Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation of the intensity of the periodic acid-Schiff (PAS) staining produced following oxidation for 1 h at 4 degrees C with 0.4 mM periodic acid in approximately 1 M hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules or vicinal diols located on hexose, 6-deoxyhexose or N-acetylhexosamine residues ('neutral sugars') of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4 mM periodic acid in approximately 1 M hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure. Studies of the mechanism of the oxidation of neutral sugars with 0.4 mM periodic acid in approximately 1 M hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4 mM periodic acid in approximately 1 M hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.
Four methods based upon the periodic acid-phenylhydrazine-Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizable vicinal diols (hexose, 6-deoxyhexose, N-acetylhexosamine) and either sialic acids or side chain O-acyl sialic acids. In the first of these procedures, the saponification-periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A-Schiff-saponification (KOH-PA-DNPH-Az-KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizable vicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A Schiff-saponification (PA-DNPH-Az-KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have an O-acyl substituent at position C7, while in the third method, the selective periodate oxidation-borohydride reduction-saponification-periodic acid oxidation-2,4-dinitrophenyl hydrazine-Azure A-Schiff-saponification (PA-Bh-KOH-PA-DNPH-Az-KOH) technique, only sialic acids with O-acyl substituents at positions C7, C8 or C9 (or which have two or three O-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A-Schiff-saponification-borohydride reduction-periodic acid oxidation-Schiff (PA-DNPH-Az-KOH-Bh-PAS), sialic acids without side chain substituents or which have O-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.
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