Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay

Leland D. Powell, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)179-185
Number of pages7
JournalAnalytical Biochemistry
Volume157
Issue number1
DOIs
StatePublished - Aug 15 1986

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Keywords

  • colorimetric assay
  • HPLC techniques
  • N-acetylneuraminic acid
  • N-glycolylneuraminic acid
  • sialic acid
  • sugar analysis
  • thiobarbituric acid

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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