Sialic acid detection

Paper chromatography was used in the early days of sialic acid detection (Blix and Lindberg 1960, Gottschalk 1960) and has now been superceded by the more sensitive thin-layer chromatographic methods. Some paper chromatographic systems and the corresponding references are noted in Table 2. 

Hydrophilic interaction chromatography on Asahipak NH2P or Excel-pak CHA-P44 with pulsed amperometric detection has been used to fractionate malto-oligosaccharides.266 The Asahipak NH2P is a polyvinyl alcohol support with a polyamine bonded phase, and the Excelpak is a sulfonated polystyrene in the Zn+2 form. Amine adsorption of sialic acid-containing oligosaccharides was performed on a Micropak AX-5 column (Varian) using acetonitrile-water-acetic acid-triethylamine.267... 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

The sensors discussed so far are based on ligands covalently bound to the polymer backbone. Other methods of detection - often referred to as mix and detect methods - work by simple noncovalent incorporation of the polymer with the ligand of interest. Reichert et al. generated liposomes of polydiacetylene with sialic acid for the same purpose of detection as Charych s surface-bound polymers, but realized that covalent functionalization of the polymer was not necessary [17]. Through simple mixing of the lipid-bound sialic acid with the polymer before sonication and liposome formation, they were able to form a functional colorimetric recognition system (Fig. 8). 

Reichert A, Nagy JO, Spevak W, Charych D (1995) Polydiacetylene liposomes functionalized with sialic-acid bind and colorimetrically detect influenza-virus. J Am Chem Soc 117 829-830... 

Scientists recently announced that a sialic acid NeuSGc (A-glycolyneur-aminic acid), normally present in red meat but not in humans, may be incorporated into the tissues of meat consumers and accumulate there, leading the consumers bodies to produce antibodies directed against it and which destroy a body s own tissues. As the occurrence of NeuSGc was also detected in the courses of cancers, there is now a discussion on whether it may contribute to development of cancer diseases as well as cardiovascular disease and diabetes. The possible connections have not been proven so far (Tangvoranuntakul et ah, 2003), yet it may turn out that diet style, i.e.. 

C. Fahr and R. Schauer, Detection of sialic acids and gangliosides with special reference to 9-0-acetylated species in basilomas and normal human skin, J. Invest. Dermatol., 116 (2001) 254-260. 

Figure 12.4 Colorimetric detection of influenza virus using polymerized liposomes containing sialic acid, (a) Photograph of liposomes to which have been added increasing amounts (from left to right) of influenza virus. Liposomes were 1 and 95% 5. To each well was added the following amounts of influenza virus (left to right) 0, 8, 16, and 32 hemagglutinin units (HAU). Reprinted from Charych et al. (1996). Copyright 1996 Elsevier Science. (See color insert.)...

In both of these cases, the ligand (sialic acid) for the analyte of interest (influenza vims) was covalently linked to the PDA backbone generated upon photopolymerization. Functional sensors based on ligands that are noncovalently incorporated into liposomes have also been reported (Charych et al. 1996 Pan and Charych 1997). Mixed liposomes as well as mixed thin films on glass containing a combination of the ganglioside GMl and diacetylene lipids detect the presence of cholera toxin, a protein that binds to GMl. 

Polythiophenes functionalized with monosaccharides have been evaluated for their ability to detect the influenza virus and E. coli (Baek et al. 2000). Copolymers of thiophene acetic acid 10 and carbohydrate-modified thiophenes 11 have been prepared via iron(III) chloride mediated polymerization. Addition of influenza virus to a sialic acid containing copolymer resulted in a blue shift of the polymer absorption maximum, resulting in an orange to red chromatic transition. Mannose-containing polythiophenes underwent color changes upon the addition of the lectin ConA or E. coli cells that contain cell surface mannose-binding receptors. A similar biotinylated pol5hhiophene afforded a streptavidin responsive material (Paid and Leclerc 1996). 

A further method for estimation of sialic acid using periodic acid has been reported by Massamiri and coworkers.121 Fonnaldehyde produced by mild, oxidative cleavage of the D-en/f/iro-glycerol-l-yl side-chain of Neu can be detected by 3-methyl-2-benzothiazolinone hydrazone, which yields a green-blue color (absorbance maximum 625 nm). This test, which is slightly more sensitive than the thiobarbi-... 

In contrast to this work on whole brain, Kemp and Stoolmiller138 used cultured mouse-neuroblastoma cells for their study of incorporation of [ lH]2-acetamido-2-deox> -D-mannose into the sialic acid moiety of gangliosides. They were able to demonstrate clearly the precursor-product relationship among CMS, GM2, and GM1. They found that cultured NB41A cells incorporated [3H]2-acetamido-2-deoxy-D-man-nose into the sialic acid moiety of GM3 in less than 10 minutes. Labeled GM2 was not detected in cells incubated for less than 30 minutes, and measurable activity did not appear in G I1 until alter 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of ganglio-tvpe gangliosides proceeds by way of GM3 —> GM2 — GM1. 

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