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Publication
Biomarkers monitoring to elucidate hyperammonemia mechanisms and urea cycle disorders
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Authors
Abstract(s)
As doenças do ciclo da ureia (DCU) são um grupo de patologias genéticas que afetam principalmente o metabolismo hepático com consequências potencialmente graves para a função cerebral. O tratamento da huperamonémia(HA) associada à disfunção do ciclo da ureia é vital e a monitorização de complicações crónicas, como a doença hepática,é também crucial. A deficiência em ornitina transcarbamilase (OTCD) é uma das DCU mais comuns e com maior risco de vida, que pode manifestar-se como uma patologia de início precoce oi tardio. Os sintomas e sinais clínicos de OTCD são heterogéneos, variando de um espetro menos grave a muito grave 9estado mental alterado, coma e morte). O presente trabalho centrou-se nesta patologia e visa aprofundar o conhecimento sobre as técnicas e modelos que permitem entender as mudanças geradas por metabolitos em DCU e HA. Considerando que a acumulação de carbamoil fosfato na OTCD result no seu metabolismo alternativo a orotato(ORA) , pretende-se realçar a importância deste biomarcador um novo método usando GC-MS com aplicação de isótopos estáveis para detetar e quantificar ORA. A combinação de HPLC e SID-GC-MS, permite a análise abrangente de inúmeros produto do metabolismo intermediário, incluindo aminoácidos e metabolitos relacionados com o ciclo da ureia. Os respetivos níveis de ORA podem ser quantificados com precisão e exatidão na urina e no plasma dos doentes usando SID-GC-MS. A incerteza dos resultados repetidos e sistemáticos. Além disso, recorreu-se a três linhas diferentes de células hepáticas para estudar o seu perfil ou proteoma caraterístico que inclui a OTC e restantes enzimas do ciclo da ureia. Para esse fim, utilizou-se a focagem isoelétrica e eletroforese em gel bidimensional. Recorreu-se também a esta metodologia para a avaliação preliminar de biomarcadores proteicos, aplicando-a a amostras de plasmas controlo e de doentes já diagnosticados com OTCO. Os resultados realçam o valor agregado de diversas técnicas abrangentes, realçando-se a aplicação de GC-MS à análise de ORA como biomarcador para o diagnóstico diferencial de DCU, ou condições associadas a HA. O ganho adquirido com o maior poder de resolução da análise 2D de proteínas numa amostra complexa como plasma humano, representa um progresso metodológico significativo facilitando futuros estudos de proteómica. Em conclusão, este trabalho contribui para o esclarecimento e compreensão dos mecanismos de ureagénese em doentes com DCU ou portadores de mutações, incluindo OTCD, visando novos planos de investigação e desenvolvimento de terapias personalizadas.
Urea cycle disorders (UCDs) are a group of genetic disorders that primarily affect liver metabolism with potentially severe consequences for brain function. Treatment of huperammonemia (HA) associated with urea cycle dysfunction is vital and monitoring for chronic complications such as liver disease is also crucial. Ornithine transcarbamylase deficiency (OTCD) is one of the most common and life-threatening UCDs, which can manifest as early or late onset disease. The clinical symptoms and signs of OTCD are heterogeneous, ranging from less severe to very severe (altered mental status, coma, and death). The present work focused on this pathology and aims to deepen the knowledge about the techniques and models that allow understanding the changes generated by metabolites in UCD and AH. Considering that carbamoyl phosphate accumulation in OTCD results in its alternative metabolism to orotate(ORA) , we aimed to highlight the importance of this biomarker a new method using GC-MS with stable isotope application to detect and quantify ORA. The combination of HPLC and SID-GC-MS allows the comprehensive analysis of numerous products of intermediate metabolism, including amino acids and urea cycle related metabolites. The respective levels of ORA can be quantified with precision and accuracy in the urine and plasma of patients using SID-GC-MS. The uncertainty of repeated and systematic results. In addition, three different liver cell lines were used to study their characteristic profile or proteome that includes OTC and other urea cycle enzymes. For this purpose, isoelectric focusing and two-dimensional gel electrophoresis were used. This methodology was also used for the preliminary evaluation of protein biomarkers, applying it to samples from control plasmas and from patients already diagnosed with OTC. The results highlight the added value of several comprehensive techniques, highlighting the application of GC-MS to the analysis of ORA as a biomarker for the differential diagnosis of UCD, or conditions associated with AH. The gain gained from the higher resolving power of 2D protein analysis in a complex sample such as human plasma represents a significant methodological progress facilitating future proteomics studies. In conclusion, this work contributes to the clarification and understanding of the mechanisms of ureagenesis in patients with UCD or carrying mutations, including OTCD, targeting new research plans and development of personalized therapies.
Urea cycle disorders (UCDs) are a group of genetic disorders that primarily affect liver metabolism with potentially severe consequences for brain function. Treatment of huperammonemia (HA) associated with urea cycle dysfunction is vital and monitoring for chronic complications such as liver disease is also crucial. Ornithine transcarbamylase deficiency (OTCD) is one of the most common and life-threatening UCDs, which can manifest as early or late onset disease. The clinical symptoms and signs of OTCD are heterogeneous, ranging from less severe to very severe (altered mental status, coma, and death). The present work focused on this pathology and aims to deepen the knowledge about the techniques and models that allow understanding the changes generated by metabolites in UCD and AH. Considering that carbamoyl phosphate accumulation in OTCD results in its alternative metabolism to orotate(ORA) , we aimed to highlight the importance of this biomarker a new method using GC-MS with stable isotope application to detect and quantify ORA. The combination of HPLC and SID-GC-MS allows the comprehensive analysis of numerous products of intermediate metabolism, including amino acids and urea cycle related metabolites. The respective levels of ORA can be quantified with precision and accuracy in the urine and plasma of patients using SID-GC-MS. The uncertainty of repeated and systematic results. In addition, three different liver cell lines were used to study their characteristic profile or proteome that includes OTC and other urea cycle enzymes. For this purpose, isoelectric focusing and two-dimensional gel electrophoresis were used. This methodology was also used for the preliminary evaluation of protein biomarkers, applying it to samples from control plasmas and from patients already diagnosed with OTC. The results highlight the added value of several comprehensive techniques, highlighting the application of GC-MS to the analysis of ORA as a biomarker for the differential diagnosis of UCD, or conditions associated with AH. The gain gained from the higher resolving power of 2D protein analysis in a complex sample such as human plasma represents a significant methodological progress facilitating future proteomics studies. In conclusion, this work contributes to the clarification and understanding of the mechanisms of ureagenesis in patients with UCD or carrying mutations, including OTCD, targeting new research plans and development of personalized therapies.
Description
Tese de mestrado, Ciências Biofarmacêuticas, 2022, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Urea cycle disorder Hyperammonemia Orotic acid OTC deficiency Biomarkers Teses de mestrado - 2022