A systematic study of nucleotide analysis of human erythrocytes using an anionic exchanger and HPLC.
A two-step procedure for rapid HPLC analysis of nucleotides from human erythrocytes is described. A strong anion exchanger Partisil-10 is used as column material. Elution of monophosphates requires about 12 min and the elution of di- and triphosphates 21 min. The elutions are performed separately and with different injections as such a procedure will save time otherwise used to re-condition the columns. In addition to the three adenylates other nucleotides such as GTP, GDP, IMP and NAD can be recorded as isolated, well-defined peaks, which can be subject to quantitative analysis. The mean value of ATP concentration for 12 healthy individuals was 1.55 +/- 0.05 mmol/l, about 25% higher than generally reported in the literature. Accurate estimates of ADP and AMP concentrations allowed calculation of mean values for the physiologically interesting ratios: adenylate energy charge (0.945 +/- 0.002) and equilibrium constant for adenylate kinase (1.205 +/- 0.053). GDP and GTP are present in concentrations that are about 4% of those for corresponding adenylates. The analysis of nucleotides in human erythrocytes is a useful way of studying erythrocyte preservation and investigating patients with hemolytic disorders.
D000227:Adenine Nucleotides / Q000097:blood; D000328:Adult; D000368:Aged; D000837:Anion Exchange Resins; D001793:Blood Preservation; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000032:analysis*; D005260:Female; D006150:Guanine Nucleotides / Q000097:blood; D006402:Hematologic Diseases / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007291:Inosine Monophosphate / Q000097:blood; D008297:Male; D009243:NAD / Q000097:blood; D011685:Purine Nucleotides / Q000097:blood*
D000227:Adenine Nucleotides; D000837:Anion Exchange Resins; D006150:Guanine Nucleotides; D011685:Purine Nucleotides; D009243:NAD; D007291:Inosine Monophosphate