Journal article
Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis
Biomedical chromatography, Vol.22(12), pp.1374-1384
12/2008
DOI: 10.1002/bmc.1068
PMID: 18814195
Abstract
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.
Details
- Title: Subtitle
- Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis
- Creators
- Ye Li - Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USAPiyanka G. de Silva - Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USALu Xi - Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USAAlissa van Winkle - Department of Biology, University of Iowa, Iowa City, IA 52242, USAJim Jung-Ching Lin - Department of Biology, University of Iowa, Iowa City, IA 52242, USASemeena Ahmed - Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USAMaxwell Lei Geng - Department of Chemistry and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA
- Resource Type
- Journal article
- Publication Details
- Biomedical chromatography, Vol.22(12), pp.1374-1384
- Publisher
- John Wiley & Sons, Ltd
- DOI
- 10.1002/bmc.1068
- PMID
- 18814195
- ISSN
- 0269-3879
- eISSN
- 1099-0801
- Number of pages
- 11
- Language
- English
- Date published
- 12/2008
- Academic Unit
- Biology; Chemistry
- Record Identifier
- 9984216679802771
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