title: Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence.
abstract: Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Shandong Normal University, Jinan 250014, People's Republic of China.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Chen
authors.forename: Qingling
authors.initials: Q
authors.lastname: Li
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Sun
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Miao
authors.initials: M
authors.lastname: Yin
authors.forename: Yanxia
authors.initials: Y
authors.lastname: Xie
authors.forename: Hongmin
authors.initials: H
authors.lastname: Li
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
mesh_terms: D000818:Animals; D017209:Apoptosis; D001204:Ascorbate Oxidase / Q000737:chemistry / Q000378:metabolism; D001205:Ascorbic Acid / Q000737:chemistry; D052160:Benzothiazoles; D003510:Cyclohexanes; D003683:Dehydroascorbic Acid / Q000737:chemistry; D046209:Electrophoresis, Microchip; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D056945:Hep G2 Cells; D006801:Humans; D007834:Lasers; D008928:Mitochondria / Q000378:metabolism*; D009569:Nitric Oxide / Q000032:analysis*; D016716:PC12 Cells; D051381:Rats; D013481:Superoxides / Q000032:analysis*
chemical_list: C562228:2-chloro-1,3-dibenzothiazolinecyclohexene; D052160:Benzothiazoles; D003510:Cyclohexanes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013481:Superoxides; D009569:Nitric Oxide; D001204:Ascorbate Oxidase; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid