Studies on the quantification of proteoglycans by the dimethylmethylene blue dye-binding method. Specificity, quantitation in synovial lavage fluid, and automation.

The dimethylmethylene blue (DMMB) dye-binding technique is widely used for the quantification of sulfated glycosaminoglycans (sGAG) and proteoglycans. We conducted further studies on this technique in our laboratory and found that concentrations of DNA and RNA in excess of 20 micrograms/ml interfered negatively with the detection of sGAGs; interference was eliminated by using DNase and RNase. Hyaluronan at 40 micrograms per ml did not interfere with the detection of sGAG. However, because of the higher concentrations of hyaluronan in synovial lavage fluid, it was necessary to treat this fluid with Streptomyces hyaluronidase in order to quantify sGAG. The DMMB assay was automated with a laboratory work station and compared to the standard method.

[1]  M. Lark,et al.  Recombinant human interleukin-1 beta-induced increase in levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid. , 1992, Arthritis and rheumatism.

[2]  A. Ratcliffe,et al.  Cartilage proteoglycan depletion in acute and chronic antigen-induced arthritis. , 1989, Arthritis and rheumatism.

[3]  G. Carroll.  Spectrophotometric measurement of proteoglycans in osteoarthritic synovial fluid. , 1987, Annals of the rheumatic diseases.

[4]  B. Caterson,et al.  Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. , 1983, The Journal of biological chemistry.

[5]  R. L. Goldberg,et al.  An improved method for determining proteoglycans synthesized by chondrocytes in culture. , 1990, Connective tissue research.

[6]  N. Blumenkrantz,et al.  New method for quantitative determination of uronic acids. , 1973, Analytical biochemistry.

[7]  D Heinegård,et al.  Structure and biology of cartilage and bone matrix noncollagenous macromolecules , 1989, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  P. Roughley,et al.  The immunologic detection and characterization of cartilage proteoglycan degradation products in synovial fluids of patients with arthritis. , 1987, Arthritis and rheumatism.

[9]  D. Templeton.  The basis and applicability of the dimethylmethylene blue binding assay for sulfated glycosaminoglycans. , 1988, Connective tissue research.

[10]  V. Hascall,et al.  [45] Proteoglycans: Isolation and Characterization , 1982 .

[11]  D. Heinegård,et al.  A method for the quantification of cartilage proteoglycan structures liberated to the synovial fluid during developing degenerative joint disease. , 1985, Scandinavian journal of clinical and laboratory investigation.

[12]  E. Thonar,et al.  Quantification of keratan sulfate in blood as a marker of cartilage catabolism. , 1985, The Journal of rheumatology.

[13]  R W Farndale,et al.  A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. , 1982, Connective tissue research.

[14]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.