Biochemical analysis of rabbit articular cartilage using an amino acid analyzer

Abstract

The automated amino acid analyzer has been used to provide a simple, rapid and accurate means of assaying the glycosaminoglycan (GAG) and collagen content, and collagen type of connective tissues. The analysis of rabbit articular cartilage using this method is reported. Cartilage slices taken from the tibial plateaus of rabbits were dried and weighed. A comparison study showed that oven-drying (110 degrees C for 24 hours) was more effective in dehydrating cartilage than vacuum dessication for 96 hours. This drying temperature could be maintained for 24 hours without destroying hexosamine. The dried samples were hydrolyzed for 4 hours in 4 N HCl at 100 degrees C; these conditions were shown to give virtually 100% release of galactosamine from purified chondroitin-6-sulphate with minimal degradation of the hexosamine. The 4-hour hydrolysates were assayed for galactosamine and glucosamine using the amino acid analyzer. From these values the amount of chondroitin sulphate and keratan sulphate were calculated. An aliquot of the 4-hour hydrolysate was hydrolyzed further in 6 N HCl for 24 hours. This destroyed hexosamine but permitted the quantitation of hydroxyproline and hydroxylysine using the amino acid analyzer. Collagen content was calculated from the hydroxyproline value and the hydroxylysine to hydroxyproline ratio was used to determine the collagen type.