Quantitative analysis of chondroitin sulfate isomers in intervertebral disk chondrocyte culture using capillary electrophoresis

• Published in: Journal of Veterinary Medical Science Vol. 63; no. 9; pp. 1039 - 1043
• Main Authors: Maeda, S. (University of Florida, Gainesville (USA)), Miyabayashi, T, Yamamoto, J.K, Roberts, G.D, Lepine, A.J, Clemmons, R.M
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 01.09.2001; Japan Science and Technology Agency
• Subjects: Animals; capillary electrophoresis; CELL CULTURE; Cells, Cultured; chondrocyte; Chondrocytes - chemistry; Chondrocytes - metabolism; chondroitin sulfate; Chondroitin Sulfates - isolation & purification; Chondroitin Sulfates - metabolism; Dogs - metabolism; ELECTROPHORESIS; Electrophoresis, Capillary - methods; Electrophoresis, Capillary - veterinary; GLYCOSAMINOGLYCANS; Intervertebral Disc - chemistry; Intervertebral Disc - metabolism; LIVESTOCK; QUANTITATIVE ANALYSIS
• ISSN: 0916-7250; 1347-7439
• DOI: 10.1292/jvms.63.1039

Chondroitin sulfate (CS) isomers, 6-sulfate (CS6) and 4-sulfate (CS4), change their ratio to each other in cartilaginous tissues with aging. In this study, a quantitative measurement method of CS6 and CS4 was developed, using capillary electrophoresis (CE). Various buffer solutions, pH, and digestion times were studied, and the use of 0.1 M Tris-HCl at pH of 8.0 allowed the isolation of CS6 and CS4 from CS most efficiently when combined with chondrotinase ABC at a concentration of 1 mU/jUg of the substrate during a 3 hr digestion period. Amounts of newly synthesized CS6 and CS4 in the interveitebral disk chondrocyte three-dimensional culture were quantified by this method after the proteoglycans were extracted by equilibrium density centrifugation.