QUANTITATION OF PROTEOGLYCANS AS GLYCOSAMINOGLYCANS IN BIOLOGICAL-FLUIDS USING AN ALCIAN BLUE DOT-BLOT ANALYSIS

A method for quantitation of intact proteoglycans as GAGs in biologica l fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of t issues has been published previously (S. Bjornsson, Anal. Biochem. 210 , 282-291, 1993). The method is based on the specific interaction betw een sulfated polymers and the tetravalent cationic dye Alcian blue at pH 1.5 in 0.4 M guanidine-HCl and in the presence of 0.25% Triton. The absorbance assay has a measuring range of 1-20 mu g of glycosaminogly can (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assa y is now described in which the Alcian blue-GAG complexes are collecte d on a polyvinylidene fluoride membrane, by filtration in a dot blot a pparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 mu l of sample and has a measuring range of 10-800 ng of GAG, corresponding to a concentration of 1-80 m g/liter, suitable for proteoglycans in biological fluids. The procedur es for chemistry, scanning, densitometry, and curve fitting were each evaluated separately. The error contributed by chemistry accounted for a minor portion of the imprecision. The imprecision contributed by sc anning was the most important source of within-run and between-run imp recision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major s ource of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as C V (SD/mean . 100) was 13.0% at 8 mg/liter. The response of the assay w as identical for six different commercial preparations of GAGs (chondr oitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulf ate, heparan sulfate, and heparin) despite differences in degree of su lfation known to exist. There was no positive or negative interference by blood plasma, apart from a slight negative interference on the qua ntitation of heparan sulfate. Analysis of 319 paired blood plasma and urine specimens from hospitalized patients showed a variation of plasm a GAGs of 0.1-17.6 and urine-GAGs of 0.0-45.6 mg/liter. There was no c orrelation between plasma and urine GAG concentrations. (C) 1998 Acade mic Press.