A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.13/2095 |
Resumo: | Asthma is a disease characterized by chronic airway inflammation and by its long term irreversible remodeling. The enzymatic peroxidation of the arachidonic acid leads to the formation of bioactive eicosanoids, key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of this disease. The present work aimed to develop an improved ultra-high pressure liquid chromatography (UHPLC)-based strategy to characterize biomarkers of lipid peroxidation: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was used to isolate the target analytes. Several experimental parameters with influence on this procedure and on the chromatographic resolution, were evaluated and optimized. The method was fully validated with compatible values under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 µL of ACN-H2O at 0.1% FA, 10 extract-discard cycles of 250 µL of sample at a pH of 5.1, elution with 2 times 50 µL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 µL min-1 in an UHPLC-PDA equipped with a BEH C18 column). The developed method was successfully applied to the urine of asthmatic patients and healthy individuals. On average, the urine of asthmatic patients present significantly higher concentrations of 11βPGF2α (112.96 ng mL-1 vs 62.56 ng mL-1 in control group), LTE4 (1.27 ng mL-1 vs 0.89 ng mL-1 in control group) and LTB4 (1.39 ng mL-1 vs 0.76 ng mL-1 in control group). These results suggest the potential of the target eicosanoids on asthma diagnosis and on the follow-up of the therapeutic response, even though a larger and more extensive study will be necessary, using a bigger number of cases, to confirm the data obtained and to guarantee a greater robustness to the approach. |
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A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturizationAsthmaBiomarkers11β-Prostaglandin F2αLeukotriene E4Leukotriene B4Micro extraction by packed sorbentUltra high pressure liquid chromatographyAsma11β-Prostaglandina F2αLeucotrieno E4Leucotrieno B4Micro extracção por sorvente empacotadoCromatografia líquida de ultra alta pressãoApplied Biochemistry.Faculdade de Ciências Exatas e da EngenhariaDomínio/Área Científica::Ciências Médicas::Biotecnologia MédicaDomínio/Área Científica::Ciências Naturais::Ciências QuímicasDomínio/Área Científica::Ciências Médicas::Medicina ClínicaAsthma is a disease characterized by chronic airway inflammation and by its long term irreversible remodeling. The enzymatic peroxidation of the arachidonic acid leads to the formation of bioactive eicosanoids, key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of this disease. The present work aimed to develop an improved ultra-high pressure liquid chromatography (UHPLC)-based strategy to characterize biomarkers of lipid peroxidation: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was used to isolate the target analytes. Several experimental parameters with influence on this procedure and on the chromatographic resolution, were evaluated and optimized. The method was fully validated with compatible values under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 µL of ACN-H2O at 0.1% FA, 10 extract-discard cycles of 250 µL of sample at a pH of 5.1, elution with 2 times 50 µL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 µL min-1 in an UHPLC-PDA equipped with a BEH C18 column). The developed method was successfully applied to the urine of asthmatic patients and healthy individuals. On average, the urine of asthmatic patients present significantly higher concentrations of 11βPGF2α (112.96 ng mL-1 vs 62.56 ng mL-1 in control group), LTE4 (1.27 ng mL-1 vs 0.89 ng mL-1 in control group) and LTB4 (1.39 ng mL-1 vs 0.76 ng mL-1 in control group). These results suggest the potential of the target eicosanoids on asthma diagnosis and on the follow-up of the therapeutic response, even though a larger and more extensive study will be necessary, using a bigger number of cases, to confirm the data obtained and to guarantee a greater robustness to the approach.A asma é uma doença caracterizada por inflamação crónica das vias aéreas e pela sua remodelação irreversível a longo prazo. A peroxidação enzimática do ácido araquidónico leva à formação de eicosanóides bioactivos, importantes mediadores da inflamação brônquica e da modulação da resposta nas vias aéreas contribuindo para a fisiopatologia desta doença. O presente trabalho teve como objectivo desenvolver uma estratégia aperfeiçoada baseada em cromatografia líquida de ultra pressão (UHPLC) para caracterizar biomarcadores da peroxidação enzimática: leucotrienos E4 (LTE4) e B4 (LTB4) e 11βprostaglandina F2a (11βPGF2α), presentes na urina de pacientes asmáticos e de indivíduos saudáveis. Foi utilizada uma microextracção por sorvente empacotado (MEPS) em modo semiautomático (eVol®) para isolar os analitos alvo. Foram avaliados e optimizados vários parâmetros experimentais com influência neste procedimento e na resolução cromatográfica. O método foi totalmente validado, com valores compatíveis em condições óptimas de extracção (sorvente R-AX, 3 ciclos de condicionamento-equilíbrio com 250 µL de ACN-H2O a 0,1% FA, com 10 ciclos de extracção-descarte de 250 µL de amostra a um pH de 5,1, eluição com 2×50 µL de MeOH e evaporação do eluato até metade do seu volume) e cromatográficas (análise de 14 minutos a uma taxa de fluxo de 300 µL min-1 num UHPLC-PDA equipado com uma coluna BEH C18). O método desenvolvido foi aplicado com sucesso às amostras alvo. Em média, os pacientes asmáticos apresentaram concentrações significativamente mais elevadas de 11βPGF2α (112,96 ng mL-1 vs 62,56 ng mL-1 em indivíduos saudáveis), LTE4 (1,27 ng mL-1 vs 0,89 ng mL-1 em indivíduos saudáveis) e LTB4 (1,39 ng mL-1 vs 0,76 ng mL-1 em indivíduos saudáveis). Estes resultados sugerem o potencial destes eicosanóides no diagnóstico da asma e no seguimento da resposta à terapêutica. Contudo, será necessário um estudo mais alargado e extensivo, usando um maior número de casos, para confirmar os dados obtidos e garantir uma maior robustez desta estratégia.Câmara, José de SousaCamacho, Irene Gomes CâmaraDigitUMaBerenguer, Pedro Henrique Fernandes da Silva2019-11-03T01:30:10Z2018-05-032018-05-03T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.13/2095TID:201922835enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-03-26T03:36:08Zoai:digituma.uma.pt:10400.13/2095Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T15:04:51.789745Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
title |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
spellingShingle |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization Berenguer, Pedro Henrique Fernandes da Silva Asthma Biomarkers 11β-Prostaglandin F2α Leukotriene E4 Leukotriene B4 Micro extraction by packed sorbent Ultra high pressure liquid chromatography Asma 11β-Prostaglandina F2α Leucotrieno E4 Leucotrieno B4 Micro extracção por sorvente empacotado Cromatografia líquida de ultra alta pressão Applied Biochemistry . Faculdade de Ciências Exatas e da Engenharia Domínio/Área Científica::Ciências Médicas::Biotecnologia Médica Domínio/Área Científica::Ciências Naturais::Ciências Químicas Domínio/Área Científica::Ciências Médicas::Medicina Clínica |
title_short |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
title_full |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
title_fullStr |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
title_full_unstemmed |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
title_sort |
A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization |
author |
Berenguer, Pedro Henrique Fernandes da Silva |
author_facet |
Berenguer, Pedro Henrique Fernandes da Silva |
author_role |
author |
dc.contributor.none.fl_str_mv |
Câmara, José de Sousa Camacho, Irene Gomes Câmara DigitUMa |
dc.contributor.author.fl_str_mv |
Berenguer, Pedro Henrique Fernandes da Silva |
dc.subject.por.fl_str_mv |
Asthma Biomarkers 11β-Prostaglandin F2α Leukotriene E4 Leukotriene B4 Micro extraction by packed sorbent Ultra high pressure liquid chromatography Asma 11β-Prostaglandina F2α Leucotrieno E4 Leucotrieno B4 Micro extracção por sorvente empacotado Cromatografia líquida de ultra alta pressão Applied Biochemistry . Faculdade de Ciências Exatas e da Engenharia Domínio/Área Científica::Ciências Médicas::Biotecnologia Médica Domínio/Área Científica::Ciências Naturais::Ciências Químicas Domínio/Área Científica::Ciências Médicas::Medicina Clínica |
topic |
Asthma Biomarkers 11β-Prostaglandin F2α Leukotriene E4 Leukotriene B4 Micro extraction by packed sorbent Ultra high pressure liquid chromatography Asma 11β-Prostaglandina F2α Leucotrieno E4 Leucotrieno B4 Micro extracção por sorvente empacotado Cromatografia líquida de ultra alta pressão Applied Biochemistry . Faculdade de Ciências Exatas e da Engenharia Domínio/Área Científica::Ciências Médicas::Biotecnologia Médica Domínio/Área Científica::Ciências Naturais::Ciências Químicas Domínio/Área Científica::Ciências Médicas::Medicina Clínica |
description |
Asthma is a disease characterized by chronic airway inflammation and by its long term irreversible remodeling. The enzymatic peroxidation of the arachidonic acid leads to the formation of bioactive eicosanoids, key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of this disease. The present work aimed to develop an improved ultra-high pressure liquid chromatography (UHPLC)-based strategy to characterize biomarkers of lipid peroxidation: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was used to isolate the target analytes. Several experimental parameters with influence on this procedure and on the chromatographic resolution, were evaluated and optimized. The method was fully validated with compatible values under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 µL of ACN-H2O at 0.1% FA, 10 extract-discard cycles of 250 µL of sample at a pH of 5.1, elution with 2 times 50 µL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 µL min-1 in an UHPLC-PDA equipped with a BEH C18 column). The developed method was successfully applied to the urine of asthmatic patients and healthy individuals. On average, the urine of asthmatic patients present significantly higher concentrations of 11βPGF2α (112.96 ng mL-1 vs 62.56 ng mL-1 in control group), LTE4 (1.27 ng mL-1 vs 0.89 ng mL-1 in control group) and LTB4 (1.39 ng mL-1 vs 0.76 ng mL-1 in control group). These results suggest the potential of the target eicosanoids on asthma diagnosis and on the follow-up of the therapeutic response, even though a larger and more extensive study will be necessary, using a bigger number of cases, to confirm the data obtained and to guarantee a greater robustness to the approach. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-05-03 2018-05-03T00:00:00Z 2019-11-03T01:30:10Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.13/2095 TID:201922835 |
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http://hdl.handle.net/10400.13/2095 |
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TID:201922835 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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