CZE separation of cellular energetically important metabolites
| Authors | |
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| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | EURO ANALYSIS 2009 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis; metabolom; energetic metabolites; |
| Description | Nucleotides are essential for viability and growth of all living cells. They have at least four important functions; storage and transport of cellular metabolic energy; activation and transfer of precursors for cellular biosynthesis; synthesis of DNA and RNA; control and regulation of cellular metabolism. Their total concentration and distribution of individual species is highly dynamic with changes in environmental conditions. Monitoring the nucleotides pool is able to verify the physiological state of the cell and predict this state due to the presence or rations to each other. Due to the importance of nucleotides for metabolism, numerous methods for their determination in biological samples have been reported, mainly HPLC, CE or TLC. The main aim of this work was to find appropriate conditions for the selective and rapid determination of purine (AMP, ADP, ATP, GMP, GDP, GTP), pyrimidine (CMP, CDP, CTP, UMP, UDP, UTP) nucleotides, adenine coenzymes (NAD+, NADH, NADP+, NADPH) and Acetyl CoA using capillary electrophoresis (CE). Because of low intra and extracellular concentration of these metabolites the capillary zone electrophoresis was combined with the online preconcentration technique field enhanced sample stacking to improve the concentration sensitivities. The determination was performed in a bare fused silica capillary using separation voltage 20 kV (positive polarity) and direct detection at 260, 280 and 340 nm. For method optimization different concentrations of phosphate buffer (from 50 to 80 mM), pH range from 5 to 7 and temperature of capillary from 16 to 25 C were tested. The best resolution was found in the pH range from 5.5 to 6 for each buffer concentration; hence this pH range was examined in detail. Metabolite samples dissolved in deionised water were injected into the capillary hydrodynamically (50 mbar, 14 s). |
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