[CITATION][C] Determination of mucin in salivary glands using sialic acids as the marker by high-performance liquid chromatography with fluorometric detection
A Kawabata, N Morimoto, Y Oda, M Kinoshita… - Analytical …, 2000 - Elsevier
A Kawabata, N Morimoto, Y Oda, M Kinoshita, R Kuroda, K Kakehi
Analytical Biochemistry, 2000•ElsevierSalivary mucin is a family of proteins that contain large amounts of O-glycosidically linked
carbohydrate chains (1). The nonreducing termini of mucin-type carbohydrate chains are
often substituted with sialic acids. The amino group of sialic acids is usually substituted with
acetyl or glycolyl groups. Furthermore, hydroxyl groups of sialic acids are also sometimes
substituted with acetyl, lactyl, and other groups (2). Salivary mucin plays an important role in
the preservation of the oral cavity and the defense from bacterial and enzymatic attack, and …
carbohydrate chains (1). The nonreducing termini of mucin-type carbohydrate chains are
often substituted with sialic acids. The amino group of sialic acids is usually substituted with
acetyl or glycolyl groups. Furthermore, hydroxyl groups of sialic acids are also sometimes
substituted with acetyl, lactyl, and other groups (2). Salivary mucin plays an important role in
the preservation of the oral cavity and the defense from bacterial and enzymatic attack, and …
Salivary mucin is a family of proteins that contain large amounts of O-glycosidically linked carbohydrate chains (1). The nonreducing termini of mucin-type carbohydrate chains are often substituted with sialic acids. The amino group of sialic acids is usually substituted with acetyl or glycolyl groups. Furthermore, hydroxyl groups of sialic acids are also sometimes substituted with acetyl, lactyl, and other groups (2).
Salivary mucin plays an important role in the preservation of the oral cavity and the defense from bacterial and enzymatic attack, and its determination is important in understanding its biological roles (1). Colorimetric methods such as thiobarbituric acid assay for sialic acids have been used for the analysis of mucin in salivary glands (3). However, they are not available for the determination of molecular species of sialic acids such as N-acetyl (NeuAc), 2 N-glycolylneuraminic acid (NeuGc)(3), and their O-acyl analogues. Hara et al. reported fluorometric high-performance liquid chromatography (HPLC) of NeuAc and NeuGc after precolumn derivatization with 1, 2-diamino-4, 5-methylenedioxybenzene (DMB)(4). Recently, Klein et al. applied this method to the analysis of various sialic acid analogues in bovine submaxillary mucin and described the nature of the fluorophore including its structure in detail (5). In the present study, we employed two different hydrolysis conditions in aqueous solutions of 2 M acetic acid and 0.05 M hydrochloric acid for the release of sialic acids from mucin samples derived from different animals. The sialic acid analogues released by acid hydrolysis were labeled with DMB in a one-pot reaction, and analyzed by HPLC. The present method allows determination of both total sialic acids and distribution of their molecular species. Materials. DMB was obtained from Dojin (Kumamoto, Japan). All reagents were of the highest grade commercially available or HPLC grade. Mucin samples used in the present study were purified from the isolated salivary glands according to the method by Tettamanti and Pigman (6). Porcine stomach mucin was obtained from Sigma-Aldrich Japan (Tokyo, Japan).
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