Radioenzymatic determination of CMP-N-acetylsialic acid and free N-acetylsialic acid in biological material.
Free N-acetylsialic acid (NeuNAc) and CMP-N-acetylsialic acids (CMP-NeuNAc) are extracted from freeze-clamped or liquid nitrogen-frozen biological material by sequential extraction with cold acetone and acetone/water. [14C]NeuNAc and [14C]CMP-NeuNAc (20,000 dpm each) are added to the frozen material to correct for small losses occurring during the subsequent steps. NeuNAc and CMP-NeuNAc are separated by anion-exchange chromatography. CMP-NeuNAc is hydrolyzed with formic acid and again chromatographed on an ion-exchange column. The NeuNAc-containing fractions (representing free NeuNAc and CMP-NeuNAc) are converted to [14C]CMP-NeuNAc in the presence of [14C]CTP and CMP-NeuNAc synthetase. [14C]CMP-NeuNAc is separated by paper chromatography and the radioactivity measured by liquid scintillation counting. The amount of NeuNAc is calculated from a calibration curve obtained with NeuNAc standards. The small amounts of [14C]NeuNAc and [14C]CMP-NeuNAc added initially do not interfere with the final assay. The method gives reliable values down to 50 pmol/assay, but the sensitivity can be easily increased by a factor of 10. Recoveries, with NeuNAc and CMP-NeuNAc added to biological extracts, were 98.3 and 98.5% for NeuNAc and CMP-NeuNAc, respectively. With this method values of 61.2 +/- 12.8 and 24.4 +/- 5.2 nmol/g wet wt were found in rat liver for free NeuNAc and CMP-NeuNAc, respectively. Values for free NeuNAc found in human blood plasma were 600 +/- 476 and 373 +/- 180 pmol/g plasma for healthy persons and patients with breast cancer, respectively. Free CMP-NeuNAc could not be found in plasma.
D000818:Animals; D001943:Breast Neoplasms / Q000097:blood; D002250:Carbon Radioisotopes; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000097:blood / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008099:Liver / Q000032:analysis; D008297:Male; D008832:Microchemistry; D000216:N-Acylneuraminate Cytidylyltransferase; D051381:Rats; D012794:Sialic Acids / Q000097:blood / Q000302:isolation & purification*; D012997:Solvents
D002250:Carbon Radioisotopes; D012794:Sialic Acids; D012997:Solvents; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase