Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay.
A simple HPLC adaptation of the periodate-TBA assay for free N-acetyl- and N-glycolylneuraminic acids greatly extends the sensitivity and increases the specificity of this standard colorimetric assay. The method, employing a C18 reverse-phase column eluted isocratically with a phosphoric acid-MeOH buffer, is linear over a range of 2 pmol to 20 nmol. Analyses can be performed directly on cell lysates and digests without prior purification of released sialic acids from contaminating salts and biological materials. Interference from 2-deoxysugars is completely eliminated as the chromophore from these compounds is completely resolved from that derived from sialic acids. The application of the technique to quantify cell-surface and total cellular TBA-reactive sialic acids on the surfaces of a variety of tumor cells is described. Additionally, the extent of desialylation of erythrocytes necessary to expose the T antigen is determined.
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry; D004912:Erythrocytes / Q000032:analysis; D006801:Humans; D051379:Mice; D008832:Microchemistry; D019158:N-Acetylneuraminic Acid; D009374:Neoplasms, Experimental / Q000032:analysis; D009438:Neuraminic Acids / Q000032:analysis*; D012794:Sialic Acids / Q000032:analysis*; D013858:Thiobarbiturates / Q000032:analysis
D000957:Antigens, Viral, Tumor; D009438:Neuraminic Acids; D012794:Sialic Acids; D013858:Thiobarbiturates; C032592:N-glycolylneuraminic acid; D019158:N-Acetylneuraminic Acid; C029684:thiobarbituric acid