C.A. Antonopoulos

Determination of sulphated disaccharides from chondroitin suphates by high-performance liquid chromatography

A sensitive method for the determination of chondroitin 4- and 6-sulphate is presented. After chondroitinase digestion of the chondroitin sulphate preparations, the obtained disaccharides are separated on a weak anion-exchange resin in a high-performance liquid chromatography system. The method was used to study 4-sulphate to 6-sulphate ratios in chondroitin sulphates prepared from bovine nasal cartilage and human nucleus pulposus. The results show clearly that these two preparations contain considerable amounts of both isomers.

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection.

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.

Separation and quantitative determination of galactosamine and glucosamine at the nanogram level by sulphonyl chloride reaction and high-performance liquid chromatography

Abstract Two chromatographical systems for the separation and determination of glucosamine and galactosamine are described. In the more rapid and less sensitive of the two systems, the hexosamines are treated with toluene sulphonyl chloride and subsequently separated in a reversed-phase system. In this mode 0.4–50 μg of the hexosamine are chromatographed within 10 min. In the second and more sensitive system, the hexosamine are treated with Dns-Cl and subsequently chromatographed in a similar reversed-phase column. With this method, the separation of 0.02–2 μg is possible within 25 min. When internal standard was used, the procedure was lengthened by ca . 10 min. Both determinations can also be used when there are large differences in the relative amounts of the two hexosamines.

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

Abstract A rapid and sensitive method for the determination of sialic acids is described. The measurement is based on isocratic high-performance liquid chromatography, whereby N-acetylneuraminic and N-glycolylneuraminic acids are separated. The total amounts of these acids can be determined after hydrolysis and per-O-benzoylation. The conditions for hydrolysis and derivatization were optimized for measurement of these sialic acids in glycoconjugates. The benzoyl derivatives were chromatographed on a reversed-phase column with 67% (v/v) aqueous acetonitrile and the eluted peaks were monitored by UV detection. The method allows the determination of picomole amounts. the reaction was shown to give linear calibration graphs over the entire range tested. i.e. , up to 160 nmol (50 μg) of each of the sialic acids.

Analysis of neutral sugars as dinitrophenyl-hydrazones by high-performance liquid chromatography

Abstract A new method for the separation and determination of neutral sugars as 2,4-dinitrophenyl derivatives is presented. After a simple derivatization procedure at 65°C for 90 min and purification of the reaction mixture from excess of reagents, the sugar derivatives were separated on a LiChrosorb Si 100 column using methanolwater in chloroform as the eluent and UV detection at 352 nm. The yield of the products is affected by several parameters. However, when the proposed conditions are closely adhered to an excellent precision is obtained. Calibration graphs are linear in the range of 0.05–3.35 nmol of each sugar injected. The method was used for the determination of neutral sugars normally found in glycoconjugates, namely fucose, xylose, mannose, galactose and glucose. Hexosamines, uronic acids, alditoles and amino acids do not interfere with the analysis. Thus, the derivatization procedure can be applied directly to unfractionated acid hydrolysate residues of glycoconjugates.

Analysis of dentine glycosaminoglycans using high-performance liquid chromatography

SummaryPuppy dentine was prepared using ultracentrifugation of tooth powder in organic density gradients. The glycosaminoglycans of the obtained tissue fraction were prepared after papain digestion andβ-elimination, using preparative chromatography on DEAE-cellulose and CPC-cellulose. These polysaccharide fractions were analyzed using highly sensitive HPLC procedures. One such HPLC procedure allowed hyaluronic acid to be determined in less than microgram amounts.The glycosaminoglycans thus prepared consisted only of chondroitin-4-sulfate, chondroitin-6-sulfate, and small amounts of highly hybridized dermatan sulfate, while the experiments failed to demonstrate even trace amounts of keratan sulfate, hyaluronic acid or heparan sulfate.

Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide

The polysaccharides of cranical cartilage were isolated by ethanol precipitation after papain digestion and beta-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39 200 on weight basis (Mw) and 31 400 on number basis (Mn).

Extraction and fractionation of proteoglycans from squid skin

The extractability of squid skin proteoglycans with solutions of varying concentrations of guanidine-HCl, urea and SDS was studied; 4 M guanidine-HCl, being the best extractant, removed 95% of the tissue proteoglycans (glycosaminoglycan uronic acid). The proteoglycans in the 4 M guanidine-HCl extract were fractionated by repeated ion exchange and gel chromatography on Sepharose CL-4B to give three main populations, all being present in about equal proportions. Two populations (Kd 0.34 and 0.56) contained only chondroitin (proteochondroitin) and the other (Kd 0.50) only oversulphated chondroitin sulphate (oversulphated proteochondroitin sulphate). Two minor populations, one containing chondroitin and chondroitin sulphate and the other chondroitin sulphate and oversulphated chondroitin sulphate, were also identified.

Isolation and chemical study of the glycosaminoglycans from squid cornea

1. Oversulphated chondroitin sulphate (ca 93% of tissue glycosaminoglycans) with average molecular weight 72,500, chondroitin sulphate (5%) and small amounts of lowsulphated chondroitin sulphate were isolated from squid cornea. 2. The sulphation pattern of oversulphated chondroitin sulphate was delta di-4S (52%), delta di-diSD (28%), delta di-6S (9%) and delta di-OSCS (11%) and that of chondroitin sulphate 49, 1, 20 and 30% respectively. 3. All glycosaminoglycans contained neutral monosaccharides, glucose being the predominant neutral monosaccharide in oversulphated chondroitin sulphate and chondroitin sulphate and fucose in low-sulphated chondroitin sulphate. 4. Although L-iduronic acid was not detected, the digestion of oversulphated chondroitin sulphate with chondroitinases ABC and AC gave unexpected results.

Study of the glycosaminoglycans from squid skin

Abstract 1. 1. Chondroitin and highly sulphated chondroitin with average molecular weights 80,000 and 40,000, respectively, were isolated from squid skin. 2. 2. l -Iduronic acid was not detected, and the digestion with chondroitinase ABC and AC gave different results. 3. 3. Both glycosaminoglycans contain neutral sugars, glucose being the predominant neutral sugar.