The assessment of chondrocyte proteoglycan metabolism using molecular sieve column chromatography as compared to three commonly utilized techniques.
OBJECTIVE
Various methods have been used to quantify [35S] sulfate incorporation into proteoglycans and separate this from unincorporated [35S] 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) precipitation, potassium acetate (KAc) precipitation, rapid filtration, modified dye-binding (AB) technique to molecular weight exclusion chromatography (PD-10) as techniques to quantitate chondrocyte proteoglycan metabolism.


METHODS
Each of the first three techniques was compared to the PD-10 technique as a 'gold standard' based on this technique's accuracy, precision and repeatability in molecular weight exclusion utilizing sieve chromatography. Articular cartilage was harvested, cultured and labeled with [35S] sulfate in a routine manner. Seventeen samples were processed and each was analyzed by the four different methodologies. Linear regression analysis was used to predict the linear relationship of PD-10 chromatography to the other methods (CPC, KAc, and AB) in the amount of proteoglycan synthesis in a pulse chase experiment.


RESULTS
The KAc and AB methods had a significant linear relationship to the PD-10 method but the CPC did not. The AB technique was much easier than the KAc method to complete although the relative economics were similar per sample, when capital equipment costs were not considered.


CONCLUSIONS
Either the KAc or AB methods compared favorably to the PD-10 method, although the AB procedure was much easier, and was more precise and more accurate. The CPC method did not compare favorably to the PD-10 method in precision or accuracy although it was easier and cost effective.
Department of Clinical Sciences, Colorado State University, Fort Collins 80523, USA.
D D
DD
Frisbie
C E
CE
Kawcak
G W
GW
Trotter
C W
CW
McIlwraith
D000423:Alcian Blue; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002594:Cetylpyridinium; D011232:Chemical Precipitation; D002845:Chromatography / Q000379:methods*; D046508:Culture Techniques; D006736:Horses; D019347:Potassium Acetate; D011509:Proteoglycans / Q000032:analysis / Q000378:metabolism*; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000378:metabolism
D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D002594:Cetylpyridinium; D019347:Potassium Acetate; D000423:Alcian Blue