Determination of N-acetyl- and N-glycolylneuraminic acids in glycoconjugates by reversed-phase high-performance liquid chromatography with ultraviolet detection

Karamanos, Nikos K.; Wikström, B.; Antonopoulos, C.A.; Hjerpe, Anders

doi:10.1016/s0021-9673(01)81521-1

Cited by 50 publications

(32 citation statements)

References 28 publications

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“…Following ion-exchange chromatography on Dowex 50 × 8 (H + type) and Dowex 1 × 8 (HCOO − type), neutral monosaccharides and hexosamines were removed (Reuter and Shauer, 1994). Benzoylation was performed according to the method of Karamanos et al (1990a), using benzoic anhydride in pyridine in the presence of p-dimethylaminopyridine. The per-O-benzoylated derivatives of Neu5Ac and Neu5Gc were carried out on a reversedphase Supelcosil LC-18 column eluted with 67% (v/v) aqueous acetonitrile and detection at 231 nm.…”

Section: Resultsmentioning

confidence: 99%

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Determination and distribution ofN-acetyl- andN-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Tzanakakis

Syrokou

Kanakis

et al. 2006

Biomed. Chromatogr.

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Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.

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Section: Resultsmentioning

confidence: 99%

“…Per-O-benzoylation was performed by the method described by Karamanos et al (1990a). In brief, 100 µL of benzoylation mixture were added to the dry hydrolysate and the obtained mixture was heated at 80°C for 20 min.…”

Section: Introductionmentioning

confidence: 99%

Determination and distribution ofN-acetyl- andN-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Tzanakakis

Syrokou

Kanakis

et al. 2006

Biomed. Chromatogr.

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“…The determination of Adi4S and Adi-6S was performed by HPLC on a Hypersil APS column using 20 mM Na,S04, 10 mM sodium acetate pH 5.0 as eluant [28], after digestion of the samples with chondroitinase AC [29], and of Adi-diS by chromatography on the same column eluted with 100 mM Na2S04, 50 mM sodium acetate pH 5.0 [18]. Detection of hydroxyproline was attempted by the method of Woessner [30] and sialic acids by the HPLC method of Karamanos et al [31]. The presence of sulphate groups on C-2 or C-3 of the uronic acid of Adi-dig was evaluated by the periodate/thiobarbituric acid reaction [32].…”

Section: Methodsmentioning

confidence: 99%

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern

Karamanos¹,

Aletras²,

Tsegenidis³

et al. 1992

European Journal of Biochemistry

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Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had M , 3.5 x lo5, contained on average six oversulphated chondroitin sulphate chains (M, 4 x lo4) bound on a polypeptide of M , 2.8 x lo4, and ohgosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), a-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2 -deoxy-3-O-(r-~-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (ddi-4s) and disulphated disaccharides (ddi-diS)[9O0h 2-acetamido-2-deoxy-3-O-(x-~-threo-4-enopyranosyluronic acid 2/3-~ulphate)-~-galactose 6-sulphate (Adi-diSD) and 10% 2-acetamido-2-deoxy-3-O-(~-~-threo-4-enopyranosyluronic acid 213-sulphate)-D-galactose 4-sulphate (ddi-diS,)] as the major disaccharides, significant amounts of trisulphated disaccharides (ddi-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(x-~-threo-4-enopyranosyluronic acid)-r>-galactose 6-sulphate (ddi-6s) and 2-acetamido-2-deoxy-3-O-(r-~-threo-4-enopyranosyluronic acid)-~-galactose (ddi-0s). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches. It has been previously described that squid skin contains two major types of proteoglycans 11, 21. One type contains only chondroitin chains and is composed of two populations Corre.sp,oitdence 10

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“…It has in this context been suggested that Sia play an important biological role, masking and protecting cancer cells by cell-bound immune reactions. Such elevated Sia levels in biological samples can be demonstrated by CE, following depolymerization under mild acidic conditions (Karamanos et al, 1990). This analysis may provide useful information on cancer development and progression Paszkowska et al, 1998;Diamantopoulou et al, 1999), although no such analysis has yet been established as clinical routine.…”

Section: Diagnostic and Biopharmaceutical Importance Of Gags/pgs Analmentioning

confidence: 99%

Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance

Karamanos

Hjerpe

1999

Biomed. Chromatogr.

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Proteoglycans are key biological macromolecules that, via their glycan constituents, participate and regulate several cellular events and physiopathological processes. Refined structures of their highly anionic glycan chains, involving sulphation pattern and uronic acid distribution through the polymeric chain, determine the interactions of proteoglycans with matrix effector molecules and are responsible for numerous effects. Analysis and structural characterization of glycans are, therefore, essential in understanding the biological functions of proteoglycans. Capillary electrophoresis with its high resolving power and sensitivity may successfully be used for the fine chemical characterization of components present in low amounts and to overcome limitations due to low amount/volume of biologic samples available. This technique is also friendly to the user and to the environment, since a very small amount of solvents (a few µL) is required. In this review the strategies used to analyse and characterize the structure of glycan chains of proteoglycans are summarized. They involve capillary electrophoretic analysis of depolymerized acidic glycan chains using specific enzymes and analysis of intact chains. The importance of this type of analysis in biologic samples and tissues and its possible diagnostic and biopharmaceutical use are also discussed. Copyright © 1999 John Wiley & Sons, Ltd.

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Determination of N-acetyl- and N-glycolylneuraminic acids in glycoconjugates by reversed-phase high-performance liquid chromatography with ultraviolet detection

Cited by 50 publications

References 28 publications

Determination and distribution ofN-acetyl- andN-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Determination and distribution ofN-acetyl- andN-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern

Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance

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