biopterines (BH4 and BH2), cyclic guanosine monophosphate (cGMP), malondialdehyde (MDA), phosphodiesterase-3 (PDE-3), total antioxidant capacity (TAC), and calcitonin gene-related peptide (CGRP) in the rat aorta. Wistar rats were divided into four groups: (1) control, (2) capsaicin (CS, 20 mg/kg), (3) capsazepine (CZ, 24 mg/kg), and (4) CZ + CS. Treatments were applied daily for 4 days before removing the thoracic aortas for testing of aortic tissue and endothelial cells. TRPV1 activation produced increases in BH4 14%, cGMP 25%, NO 29%, and TAC 59.2% in comparison to the controls. BH2 and MDA increased with CZ. CGRP shows a tendency to decrease with CZ. The analysis by immunocytochemistry confirmed that the TRPV1 is present in aortic endothelial cells. Aortic endothelial cells were obtained from healthy rats and cultured to directly explore the effects of CS and CZ. The activation of the TRPV1 (CS 30 µM) produced increases in BH4 17%, NO 36.6%, TAC 56.3%, and CGRP 65%, when compared to controls. BH2 decreased with CZ + CS. CS effects were diminished by CZ in cells and in the tissue. We conclude that the TRPV1 is a structure present in the membrane of aortic endothelial cells and that it participates in the production of NO. The importance of the TRPV1 should be considered in vascular reactivity studies.

Immunological impact of tetrahydrobiopterin on the central nervous system in a murine model of rabies virus infection.

Currently, the Milwaukee protocol presents healing results in human beings affected by the rabies virus. However, there are many points to clarify on the action of drugs and the immune mechanism involved in the evolution of the disease. One of the drugs used is biopterin, which is an important cofactor for nitric oxide, important for preventing vasospasm. Thus, we describe the effect of biopterin on some inflammatory factors in a rabies virus infection developed in an animal model. The immunological mediators studied in animals infected with rabies virus submitted to doses of sapropterin were Anti-RABV, IL-6, IL-2, IL-17a, INF-gamma and Anti-iNOS. It is suggested that the medication in the context of a RABV infection already installed, had the effect of modulating the inflammatory mechanisms mainly linked to the permeability of the blood-brain barrier and the migration of cytotoxic cells.

Arginase inhibition by (-)-Epicatechin reverses endothelial cell aging.

Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (-)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 µM), Epi (1 µM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity.

[Early diagnosis of phenylketonuria. Physiopathology of the neuronal damage and therapeutic options]. / Fenilcetonuria de diagnóstico precoz. Bases fisiopatológicas del daño neuronal y opciones terapéuticas.

Phenylketonuria, also known as PKU, is the most frequent congenital inborn error of metabolism. The severe form or classic PKU untreated causes intellectual disability, although with the early detection programs in the neonatal period, diagnosis and treatment prevent the appearance of the symptoms. Despite early diagnosis and treatment we have observed some neurotoxicity in treated PKU patients. We analyzed the factors involved apart from the toxicity due to the high cerebral concentrations of phenylalanine (Phe), the defects of synthesis of neurotransmitters, the alteration of cerebral myelination, the effect of the elevation of Phe in the processes of transport and distribution of neutral amino acids with an abnormal synthesis of brain proteins, plasma and cerebral tyrosine deficiency, the neurotoxicity of Phe metabolites, the defect of cholesterol biosynthesis or the increase of oxidative stress. White matter alterations in early treated PKU patients have an important role in neurological manifestations. The treatment of PKU is for life and is based on the reduction of foods containing Phe combined with the administration of a special formula or tetrahydrobiopterin (BH4) treatment. New therapeutic options will be analyzed.

La fenilcetonuria, también conocida como PKU, es el error congénito más frecuente del metabolismo de los aminoácidos. La forma grave o PKU clásica no tratada, causa una discapacidad intelectual, aunque los programas de detección en el período neonatal, el diagnóstico y el tratamiento evitan la aparición de los síntomas. A pesar de un diagnóstico y tratamiento temprano hemos observado cierta neurotoxicidad en los pacientes con PKU tratados. Analizamos los demás factores implicados, aparte de la toxicidad por las elevadas concentraciones cerebrales de fenilalanina (Phe), se revisan los defectos de síntesis de neurotransmisores, las alteración de la mielinización cerebral, el efecto de la elevación de Phe en los procesos de transporte y distribución de los aminoácidos neutros con una síntesis anómala de proteínas cerebrales, la deficiencia plasmática y cerebral de tirosina, la neurotoxicidad de los metabolitos de Phe, el defecto de la biosíntesis del colesterol o el aumento del estrés oxidativo. Las alteraciones de la sustancia blanca en los pacientes con PKU tienen un papel importante en las manifestaciones neurológicas. El tratamiento de la PKU es para toda la vida y se basa en la reducción del aporte de alimentos que contienen Phe combinado con la administración de una fórmula especial, o en el tratamiento con tetrahidrobiopterina (BH4). Se analizan nuevas opciones terapéuticas.

The Role of the Activation of the TRPV1 Receptor and of Nitric Oxide in Changes in Endothelial and Cardiac Function and Biomarker Levels in Hypertensive Rats.

The purpose of the present study was to analyze the actions of transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin (CS) and of its antagonist capsazepine (CZ), on cardiac function as well as endothelial biomarkers and some parameters related with nitric oxide (NO) release in L-NG-nitroarginine methyl ester (L-NAME)-induced hypertensive rats. NO has been implicated in the pathophysiology of systemic arterial hypertension (SAHT). We analyzed the levels of nitric oxide (NO), tetrahydrobiopterin (BH4), malondialdehyde (MDA), total antioxidant capacity (TAC), cyclic guanosin monophosphate (cGMP), phosphodiesterase-3 (PDE-3), and the expression of endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GTPCH-1), protein kinase B (AKT), and TRPV1 in serum and cardiac tissue of normotensive (118±3 mmHg) and hypertensive (H) rats (165 ± 4 mmHg). Cardiac mechanical performance (CMP) was calculated and NO was quantified in the coronary effluent in the Langendorff isolated heart model. In hypertensive rats capsaicin increased the levels of NO, BH4, cGMP, and TAC, and reduced PDE-3 and MDA. Expressions of eNOS, GTPCH-1, and TRPV1 were increased, while AKT was decreased. Capsazepine diminished these effects. In the hypertensive heart, CMP improved with the CS treatment. In conclusion, the activation of TRPV1 in H rats may be an alternative mechanism for the improvement of cardiac function and systemic levels of biomarkers related to the bioavailability of NO.

Fenilcetonuria de diagnóstico precoz: Bases fisiopatológicas del daño neuronal y opciones terapéuticas / Early diagnosis of phenylketonuria. Physiopathology of the neuronal damage and therapeutic options

La fenilcetonuria, también conocida como PKU, es el error congénito más frecuente del metabolismo de los aminoácidos. La forma grave o PKU clásica no tratada, causa una discapacidad intelectual, aunque los programas de detección en el período neonatal, el diagnóstico y el tratamiento evitan la aparición de los síntomas. A pesar de un diagnóstico y tratamiento temprano hemos observado cierta neurotoxicidad en los pacientes con PKU tratados. Analizamos los demás factores implicados, aparte de la toxicidad por las elevadas concentraciones cerebrales de fenilalanina (Phe), se revisan los defectos de síntesis de neurotransmisores, las alteración de la mielinización cerebral, el efecto de la elevación de Phe en los procesos de transporte y distribución de los aminoácidos neutros con una síntesis anómala de proteínas cerebrales, la deficiencia plasmática y cerebral de tirosina, la neurotoxicidad de los metabolitos de Phe, el defecto de la biosíntesis del colesterol o el aumento del estrés oxidativo. Las alteraciones de la sustancia blanca en los pacientes con PKU tienen un papel importante en las manifestaciones neurológicas. El tratamiento de la PKU es para toda la vida y se basa en la reducción del aporte de alimentos que contienen Phe combinado con la administración de una fórmula especial, o en el tratamiento con tetrahidrobiopterina (BH4). Se analizan nuevas opciones terapéuticas.

Phenylketonuria, also known as PKU, is the most frequent congenital inborn error of metabolism. The severe form or classic PKU untreated causes intellectual disability, although with the early detection programs in the neonatal period, diagnosis and treatment prevent the appearance of the symptoms. Despite early diagnosis and treatment we have observed some neurotoxicity in treated PKU patients. We analyzed the factors involved apart from the toxicity due to the high cerebral concentrations of phenylalanine (Phe), the defects of synthesis of neurotransmitters, the alteration of cerebral myelination, the effect of the elevation of Phe in the processes of transport and distribution of neutral amino acids with an abnormal synthesis of brain proteins, plasma and cerebral tyrosine deficiency, the neurotoxicity of Phe metabolites, the defect of cholesterol biosynthesis or the increase of oxidative stress. White matter alterations in early treated PKU patients have an important role in neurological manifestations. The treatment of PKU is for life and is based on the reduction of foods containing Phe combined with the administration of a special formula or tetrahydrobiopterin (BH4) treatment. New therapeutic options will be analyzed.

Genotype-phenotype correlations and BH4 estimated responsiveness in patients with phenylketonuria from Rio de Janeiro, Southeast Brazil.

BACKGROUND: Genetic heterogeneity and compound heterozygosis give rise to a continuous spectrum of phenylalanine hydroxylase deficiency and metabolic phenotypes in phenylketonuria (PKU). The most used parameters for evaluating phenotype in PKU are pretreatment phenylalanine (Phe) levels, tolerance for dietary Phe, and Phe overloading test. Phenotype can vary from a "classic" (severe) form to mild hyperphenylalaninemia, which does not require dietary treatment. A subset of patients is responsive to treatment by the cofactor tetrahydrobiopterin (BH4 ). Genotypes of PKU patients from Rio de Janeiro, Brazil, were compared to predicted and observed phenotypes. Genotype-based estimations of responsiveness to BH4 were also conducted. METHODS: Phenotype was defined by pretreatment Phe levels. A standard prediction system based on arbitrary assigned values was employed to measure genotype-phenotype concordance. Patients were also estimated as BH4 -responders according to the responsiveness previously reported for their mutations and genotypes. RESULTS: A 48.3% concordance rate between genotype-predicted and observed phenotypes was found. When the predicted phenotypes included those reported at the BIOPKU database, the concordance rate reached 77%. A total of 18 genotypes from 30 patients (29.4%) were estimated as of potential or probable BH4 responsiveness. Inconsistencies were observed in genotypic combinations including the common "moderate" mutations p.R261Q, p.V388M, and p.I65T and the mild mutations p.L48S, p.R68S, and p.L249F. CONCLUSION: The high discordance rate between genotype-predicted and observed metabolic phenotypes in this study seems to be due partially to the high frequency of the so-called "moderate" common mutations, p.R261Q, p.V388M, and p.I65T, which are reported to be associated to erratic or more severe than expected metabolic phenotypes. Although our results of BH4 estimated responsiveness must be regarded as tentative, it should be emphasized that genotyping and genotype-phenotype association studies are important in selecting patients to be offered a BH4 overload test, especially in low-resource settings like Brazil.

De novo tetrahydrobiopterin biosynthesis is impaired in the inflammed striatum of parkin(-/-) mice.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disease, is primarily characterized by neurodegeneration in the substantia nigra pars compacta, resulting in motor impairment. Loss-of-function mutations in parkin are the major cause of the early onset familial form of the disease. Although rodents deficient in parkin (parkin(-/-) ) have some dopaminergic system dysfunction associated with central oxidative stress and energy metabolism deficiencies, these animals only display nigrostriatal pathway degeneration under inflammatory conditions. This study investigated the impact of the inflammatory stimulus induced by lypopolisaccharide (LPS) on tetrahydrobiopterin (BH4) synthesizing enzymes (de novo and salvage pathways), since this cofactor is essential for dopamine synthesis. The mitochondrial content and architecture was investigated in the striatum of LPS-exposed parkin(-/-) mice. As expected, the LPS (0.33 mg/kg; i.p.) challenge compromised spontaneous locomotion and social interaction with juvenile parkin(-/-) and WT mice. Moreover, the genotype impacted the kinetics of the investigation of the juvenile. The inflammatory scenario did not induce apparent changes in mitochondrial ultrastructure; however, it increased the quantity of mitochondria, which were of smaller size, and provoked the perinuclear distribution of the organelle. Furthermore, the BH4 de novo biosynthetic pathway failed to be up-regulated in the LPS challenge, a well-known stimulus for its activation. The LPS treatment increased sepiapterin reductase (SPR) expression, suggesting compensation by the salvage pathway. This might indicate that dopamine synthesis is compromised in parkin(-/-) mice under inflammatory conditions. Finally, this scenario impaired the striatal expression of the transcription factor BDNF, possibly favoring cell death.

Simplified HPLC methodology for quantifying biological pterins by selective oxidation.

Tetrahydrobiopterin (BH4) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC). In this work, a method was established in order to quantify BH4 and BH2 by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35°C at a flow rate of 0.9mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method. The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH4/BH2 ratio in biological samples.

Role for Tetrahydrobiopterin in the Fetoplacental Endothelial Dysfunction in Maternal Supraphysiological Hypercholesterolemia.

Maternal physiological hypercholesterolemia occurs during pregnancy, ensuring normal fetal development. In some cases, the maternal plasma cholesterol level increases to above this physiological range, leading to maternal supraphysiological hypercholesterolemia (MSPH). This condition results in endothelial dysfunction and atherosclerosis in the fetal and placental vasculature. The fetal and placental endothelial dysfunction is related to alterations in the L-arginine/nitric oxide (NO) pathway and the arginase/urea pathway and results in reduced NO production. The level of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), is reduced in nonpregnant women who have hypercholesterolemia, which favors the generation of the superoxide anion rather than NO (from eNOS), causing endothelial dysfunction. However, it is unknown whether MSPH is associated with changes in the level or metabolism of BH4; as a result, eNOS function is not well understood. This review summarizes the available information on the potential link between MSPH and BH4 in causing human fetoplacental vascular endothelial dysfunction, which may be crucial for understanding the deleterious effects of MSPH on fetal growth and development.

Insulin reverses D-glucose-increased nitric oxide and reactive oxygen species generation in human umbilical vein endothelial cells.

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose-alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0-1000 µmol/L) was measured in response to 5-25 mmol/L D-glucose (0-36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose-increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose-increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2•-) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2•- generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose-increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose-reduced BH4 level. Insulin and tempol blocked the high D-glucose-increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2•-/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Phenylalanine hydroxylase deficiency in Mexico: genotype-phenotype correlations, BH4 responsiveness and evidence of a founder effect.

The mutational spectrum of the phenylalanine hydroxylase gene (PAH) in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype-phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype-phenotype correlations and genotype-based prediction of BH4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5G>T being the most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1A>T and c.969+6T>C. The genotype-phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH4 testing and therapy.

Sapropterin is safe and effective in patients less than 4-years-old with BH4-responsive phenylalanine hydrolase deficiency.

OBJECTIVE: To assess the safety and efficacy of tetrahydrobiopterin therapy with sapropterin to treat tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency in children aged <4 years compared with those aged ≥4 years. STUDY DESIGN: We analyzed a longitudinal follow-up study conducted in all patients with BH4-responsive PAH deficiency throughout Japan. At the end of 2011, 43 patients were receiving sapropterin, of whom 21 were aged <4 years at the initiation of treatment. The starting dose of sapropterin was ≥10 mg/kg/day in 11 of these 21 patients. The duration of follow-up was ≥4 years in 6 of those 11 patients; 3 of these 6 were followed for ≥10 years. Nine patients were receiving sapropterin monotherapy at the end of 2011. RESULTS: Serum phenylalanine level was maintained within the recommended optimal control range in all 21 patients who started sapropterin treatment before age 4 years. Only 1 nonserious adverse drug reaction occurred, an elevated alanine aminotransferase level in 1 patient. No significant abnormal behavior related to nerve disorders was reported. CONCLUSION: Sapropterin therapy initiated before age 4 years was effective in maintaining serum phenylalanine level within the favorable range and was safe in Japanese patients with BH4-responsive PAH deficiency.

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

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

Sapropterina no tratamento da hiperfenilalaninemia com deficiência de BH4 / Sapropterin in the treatment of hyperphenylalaninemia with BH4 deficiency

Fenilcetonúria (FNC) é uma doença genética, autossômica recessiva, causada por mutações no gene localizado no cromossomo 12q22-q24, o qual codifica a enzima hepática fenilalanina-hidroxilase (FAH). Sua ausência ou deficiência impede a conversão hepática de fenilalanina (FAL), um dos aminoácidos essenciais e mais comuns do organismo, em tirosina, causando acúmulo de FAL no sangue e em outros tecidos. O aumento de fenilalanina no sangue em 98% dos casos é devido a mutações na codificação genética para a enzima fenilalanina-hidroxilase, enquanto 2% são devidos a defeitos no metabolismo da tetrahidrobiopterina (BH4), que é um cofator essencial para a atividade da fenilalanina-hidroxilase. A principal característica da doença não tratada é retardo mental, com piora durante a fase de desenvolvimento do cérebro e que se estabilizaria com a maturação completa deste órgão. O quociente de inteligência (QI) mede a extensão deste retardo e varia de leve a gravemente prejudicado. A HFA não tratada resulta em progressivo retardo mental, com QI < 50. A piora está relacionada aos níveis sanguíneos de FAL. Caso a doença seja diagnosticada logo após o nascimento e o paciente for mantido em dieta restrita em FAL, os sintomas podem ser prevenidos e a criança pode ter desenvolvimento e expectativa de vidas normais. Nesse sentido, o rastreamento no Brasil é realizado pelo teste do pezinho, cuja necessidade consta no Estatuto da Criança e do Adolescente, e está regulamentado pela portaria que estabeleceu o Programa Nacional de Triagem Neonatal para diagnóstico precoce de fenilcetonúria. No Protocolo Clínico e Diretrizes Terapêuticas do Ministério da Saúde para Fenilcetonúria foram incluídos os pacientes com níveis de FAL≥ 10mg/dl (600 µmol/l) em dieta normal 1,14 e todos os que apresentarem níveis de FAL entre 8 e 10 mg/dl persistentes (pelo menos em 3 dosagens consecutivas, semanais, em dieta normal). Dieta restrita em FAL é eficaz em reduzir os níveis sanguíneos de FAL e melhorar o QI e o prognóstico neuropsicológico dos pacientes com HFA. O tratamento deve ser iniciado tão cedo quanto possível, idealmente até o 10º dia de vida. O aleitamento materno deve ser encorajado e associado ao uso de fórmula isenta de FAL. Os níveis de FAL devem ser diminuídos rapidamente. Além da dieta, o tratamento clínico recomendado pelo PCDT do Ministério da Saúde para o controle metabólico dos pacientes é a utilização de fórmulas alimentares especiais. As fórmulas são medicamentos que devem conter as quantidades recomendadas de vitaminas e sais minerais adequadas à faixa etária do paciente. Sapropterina é uma forma sintética oral de BH4 (BH4 supplementation sapropterin dihydrochloride). Há relatos de casos de pessoas com FCN que apresentaram boa resposta após o uso de doses farmacológicas de BH4, com redução dos níveis de FAL. Todos tinham mutação no gene FAH. Pessoas com resposta ao BH4 são identificadas inicialmente por um teste com teste de tolerância a BH4. Resposta positiva é considerada como uma redução de 30% ou mais na concentração de FAL, 24 horas após a administração de BH4. A variação na intensidade da resposta é independente da gravidade da FCN, da dose de BH4 empregada no teste de tolerância, duração do teste e genótipo. Pessoas com mesmo genótipo mostram respostas diferentes. Há poucos resultados de uso de longa duração de BH4 que mostram que pode haver relaxamento da restrição dietética sem efeitos adversos. A maioria dos indivíduos dos estudos apresentava doença moderada ou leve. A Secretaria-Executiva da CONITEC realizou busca na literatura por artigos científicos, com o objetivo de localizar a melhor evidência científica disponível sobre o tema. A CONITEC em sua 14ª reunião ordinária realizada no dia 04 de abril de 2013, recomendou a não incorporação no SUS da sapropterina para o tratamento de hiperfenilalaninemia (HFA) com deficiência em tetrahidrobiopterina (BH4). Considerou-se que os estudos, a maioria de baixa qualidade metodológica, não conseguiram comprovar a superioridade do tratamento, principalmente no que diz respeito à ausência de dados específicos para o subgrupo com deficiência de BH4. Os membros da CONITEC presentes na 15ª reunião do plenário do dia 09/05/2013 deliberaram, por unanimidade, por não recomendar a sapropterina para o tratamento de hiperfenilalaninemia (HFA) com deficiência em tetrahidrobiopterina (BH4). A Portaria nº 34, de 6 de agosto de 2013 - Torna pública a decisão de não incorporar o medicamento sapropterina no tratamento da hiperfenilalaninemia com deficiência de BH4 no Sistema Único de Saúde (SUS).

Sapropterin dihydrochloride for phenylketonuria.

BACKGROUND: Phenylketonuria results from a deficiency of the enzyme phenylalanine hydroxylase. Dietary restriction of phenylalanine keeps blood phenylalanine concentration low. Most natural foods are excluded from diet and supplements are used to supply other nutrients. Recent publications report a decrease in blood phenylalanine concentration in some patients treated with sapropterin dihydrochloride. We examined the evidence for the use of sapropterin dihydrochloride to treat phenylketonuria.   OBJECTIVES: To assess the safety and efficacy of sapropterin dihydrochloride in lowering blood phenylalanine concentration in people with phenylketonuria. SEARCH METHODS: We identified relevant trials from the Group's Inborn Errors of Metabolism Trials Register. Date of last search: 29 June 2012.We also searched ClinicalTrials.gov and Current controlled trials. Last search: 23 July 2012.We contacted the manufacturers of the drug (BioMarin Pharmaceutical Inc.) for information regarding any unpublished trials. SELECTION CRITERIA: Randomized controlled trials comparing sapropterin with no supplementation or placebo in people with phenylketonuria due to phenylalanine hydroxylase deficiency. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trials and extracted outcome data. MAIN RESULTS: Two placebo-controlled trials were included. One trial administered 10 mg/kg/day sapropterin in 89 children and adults with phenylketonuria whose diets were not restricted and who had previously responded to saproterin.This trial measured change in blood phenylalanine concentration. The second trial screened 90 children (4 to 12 years) with phenylketonuria whose diet was restricted, for responsiveness to sapropterin. Forty-six responders entered the placebo-controlled part of the trial and received 20 mg/kg/day sapropterin. This trial measured change in both phenylalanine concentration and protein tolerance. Both trials reported adverse events. The trials showed an overall low risk of bias; but both are Biomarin-sponsored. One trial showed a significant lowering in blood phenylalanine concentration in the sapropterin group (10 mg/kg/day), mean difference -238.80 µmol/L (95% confidence interval -343.09 to -134.51); a second trial (20 mg/kg/day sapropterin) showed a non-significant difference, mean difference -51.90 µmol/L (95% confidence interval -197.27 to 93.47). The second trial also reported a significant increase in phenylalanine tolerance, mean difference18.00 mg/kg/day (95% confidence interval 12.28 to 23.72) in the 20 mg/kg/day sapropterin group. AUTHORS' CONCLUSIONS: There is evidence of short-term benefit from using sapropterin in some patients with sapropterin-responsive forms of phenylketonuria; blood phenylalanine concentration is lowered and protein tolerance increased. There are no serious adverse events associated with using sapropterin in the short term.There is no evidence on the long-term effects of sapropterin and no clear evidence of effectiveness in severe phenylketonuria.

Determination of six pterins in urine by LC-MS/MS.

BACKGROUND: The present work describes an analytical method for urinary pterins by LC-MS/MS, with emphasis on the separation of 6- and 7-positional isomers of bio- and neopterins. RESULTS: Urine sample preparation consisted of oxidation by MnO(2), filtration and direct dilution in the mobile phase. The method was validated in urine spiked at five concentration levels with true triplicates of each level. Separation of the pterins, including the positional isomers, was achieved by employing a LUNA amino column. Six pterins were quantified (pterin, isoxanthopterin, 6-biopterin, 7-biopterin, 6-neopterin, 7-neopterin) and a linear behavior was observed; LOD varied from 7 to 360 pg/ml and correlation coefficients above 0.98 were obtained for all pterins. In addition, pterin levels were evaluated in 41 urine samples of healthy subjects, in ten urine samples of patients with classical phenylketonuria (PKU) and in one with atypical PKU. CONCLUSION: The proposed method allowed to identify, separate and quantify six pterins in urine, using a simple and rapid sample preparation. The atypical PKU was unequivocally differentiated from the classical form, demonstrating that this method could be very useful for characterization and follow-up of diseases.