ABSTRACT
The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS2 . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS2 enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL-1 ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.
Subject(s)
Peptides/analysis , Steroids/analysis , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Doping in Sports/prevention & control , Humans , Limit of Detection , Peptides/urine , Solid Phase Extraction/methods , Steroids/urineABSTRACT
Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.
Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.
Subject(s)
Animals , Cattle , Cattle/anatomy & histology , Cattle/genetics , Epigenesis, Genetic , Reproductive Techniques, Assisted/veterinaryABSTRACT
Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.(AU)
Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.(AU)
Subject(s)
Animals , Cattle , Cattle/anatomy & histology , Cattle/genetics , Epigenesis, Genetic , Reproductive Techniques, Assisted/veterinaryABSTRACT
El objetivo del presente trabajo fué evaluar el efecto de una dosis diaria de L-Carnitina de 80mg/Kg de peso corporal, sobre el perfil lipídico y la progresión de lesiones ateromatosas en la pared aórtica, empleando como modelo experimental conejos de la raza Nueva Zelanda e híbridos Nueva Zelanda-California, sometidos a una dieta rica en colesterol. Se observó, que la terapia con L-carnitina fue capaz de modular los niveles de lípidos plasmáticos dependiendo de sus valores iniciales, al mismo tiempo que detuvo parcialmente la progresión de la lesión ateromatosa. Nuevos resultados indican que la L-carnitina ejerce un efecto endotelio-protector, en el modelo experimental empleado en este estudio