Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine

George H. Thomas, Jane Scocca, Carol S. Miller, Linda W. Reynolds

Research output: Contribution to journalArticle

Abstract

Human skin fibroblasts incubated for 72 h in medium containing 10 mM N-acetyl-d-mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10% trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N-acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N-acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N-acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N-acetylmannosamine-enriched medium and incubated in regular medium, more than 80% of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N-acetylmannosamine store large amounts of N-acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.

Original languageEnglish (US)
Pages (from-to)37-43
Number of pages7
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume846
Issue number1
DOIs
StatePublished - Jul 30 1985

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N-Acetylneuraminic Acid
Fibroblasts
N-acetylneuraminate lyase
Trichloroacetic Acid
N-acetylmannosamine
Solubility
Anions
Cultured Cells
Mass Spectrometry
Proteins
Hydrolysis
Skin
Acids

Keywords

  • (Human fibroblast)
  • N-Acetylmannosamine
  • N-Acetylneuraminic acid

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine. / Thomas, George H.; Scocca, Jane; Miller, Carol S.; Reynolds, Linda W.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 846, No. 1, 30.07.1985, p. 37-43.

Research output: Contribution to journalArticle

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abstract = "Human skin fibroblasts incubated for 72 h in medium containing 10 mM N-acetyl-d-mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10{\%} trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N-acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N-acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N-acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N-acetylmannosamine-enriched medium and incubated in regular medium, more than 80{\%} of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N-acetylmannosamine store large amounts of N-acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.",
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