Journal of Pharmaceutical and Biomedical Analysis 2005-09-15

Human red blood cells targeted metabolome analysis of glycolysis cycle metabolites by capillary electrophoresis using an indirect photometric detection method.

Michał J Markuszewski, Marta Szczykowska, Danuta Siluk, Roman Kaliszan

Index: J. Pharm. Biomed. Anal. 39 , 636, (2005)

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Abstract

A capillary electrophoretic (CE) method with UV indirect spectrophotometric detection for determination of the main compounds of glycolysis in human erythrocytes has been elaborated. Blood samples for electrophoretic analysis were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration with a centrifuge equipped with filter devices: Mr cut off 5000. Using 20 mM 2,6-pyridinedicarboxylic acid (PDC) as a highly UV absorbing carrier electrolyte and 4 mM cetyltrimetylammonium bromide (CTAB), at the resulting pH 12.3, nine of the glycolysis intermediates were separated and characterized. The repeatability and linearity of the method was assessed with percent relative standard deviation (%R.S.D.) for migration time ranging from 0.3 to 1.9% and correlation coefficients of 0.991-0.999 for the studied concentration range. Limits of detection (LOD) for the analyzed metabolites were in the range of 6.25 x 10(-6) to 5.0 x 10(-5) M. The optimized CE method was used to compare metabolome content of red blood cells of 22 healthy volunteers. Mean metabolite concentrations in erythrocytes ranged from 49.6 microM for fructose-6-phosphate (F-6-P) to 3.1 mM for 2,3-diphospho-D-glyceric acid (2,3-DPG). The method can be readily applied in clinical, pathophysiological and epidemiological studies.