Quantitation of proteoglycans as glycosaminoglycans in biological fluids using an alcian blue dot blot analysis

Abstract

A method for quantitation of intact proteoglycans as GAGs in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published previously (S. Björnsson, Anal. Biochem. 210, 282-291, 1993). The method is based on the specific interaction between 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 microgram of glycosaminoglycan (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assay is now described in which the Alcian blue-GAG complexes are collected on a polyvinylidene fluoride membrane, by filtration in a dot blot apparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 microliter of sample and has a measuring range of 10-800 ng of GAG, corresponding to a concentration of 1-80 mg/liter, suitable for proteoglycans in biological fluids. The procedures 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 scanning was the most important source of within-run and between-run imprecision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major source of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as CV (SD/mean . 100) was 13.0% at 8 mg/liter. The response of the assay was identical for six different commercial preparations of GAGs (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin) despite differences in degree of sulfation known to exist. There was no positive or negative interference by blood plasma, apart from a slight negative interference on the quantitation of heparan sulfate. Analysis of 319 paired blood plasma and urine specimens from hospitalized patients showed a variation of plasma GAGs of 0.1-17.6 and urine-GAGs of 0.0-45.6 mg/liter. There was no correlation between plasma and urine GAG concentrations.