A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol.

An alternative primary amine assay utilizing 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione (fluorescamine) is compared with the frequently applied trinitrobenzenesulfonic acid assay as a means of estimating protein primary amine modification with cyanuric chloride attachment of polyethylene glycol (PEG). The results of the two assays are compared for three different proteins and several advantages of the fluorescamine assay are emphasized. In particular, the fluorescamine assay was found to be unaffected by free PEG up to 0.08% in the assay mixture. The fluorescamine assay can be used to measure the partition coefficients of native or PEG-modified protein in two-phase aqueous polymer systems.

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins.

A differential diagnostic scheme is described for the division of colonic epithelial glycoproteins into eleven histochemically distinct classes. The scheme depends upon the use of seven histochemical techniques which, collectively, permit the differential staining of O-sulphate ester, sialic acid and its side chain O-acyl variants and vicinal diols located on carbohydrate residues other than sialic acids. Elements of the scheme also provide a general approach to the classification of epithelial glycoproteins in anatomic sites other than the colon. Application of the scheme permitted the classification of the epithelial glycoproteins in the mucosa 0.5-5.0 cm from human colonic tumours and provided direct confirmation of previous observations that changes from normal in the relative proportions of either side chain O-acylated sialic acids or sialic acids and O-sulphate esters can occur independently of one another.

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. III. Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours.

Histochemical, chemical and histological studies were performed on 26 specimens of human colonic tumours and 62 specimens of mucosa taken at distances of 0.5-5.0 cm from the tumour. The tumour glycoproteins were divided almost equally between three anionic types, sulphomucin, sialomucin and mixed sialomucin and sulphomucin. All showed a reduction in staining for side chain O-acylated sialic acid. In 56% of the tumours, this was accompanied by loss of glycoprotein while, in 44%, abundant mucin was still present. Histochemical examination of the mucosal specimens indicated that in 24.2% the side chain O-acylated sialic acids did not differ from normal. In 41.9% there was a focal change and in 33.9% there was a generalized field reduction in the proportion of side chain O-acyl sialic acids. The latter were subdivided into moderate and severe. Chemical analyses correlated well with the histochemical classification of the mucosal specimens and showed that, on average, the classifications focal and severe field change were not due to sampling error. Forty-five per cent of the cases showed only focal change and 40% only field change. Mucosal specimens associated with 60% of the moderately differentiated tumours showed only focal change while those associated with 75% of well-differentiated tumours showed only field change. Abnormal patterns of staining for side chain O-acylated sialic acids (a) were largely independent of the distance from the tumour, (b) occurred in the presence of a normal pattern of staining for sialomucins and sulphomucins and (c) were associated with 61.4% of the specimens that showed no discernible evidence of histological abnormality. In contrast, only one specimen showed evidence of histological change without a corresponding change in O-acylated sialic acids. The data suggest that abnormal patterns of staining for O-acylated sialic acids may represent premalignant change but their precise significance and specificity requires further studies of non-neoplastic diseases of the colon.

Chemical and histochemical studies of normal and diseased gastrointestinal tract. IV. A comparison of histochemical and chemical methods for the estimation of side chain O-acylated sialic acids.

Statistically significant correlations were obtained between a chemical assay for the proportion of colonic epithelial glycoprotein sialic acids with side chain O-acyl substituents and two histochemical methods, the PBT-KOH-PAS sequence (rs = 0.7485 for N = 31, P = 0.01, one-sided test) and the PAPT-KOH-Bh-PAS procedure (rs = 0.7024 for N = 34). A positive correlation (rs = 0.8654 for N = 30, P = 0.01) was also obtained between the results of the two histochemical procedures. It is concluded that, on average, histochemical observations are a reliable semiquantitative comparative method for the estimation of side chain O-acetylated sialic acids.

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures.

Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Culling et al. (1976), have been devised for the histochemical identification of side-chain O-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiff-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chain vicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chain O-acylated sialic acids.

Histochemical studies of the mechanism of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure.

Histochemical investigations of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure were carried out on tissues containing carbohydrate macromolecules known to produce on periodate oxidation, only sialic acid monoaldehydes or hexosedialdehydes or mixtures of the two. The results indicated that the PAPS reaction is a generalized phenomenon independent of the hydrazine or hydrazide used, the nature of the Schiff reagent or the presence of anionic groups. It is proposed that phenylhydrazine condenses with periodate-engendered sialic acid monoaldehydes to yield the corresponding phenylhydrazone and with periodate-engendered dialdehydes to yield the corresponding morpholine or azido derivatives. Subsequent Schiff treatment results in the reversal of the blockade of sialic acid monoaldehydes but not of the dialdehydes, thus leading to selective Schiff staining of sialic acid residues.

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. I. A comparison between histologically normal colon, colonic tumours, ulcerative colitis and diverticular disease of the colon.

Chemical and histochemical methods were used to compare the epithelial glycoproteins from formalin-fixed surgical specimens of normal human large intestine, colonic tumours, ulcerative colitis and diverticular disease. All the epithelial glycoproteins contained fucose, galactose, glucosamine, galactosamine and, in addition, sialic acids both with and without O-acyl substituents in the side chain and/or at position C4. The glycoproteins of the normal ascending and descending colons differed significantly with respect to the percentage of the sialic acids released following digestion of the de-O-acylated glycoprotein with Vibrio cholera neuraminidase and to the molar fucose-sialic acid ratio. Statistical analysis of the chemical data showed that (a) compared to normal, the sialic acids of the tumour and ulcerative colitis glycoproteins from the descending colon were significantly less substituted in the side chain and at position C4; (b) the O-acetyl substitution pattern of the sialic acids of the ulcerative colitis glycoproteins from the ascending colon and the quantitative composition of the carbohydrate prosthetic groups of the ulcerative colitis glycoproteins from both ascending and descending colons differed from normal; (c) it was not always possible to distinguish between the ulcerative colitis and tumour glycoproteins on the basis of the O-acetyl substitution pattern of their sialic acids; and (d), there were minor differences between normal glycoproteins and those from cases of diverticular disease.

Removal of O-acetylated sialic acids from rat colonic epithelial glycoproteins by cell-free extracts of rat faeces.

Sterile, cell-free, extracts of freshly defaecated Wistar rat faeces in a pH 7.0 "minimal medium" contain neuraminidase(s), capable of removing sialic acids both with and without side-chain substituents from bovine submandibular mucin and rat colonic epithelial glycoproteins, and an esterase which removes O-acetyl substituents from the side chain of sialic acid residues. Studies of the removal of sialic acids from bovine submandibular mucin and rat colonic epithelial glycoproteins indicated that (i) the faecal enzymes removed a greater proportion of the sialic acid of both the de-O-acetylated and native glycoproteins than was removed with Vibrio cholera neuraminidase, (ii) sialic acids were removed more rapidly from de-O-acetylated glycoproteins, and (iii) the resistance to removal of sialic acids was apparently dependent at least in part upon the O-acetyl sialic acid content of the substrate.

Differences between the O-acetylated sialic acids of the epithelial mucins of human colonic tumors and normal controls: a correlative chemical and histochemical study.

Chemical analyses, together with histochemical assessments, were carried out on specimens of adenocarcinoma of the colon and histologically normal colonic epithelium (from resection margins from cases of carcinoma of the colon). In the epithelial glycoproteins of the normal tissue, both chemical and histochemical investigations indicated that the great majority of the sialic acids contained a side-chain O-acyl substituent located at position C8, whereas the side-chain substitution of the sialic acids of tumor glycoproteins was markedly reduced. Chemical analysis of the normal glycoproteins indicated that the great majority of the sialic acids were resistant to digestion with Vibrio cholerae neuraminidase, presumably due to an ester substituent at C4. The sialic acids of the tumor glycoproteins were significantly different from normal, in that they were less resistant to digestion with neuraminidase (p greater than 0.01), and therefore had a lower percentage of substitution at C4 (p greater than 0.01).

A correlative chemical and histochemical study of the O-acetylated sialic acids of human colonic epithelial glycoproteins in formalin fixed paraffin embedded tissues.

Procedures are described for the isolation and identification of epithelial glycoproteins from formalin fixed, paraffin embedded specimens of human large intestine. The side chain O-acetylation patterns of the sialic acids of these glycoproteins were surprisingly similar to those of purified glycoproteins prepared from epithelial cells obtained from the same tissue before fixation. These results were consistent with those obtained by histochemical procedures performed on representative sections taken from the same tissue blocks. The methodology described permits a direct correlation of chemical and histochemical results obtained from the study of colonic epithelial glycoproteins of both normal and diseased tissues. It eliminates some of the difficulties associated with interpretation of the results by either discipline and may provide new information which would be unavailable by either chemistry or histochemistry alone.

A method for the simultaneous estimation of free and ketosidically bound sialic acids.

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.

Differences in chemical composition between the epithelial glycoproteins of the upper and lower halves of rat colon.

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.

Noradrenergic sensitivity of the cerebral cortex after chronic ethanol ingestion and withdrawal.

The effects of both chronic ethanol treatment and 3 days of ethanol withdrawal on the cyclic adenosine 3':5'-monophosphate (cAMP) response of cerebral cortical slices to norepinephrine (NE) were studied. ED50 cAMP responses for each group were determined using graded doses of NE. Chronic ethanol fed rats displayed subsensitivity to NE. There was a 4.3-fold shift of the dose-response curve to the right. Rats chronically fed ethanol and then put on 3 days of ethanol withdrawal developed supersensitivity. There was a 2.4-fold shift of the dose-response curve to the left. There was no difference in the maximal cAMP response observed in either the chronic alcohol or the chronic alcohol three day withdrawal experiments. It is possible that the biphasic modifications of effectors by ethanol ingestion and withdrawal from a subsensitive to a supersenitive is the result of one basic phenomenon.

The demonstration of O-acetylated sialic acids in colonic epithelial glycoproteins.

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.