Capillary electrophoretic method for nucleotide analysis in cells: application on inherited metabolic disorders.
Purine and pyrimidine nucleotides influence many metabolic pathways and their analogs have been widely used in medicine. A capillary electrophoretic method was developed for measuring intracellular nucleotides. The final BGE consisted of 40 mM citric acid with addition of 0.8 mM CTAB titrated by gamma-aminobutyric acid to pH 4.4. The electrophoretic separations were carried out in an uncoated silica capillary (id/od - 75/375 microm; effective/total length - 90/97 cm). The method allows a complete separation of 21 nucleotides and deoxynucleotides within 15 min with separation efficiencies up to 400,000 theoretical plates per meter. Due to the use of an acidic separation medium, the method offers a high selectivity toward the studied analytes versus possible interferences from matrices. Sample preparation was optimized in order to shorten work-time and prevent analyte degradation. The method was applied for analyzing nucleotides in human erythrocytes and Chinese hamster ovary cells. Diagnostic potential for inherited metabolic disorders of nucleotide metabolism is presented.
Friedecký
David
D
Department of Clinical Biochemistry, Laboratory for Inherited Metabolic Disorders, University Hospital and Palacký University Olomouc, Olomouc, Czech Republic.
Tomková
Jana
J
Maier
Vitezslav
V
Janost'áková
Anna
A
Procházka
Martin
M
Adam
Tomás
T
D000818:Animals; D016466:CHO Cells; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011685:Purine Nucleotides / Q000032:analysis*; D011742:Pyrimidine Nucleotides / Q000032:analysis*; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
D002593:Cetrimonium Compounds; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D019343:Citric Acid; D005680:gamma-Aminobutyric Acid; D000077286:Cetrimonium