Sialic acid histochemical

D. Volz, P. E. Reid, C. M. Park, D. A. Owen, and W. L. Dunn, Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulfate ester. I. Methods based upon the selective periodate oxidation of sialic acids, Histochem. J., 19 (1987) 249-256 Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulfate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction, Histochem. J., 19 (1987) 257-263. 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

Histochemically, mucopolysaccharides outlining the epidermal cells can easily be seen (B28, G9, S23, S25, W19). In addition, Flesch and Esoda (F14) found evidence of a glycoproteolipid in normal stratum corneum and increased amounts in psoriatic scale. They could find no sialic acid in the material, it was resistant to hyaluronidase, and they proposed that a lack of proper breakdown of this material was important in the pathophysiology of the disease. 

Acetylation of mucin-bound and membrane-bound sialic acids makes them more resistant towards sialidase action [8,33,245,853,854]. This may be one of the reasons why intestinal mucins, especially of the colon, are often O-acetylated (ref. [245], and section 8.4.2). The high level of 0-acetylation of sialic acids observed in the endothelia of blood vessels, e.g, in liver, detected by histochemical methods using influenza C virus hemagglutinin, is assumed to have a similar function [234,235,730]. 

Periodate in aqueous perchloric acid oxidizes the glycuronic acid residues of glycosaminoglycuronans with sufficient selectivity that the reagent has been proposed for histochemical classification. Several other histochemical applications of periodate for the detection of carbohydrates and sialic acids specifically,have been described. 

The findings made with tissue fractionation techniques conflict with histochemical studies of Koenig, who claims that gangliosides are mainly in lysosomes. These discrepancies may result from a variety of causes (1) the staining method used to identify the glycolip-id may be nonspecific and stain sialic acid-containing proteins (2) brain lysosomes may not be separated from microsomes and (3) possibly most of the glycoprotein is in the microsomes, but only that in the lysosomes is detectable by histochemical techniques. 

The chemistry and analysis of sialic acids have been reviewed. Picomole quantities of sialic acids have been measured by the fluorescence produced by the periodate-oxidized acid in the thiobarbituric acid reaction. " Contamination of the sialic acids with 2-deoxy-D-eryt/iro-pentose (derived from cellular material) could be detected by a downfield shift of the excitation maximum. Fluorescent derivatives are also produced when free sialic acids react with pyridoxamine, a procedure that compares favourably with the thiobarbituric acid reaction for determining sialic acids. Keto-acids e.g. pyruvic acid) interfere with the determination, but 2-deoxy-D-arabmo-hexose and 2-deoxy-D-c/-ytliro-pentose do not. A nonfading chromophore is produced when DMSO is used instead of n-butanol in the thiobarbituric acid ssay for sialic acids. A new histochemical method for the visualization and identification of unmodified or 0-acylated sialic acids has been reported. ... 

Some histochemical evidence for sialic acids in lizards, teiids and dibamids (Lacertilia, Teiidae, and Ophiadae), cobras and vipers Elipidae and Viperidae) has been reported, but isolation of sialic acids is still outstanding (Lopes et al. 1973, 1974). 

Sialic acid is apparently ubiquitous in tissues, but is only demonstrable, histochemically, in certain well defined areas. Principally these are the epithelial secretions, although basement membranes (e.g. of the kidney glomeruli), are clearly delineated by those techniques appropriate for the demonstration of sialic acids. Sialic acids occurring in other glycoproteins (e.g. plasma proteins, cell membrane components) may contribute to the diffuse background staining, but due to their low concentration in tissue sections are not readily defined, on a... 

Techniques for the demonstration, investigation and identification of sialic acids, are based primarily upon the presence of two, histochemically reactive structural features in the molecule. These are the carboxyl group located at... 

An alternative to methods based upon the chemical reactions of carboxyl groups or vicinal diols is the use of lectins labelled with peroxidase or fluorescent tags. Such procedures have been little exploited in the histochemical investigation of sialic acids and indeed have specificity problems peculiar to the mechanism of their reaction but they would appear to be a promising approach to the identification of sialic acids. 

Digestion with a neuraminidase is the most commonly used histochemical technique for the identification of sialic acids and when positive is the most specific. In this procedure tissue sections are incubated with the neuraminidase and the intensity of their staining with Alcian blue pH 2.5 is compared to that of a serial section incubated under identical conditions, with the buffer but without neuraminidase and also to that of a serial section subjected to no pretreatment. Reduction in staining, in the enzyme treated section only, is evidence for the presence of sialic acids but a negative result (no reduction in staining) cannot be... 

The C.E.C. methods developed by Scott and his colleagues (Kelly et al. 1963, Scott and Dorling 1965) whereby a selective staining of various anionic groups is achieved by the use of staining baths containing various concentrations of salts are not in routine use for the histochemical identification of sialic acid. 

An example of the possibilites of the use of these techniques in the future is the paper by Stoward, Spicer, and Miller (1980) in which they use a peanut lectin— PAPS technique to identify galactose as the sub-terminal group, following removal of sialic acid with neuraminidase in, for example, the secretory glycoprotein in mouse duodenal cells. With the number of lectins now available commercially, some of which can be obtained with a PAPS label already attached, it can be anticipated that the histochemical sequencing of carbohydrate chains is well within the realm of possibility in the near future. 

Histochemical studies have revealed the presence of sialic acid in cephalic glands of several reptilian species, Ameiva ameiva (Lacertilia, Teiidae), Micrurus corallinus corallinus (Ophiadea, Elapidae), and o/Zi-rops jararaca (Ophiadia, Viperidae) (Lopes and Valeri, 1972 Lopes et al.y 1973 1974). 

The lysosomes of L cells contain considerable amounts of bound sialic acid, despite the presence of neuraminidase in these organelles. The high level might be expected because lysosomal membrane is derived in part from the plasma membrane (de Duve and Wattiaux, 1966). The phospholipids and cholesterol contents of lysosomes and plasma membrane of rat liver are similar. (Thines-Sempoux, 1967). Histochemical studies of Rambourg (1969) indicate that the lysosomes are rich in carbohydrate. It has also been shown that radioactive d-... 

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