Valores e Desenvolvimento Humano

A primeira parte desse Relatório de Desenvolvimento Humano do Brasil 2009/2010 começa com a descrição de um amplo processo de consulta aberta à sociedade, denominada Brasil Ponto a Ponto, para a escolha do tema do relatório. A Campanha Brasil Ponto a Ponto teve por objetivo estimular o debate em todo o país sobre o que precisa ser mudado no Brasil para melhorar a vida das pessoas. A campanha foi aberta a todos os brasileiros, que poderiam participar respondendo à pergunta: O que precisa mudar no Brasil para a sua vida melhorar de verdade

Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2–16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2–0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia

Metabolomic studies in the inborn error of metabolism alkaptonuria reveal new biotransformations in tyrosine metabolism

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd(−/−)) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd(−/−) AKU (n = 15) and Hgd(+/−) non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd(−/−) were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of (13)C-labelled HGA to Hgd(−/−)(n = 4) and Hgd(+/−)(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd(−/−) mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd(−/−) were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the (13)C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism

Impact of Nitisinone on the Cerebrospinal Fluid Metabolome of a Murine Model of Alkaptonuria

BackgroundNitisinone-induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and/or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone.Methods17 CSF samples were collected from BALB/c Hgd-/- mice (n = 8, treated with nitisinone-4 mg/L and n = 9, no treatment). Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3 min retention time window) were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini-Hochberg false-discovery rate adjustment.ResultsL-Tyrosine, N-acetyl-L-tyrosine, γ-glutamyl-L-tyrosine, p-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold change 2.6-6.9, 3/5 were significant p < 0.05) in the mice that received nitisinone. Several other metabolites of interest were matched, but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol, and octopamine.ConclusionsEvaluation of the CSF metabolome of a murine model of AKU revealed a significant increase in the abundance of a limited number of metabolites following treatment with nitisinone. Further work is required to understand the significance of these findings and the mechanisms by which the altered metabolite abundances occur

Evaluation of the serum metabolome of patients with alkaptonuria before and after two years of treatment with nitisinone using LC-QTOF-MS.

BackgroundThe homogentisic acid-lowering therapy nitisinone is being evaluated for the treatment of alkaptonuria (AKU) at the National Centre for AKU. Beyond hypertyrosinemia, the wider metabolic consequences of its use are largely unknown. The aim of this work was to evaluate the impact of nitisinone on the serum metabolome of patients with AKU after 12 and 24 months of treatment.MethodsDeproteinized serum from 25 patients with AKU (mean age[±SD] 51.1 ± 14.9 years, 12 male) was analyzed using the 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, UK). Raw data were processed using a batch targeted feature extraction algorithm and an accurate mass retention time database containing 469 intermediary metabolites (MW 72-785). Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability (ResultsEight metabolites increased in abundance (log2 fold change [FC] 2.1-15.2, P 2 FC 1.5-15.5, P ConclusionsEvaluation of the serum metabolome of patients with AKU showed a significant difference in the abundance of several metabolites following treatment with nitisinone, including a number that have not been previously reported; several of these were not related to the tyrosine metabolic pathway.SynopsisNitisinone therapy has a significant impact on several metabolites beyond the tyrosine metabolic pathway, several of which appear to be related to the redox state of the cell

The effect of nitisinone on homogentisic acid and tyrosine: a two-year survey of patients attending the National Alkaptonuria Centre, Liverpool

Background Alkaptonuria is a rare, debilitating autosomal recessive disorder affecting tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase leads to increased homogentisic acid which is deposited as ochronotic pigment. Clinical sequelae include severe early onset osteoarthritis, increased renal and prostate stone formation and cardiac complications. Treatment has been largely based on analgaesia and arthroplasty. The National Alkaptonuria Centre in Liverpool has been using 2 mg nitisinone (NTBC) off-license for all patients in the United Kingdom with alkaptonuria and monitoring the tyrosine metabolite profiles. Methods Patients with confirmed alkaptonuria are commenced on 2 mg dose (alternative days) of NTBC for three months with daily dose thereafter. Metabolite measurement by LC-MS/MS is performed at baseline, day 4, three-months, six-months and one-year post-commencing NTBC. Thereafter, monitoring and clinical assessments are performed annually. Results Urine homogentisic acid concentration decreased from a mean baseline 20,557 µmol/24 h (95th percentile confidence interval 18,446–22,669 µmol/24 h) by on average 95.4% by six months, 94.8% at one year and 94.1% at two year monitoring. A concurrent reduction in serum homogentisic acid concentration of 83.2% compared to baseline was also measured. Serum tyrosine increased from normal adult reference interval to a mean ± SD of 594 ± 184 µmol /L at year-two monitoring with an increased urinary excretion from 103 ± 81 µmol /24 h at baseline to 1071 ± 726 µmol /24 h two years from therapy. Conclusions The data presented represent the first longitudinal survey of NTBC use in an NHS service setting and demonstrate the sustained effect of NTBC on the tyrosine metabolite profile

Studies in alkaptonuria reveal new roles beyond drug clearance for phase I and II biotransformations in tyrosine metabolism

AbstractBackground and Purposealkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of the enzyme homogentisate 1,2-dioxygenase (HGD). The primary biochemical consequence of HGD-deficiency is increased circulating homogentisic acid (HGA), which is central to AKU disease pathology. The aim of this study was to investigate the wider metabolic consequences of targeted Hgd disruption.Experimental Approachthe first metabolomic analysis of the Hgd−/− AKU mouse model was performed. Urinary metabolites altered in Hgd−/− were further validated by showing that the HGA-lowering drug nitisinone reversed their direction of alteration in AKUKey Resultscomparison of Hgd−/− (AKU) versus Hgd+/− (heterozygous control) urine revealed increases in HGA and a group of 8 previously unreported HGA-derived transformation products from phase I and II metabolism. HGA biotransformation products HGA-sulfate, HGA-glucuronide, HGA-hydrate and hydroxymethyl-HGA were also decreased in urine from both mice and patients with AKU on the HGA-lowering agent nitisinone. Hgd knockout also revealed a host of previously unrecognised associations between tyrosine, purine and TCA cycle metabolic pathways.Conclusion and ImplicationsAKU is rare, but our findings further what is currently understood about tyrosine metabolism more generally, and show for the first time that phase I and II detoxification is recruited to prevent accumulation of endogenously-produced metabolites in inborn errors of metabolism. The data highlight the misconception that phase I and II metabolic biotransformations are reserved solely for drug clearance; these are ancient mechanisms, which represent new potential treatment targets in inherited metabolic diseases.Abstract FigureBullet point summaryWhat is already known Increased circulating homogentisic acid is central to disease pathology in the inherited metabolic disease alkaptonuriaThe Hgd knockout mouse, created in our laboratory, accurately models human alkaptonuriaWhat this study adds Phase I and II biotransformations are recruited in alkaptonuria for detoxification of homogentisic acidThese data challenge misconceptions that phase I and II metabolism is solely for drug clearanceClinical significance Phase I and II metabolic processes represent new treatment targets in inherited metabolic diseasesThe molecular pathology of AKU extends much further than the known alteration to tyrosine metabolism</jats:sec

Reversal of ochronotic pigmentation in alkaptonuria following nitisinone therapy: Analysis of data from the United Kingdom National Alkaptonuria Centre.

BackgroundIncreased homogentisic acid (HGA) causes ochronosis. Nitisinone decreases HGA. The aim was to study the effect of nitisinone on the ochronosis progression.MethodsPhotographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at V-1 (pre-baseline visit), V0 (baseline visit when 2 mg nitisinone was commenced), and yearly at V1, V2, and V3 visits. Photographs were inspected for evolution of ochronotic pigment and also scored categorically to derive eye, ear, and combined ochronosis scores. An ear cartilage biopsy was also carried out at V0 and one year after V3 (V4) and ochronotic pigment was assessed and quantitated. Visits were compared for changes in pigment. Fasting blood and 24-hour urine samples were collected for measurement of HGA.ResultsThere were 80 AKU patients at V0, and 52, 47, and 40 at V1, V2, and V3 in the group with variable numbers (VAR Group) respectively; 23 patients attended once before V0, in the V-1 visit. Photographs of patients show increase in eye pigment between V-1 and V0, followed by decrease post-nitisinone at V1, V2, and V3. Ear and combined ochronosis semiquantitative scoring showed an increase between V-1 and V0 (P P P ConclusionsNitisinone decreases HGA and partially reverses ochronosis

A Comprehensive LC-QTOF-MS Metabolic Phenotyping Strategy: Application to Alkaptonuria.

BACKGROUND:Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS:A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS:Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS:The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU

ICTV Virus Taxonomy Profile: Ophioviridae

[EN] The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.Production of this summary, the online chapter and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).Garcia, M.; Dal Bo, E.; Da Graca, JV.; Gago Zachert, SP.; Hammond, J.; Moreno, P.; Natsuaki, T.... (2017). ICTV Virus Taxonomy Profile: Ophioviridae. Journal of General Virology. 98(6):1161-1162. doi:10.1099/jgv.0.000836S1161116298