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.
A new general method has been developed for the specific histochemical identification of O-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose and N-acetylhexosamine with an ester substituent(s) located on a potential vicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA*-Bh-PAS) method] the initial PA-Bh treatment renders vicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from both O-acyl sugars and O-acyl sialic acids by saponification (KOH), sialic acid vicinal diols are selectively removed by the PA*-Bh sequence and O-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections. Using the PA-Bh-KOH-PA*-Bh-PAS method we have demonstrated, for the first time, that O-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence of O-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars and O-acyl sialic acids are selected.
Five new methods, based upon the selective oxidation of sialic acid residues with 0.4 mM periodic acid in approximately 1 M hydrochloric acid at 4 degree C for 1 h (PA), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. In the first of these, the selective periodate oxidation-borohydride reduction-saponification-selective periodate oxidation-Thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-T-KOH-Bh-PAS) technique, sialic acids with O-acyl substituents at C7, C8 or C9 (or which have two of three side chain O-acyl substituents) stain blue while neutral sugars with periodate-sensitive vicinal diols (hexose, 6-deoxyhexose, and N-acetylhexosamine) stain magenta. The second method, the saponification-selective periodate oxidation-Thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (KOH-PA-T-KOH-Bh-PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation-Thionin Schiff-borohydride reduction-periodic acid-Schiff-saponification (PA-T-Bh-PAS-KOH) method, sialic acids without side chain substituents (or which have an O-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation-borohydride reduction-Alcian Blue pH 1.0-periodic acid-Schiff (KOH-PA-Bh-AB1.0-PAS) technique, O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple.(ABSTRACT TRUNCATED AT 250 WORDS)
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