Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids

Journal Article · Tue Oct 01 00:00:00 EDT 1985 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(85)90438-5· OSTI ID:5999882

Diaz, S; Varki, A

The parent sialic acid N-acetylneuraminic acid can be modified or substituted in various ways, giving rise to a family of more than 25 compounds. The definitive identification of these compounds has previously required isolation of nanomole amounts for mass spectrometry or NMR. We have explored the possibility of using the known metabolic precursors of the sialic acids, particularly N-acetyl-(6-3H)mannosamine, to label and identify various forms of sialic acids in tissue culture cells. Firstly, we defined several variables that affect the labeling of sialic acids with N-acetyl-(6-3H)mannosamine. Secondly, we have devised a simple screening method to identify cell lines that synthesize substituted or modified sialic acids. We next demonstrate that it is possible to definitively identify the natures of the various labeled sialic acids without the use of mass spectrometry, even though they are present only in tracer amounts. The methods used include paper chromatography, analytical de-O-acetylation, periodate release of the 9-3H as (3H)formaldehyde (which is subsequently converted to a specific 3H-labeled chromophore), acylneuraminate pyruvate lyase treatment with identification of (3H)acylmannosamines, gas-liquid chromatography with radioactive detection, and two new high-pressure liquid chromatography methods utilizing the amine-adsorption:ion suppression and ion-pair principles. The use of an internal N-acetyl-(4-14C)neuraminic acid standard in each of these methods assures precision and accuracy. The combined use of these methods now allows the identification of radioactive tracer amounts of the various types of sialic acids in well-defined populations of tissue culture cells; it may also allow the identification of hitherto unknown forms of sialic acids.

• Research Organization: Univ. of California, San Diego
• OSTI ID: 5999882
• Journal Information: Anal. Biochem.; (United States), Vol. 1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
SIALIC ACID
LABELLING
CELL CULTURES
GLUCOSAMINE
HEXOSAMINES
IN VIVO
MICE
TRITIUM COMPOUNDS
AMINES
ANIMALS
CARBOHYDRATES
HEXOSES
LABELLED COMPOUNDS
MAMMALS
MONOSACCHARIDES
ORGANIC COMPOUNDS
RODENTS
SACCHARIDES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques
400702 - Radiochemistry & Nuclear Chemistry- Properties of Radioactive Materials