Quantitation of glycosaminoglycan metabolism in anatomically intact articular cartilage of the mouse patella: in vitro and in vivo studies with 35S-sulfate, 3H-glucosamine, and 3H-acetate

Abstract

We investigated the usefulness of the whole mouse patella to quantitate the synthesis of the glycosaminoglycan (GAG) backbone and its sulfation by intact murine articular cartilage, both in vitro and in vivo. Using 35S-sulfate, 3H-glucosamine, or 3H-acetate as precursors of GAG synthesis, it was found that more than 90% of the incorporated radioactivity was confined to the patellar cartilage layer compared to the whole patella. Overnight papain digestion was enough to liberate more than 95% of the incorporated radiolabels, except for 3H-acetate for which 15-25% was not digestible. Comparison of radioactivity in the patella and that in quantitatively isolated GAGs revealed that for 35S-sulfate incorporation studies the whole patella can be used as a reliable measure for sulfated GAG synthesis. The situation was different for the GAG backbone precursors 3H-glucosamine and 3H-acetate; more than 50% of the 3H labels were incorporated into compounds other than GAGs or non-covalently associated with matrix components. Hence, in studying GAG-backbone metabolism, polysaccharides must be isolated quantitatively from cartilage. In vivo studies made it clear that both 35S-sulfate and 3H-glucosamine are incorporated into patellar GAGs in amounts high enough to enable proper quantitation and that the route of administration (intraperitoneally or intravenously) is of minor importance. Due to its low specificity for cartilage GAGs, 3H-acetate is not suitable for such studies.