Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis.
Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.
Li
Ye
Y
Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA.
de Silva
Piyanka G
PG
Xi
Lu
L
van Winkle
Alissa
A
Lin
Jim Jung-Ching
JJ
Ahmed
Semeena
S
Geng
Maxwell Lei
ML
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods*; D005415:Flavins / Q000302:isolation & purification*; D009243:NAD / Q000302:isolation & purification; D009249:NADP / Q000302:isolation & purification; D009536:Niacinamide / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D005415:Flavins; D009243:NAD; D009536:Niacinamide; D009249:NADP