A simple method for the determination of the O-acetyl substitution pattern of the sialic acids of colonic epithelial glycoprotein.
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Biomedical subjects
Publications and source records attributed to C F Culling.
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Various methods for the estimation of free and ketosidically bound sialic acid were investigated for accuracy and specificity. It was found that oxidation with 0.025 M sodium metaperiodate in pH 7.0 phosphate buffer at room temperature for 20 min provided a simple, rapid, sensitive method whereby both the free and the ketosidically bound acid present in a mixture could be quantitatively analyzed. On completion of the oxidation step, the bound sialic acid is estimated with resorcinol reagent and the free sialic acid with thiobarbituric acid reagent. Oxidation under these conditions permitted a facile analysis of mixtures of bovine submaxillary mucin and free N-acetylneuraminic acid, whereas the Warren thiobarbituric acid procedure gave an erroneous value for free sialic acid.
The effects of potassium hydroxide and soidum methoxide treatments upon the Alcian blue staining and neuraminidase lability of certain neuraminidase resistant epithelial mucins have been studied. The results were interpreted as indicating that while the mucins of rat colon and rabbit Brunner's gland contain only 4-0-acetyl sialic acid, human colonic epithelial mucins may contain some sialic acid with esters at the C1 carboxyl group.
A new histochemical method is described for the differentiation of mucins that utilizes two different Schiff reagents and allows single section identification of side chain O-acylated, and nonacylated, sialic acids in contrasting colors. In the event of mucins containing only one type of sialic acid, it may allow their specific identification (e.g., C7 or C8 side chain O-acylated). It has been shown to be useful in the identification of some metastases from adenocarcinomas of colon (where the primary is potassium hydroxide/periodic acid-Schiff positive) and should prove of great value in the investigation of diseases of the gastrointestinal tract and particularly those of the colon. It should also be valuable in the general field of epithelial mucin histochemistry, particularly for those mucins of the salivary and parotid glands, etc.
Epithelial mucins of the normal terminal ileum, caecum, colon, and rectum of man are unique in that they alone exhibit staining following the periodate-borohydride/KOH/PAS technique. Application of this technique enables one to differentiate those mucin-producting metastases arising from adenocarcinoma of the lower gastrointestinal tract from those arising elsewhere, and may occasionally be useful in determining the site of the primary tumour when it is in doubt. Furthermore, it was found to be especially useful in distinguishing between primary adenocarcinoma of the lung and metastases in the lung from adenocarcinoma of the lower gastrointestinal tract.
Glycoproteins have been isolated from a 1 M sodium chloride extract of the colonic epithelial cells of man and rat by a combination of Agarose gel and DEAE-cellulose chromatography; The glycoproteins contain O-acetylated or O-esterified sialic acids of at least four types: (a) unsubstituted or possible at position C-9, (b) substituted at C-4 and possibly at C-9, (c) substituted at C-7 and/or C-8, and (d) substituted at C-4 as well as at C-7 and/or C-8.
Analysis of the apparently homogeneous sulfated sialoglycoproteins isolated from the epithelial cells of the upper (proximal to the ileum) and lower (proximal to the rectum) halves of Wistar rat colon showed that whereas they were similar in overall carbohydrate and amino acid composition, they differed significantly in the O-acetyl substitution pattern of their constituent sialic acids. The glycoprotein from the upper half of the colon was found to contain a larger percentage of sialic acid substitued at C7 and/or C8, as well as at C4 and C7 and/or C8; it contained less sialic acid which was unsubstituted as well as less subsituted at C4 alone. It has been shown that differences in substitution at C7 and/or C8 can be detected by analysis of the 105 000 g supernatants prepared from homogenates of isolated epithelial cells or fresh or formol calcium fixed whole colonic tissue.
Specimens from various types of Paget disease, other tumors, and certain normal tissues were examined with a battery of histochemical techniques, including the sodium borohydride-potassium hydroxide-PAS method that specifically stains certain sialomucins that are found in terminal parts of the ileum and of the colon. These sialomucins were present in normal anal ducts but were not present in transitional or anal-covering epithelium. A case of perianal Paget disease showed strongly positive staining, both in the underlying mucinous adenocarcinoma and in Paget cells of the affected anal and perianal skin. In contrast, stains of other forms of Paget disease were totally negative with this technique, as well as malignant melanoma and Bowen disease. These results support the theory that Paget disease represents epidermal invasion by malignant cells from underlying tumor.
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