Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion.
Anal Chim Acta
; 999: 107-113, 2018 Jan 25.
Article
en En
| MEDLINE
| ID: mdl-29254560
Superoxide anion (O2.-), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O2.- level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O2.- assay by monitoring the unique product 2-OH-E+ of HE/O2.- reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O2.- formation. By the detection of 2-OH-E+, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O2.-. Firstly, we demonstrated the proportionality of HE/O2.- reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E+. Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O2.-. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O2.- generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O2.- assay method is more sensitive, selective and accurate.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
/
Cromatografía Líquida de Alta Presión
/
Superóxidos
Límite:
Animals
Idioma:
En
Revista:
Anal Chim Acta
Año:
2018
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Países Bajos