chondroitin-sulfates and dansyl-hydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.

Topics: Calibration; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Dansyl Compounds; Disaccharides; Escherichia coli; Glycosaminoglycans; Hyaluronic Acid; Hydrazines; Monosaccharides; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Glycosaminoglycan (GAG) was extracted from monkey periodontium, consisting of gingiva, periodontal ligament, alveolar bone and cementum, and from dental pulp and dentin by digestion with Pronase E. Unsaturated disaccharide isomers formed by chondroitinase AC digestion from chondroitin sulfate were labeled with dansylhydrazine and analyzed by high-performance liquid chromatography. These tissues showed different molar ratios of the unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lowest in the dental pulp, followed by the gingiva, periodontal ligament, dentin, alveolar bone, and cementum, in that order. It was greater in the calcified than in the uncalcified tissues.

Topics: Alveolar Process; Animals; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dansyl Compounds; Dental Cementum; Dental Pulp; Dentin; Female; Gingiva; Histocytochemistry; Hydrazines; Isomerism; Macaca fascicularis; Periodontal Ligament; Periodontium; Pronase

Topics: Animals; Chondroitin; Chondroitin Sulfates; Chromatography, Thin Layer; Dansyl Compounds; Disaccharides; Hydrazines; Rabbits

Chondroitin sulfate isomers (ChS isomers) were digested with chondroitinase ABC, and the unsaturated disaccharides produced were converted into fluorescent dansylhydrazine derivatives and analyzed by HPLC. The proposed method was applied to the determination of relative amounts of ChS isomers in normal human urine and analytical results showed that chondroitin-6-sulfate and chondroitin-4-sulfate were excreted in large amounts. Moreover, this method was used for the identification of urinary ChS isomers which were extracted from a cellulose acetate strip after carrying out electrophoresis.

Topics: Chondroitin; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dansyl Compounds; Disaccharides; Fluorescent Dyes; Humans; Hydrazines; Indicators and Reagents; Isomerism; Spectrometry, Fluorescence