TY - JOUR
T1 - Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine
AU - Thomas, George H.
AU - Scocca, Jane
AU - Miller, Carol S.
AU - Reynolds, Linda W.
N1 - Funding Information:
This research was supported by NIH Grant HD-10981. Mass spectral determinations were carried out by Ms. Sharon Pallante at the Middle Atlantic Mass Spectrometry Laboratory, a National Science Foundation shared instrumentation facility.
PY - 1985/7/30
Y1 - 1985/7/30
N2 - 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.
AB - 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.
KW - (Human fibroblast)
KW - N-Acetylmannosamine
KW - N-Acetylneuraminic acid
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U2 - 10.1016/0167-4889(85)90107-7
DO - 10.1016/0167-4889(85)90107-7
M3 - Article
C2 - 4016156
AN - SCOPUS:0022262794
SN - 0167-4889
VL - 846
SP - 37
EP - 43
JO - BBA - Molecular Cell Research
JF - BBA - Molecular Cell Research
IS - 1
ER -