THE ASSESSMENT OF CHONDROCYTE PROTEOGLYCAN METABOLISM USING MOLECULAR-SIEVE COLUMN CHROMATOGRAPHY AS COMPARED TO 3 COMMONLY UTILIZED TECHNIQUES

Objective: Various methods have been used to quantify [S-35] sulfate i ncorporation into proteoglycans and separate this from unincorporated [S-35] sulfate. The methods currently used have not been compared for accuracy or precision, ease of completion and relative economics. This experiment evaluated and compared cetylpyridinium chloride (CPC) prec ipitation, potassium acetate (KAc) precipitation. rapid filtration, mo dified dye-binding (AB) technique to molecular weight exclusion chroma tography (PD-10) as techniques to quantitate chondrocyte proteoglycan metabolism. Methods: Each of the first three techniques was compared t o the PD-10 technique as a 'gold standard' based on this technique's a ccuracy, precision and repeatability in molecular weight exclusion uti lizing sieve chromatography. Articular cartilage was harvested, cultur ed and labeled with [S-35] sulfate in a routine manner. Seventeen samp les were processed and each was analyzed by the four different methodo logies. Linear regression analysis was used to predict the linear rela tionship of PD-10 chromatography to the other methods (CPC, KAc, and A B) in the amount of proteoglycan synthesis in a pulse chase experiment . Results: The KAc and AB methods had a significant linear relationshi p to the PD-10 method but the CPC did not. The AB technique was much e asier than the KAc method to complete although the relative economics were similar per sample, when capital equipment costs were not conside red. Conclusions: Either the KAc or AB methods compared favorably to t he PD-10 method, although the AB procedure was much easier, and was mo re precise and more accurate. The CPC method did not compare favorably to the PD-10 method in precision or accuracy although it was easier a nd cost effective.