An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes.

The effect of cytokines (interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor alpha) and several anti-arthritic drugs on glycosaminoglycan synthesis and secretion into medium by bovine articular chondrocytes was examined. Sulfated glycosaminoglycans (S-GAG) were measured by a modified 1,9-dimethylmethylene blue (DMB) dye binding assay. Hyaluronate (HA) was measured by an inhibition ELISA based on specific binding to a proteoglycan. All three cytokines caused a dose-dependent decrease in S-GAG production and a dose-dependent increase in HA production. Non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin, naproxen, and piroxicam, 1 microM) could not reverse the effect of IL-1 alpha on inhibiting S-GAG and stimulating HA synthesis. The anti-inflammatory steroid (dexamethasone, 1 microM) depressed HA synthesis by 50-70% in the absence or presence of IL-1 alpha. Dexamethasone depressed S-GAG synthesis by 20-30% in the absence or presence of IL-1 alpha. Therefore, none of the tested anti-rheumatic drugs reversed the cytokine mediated changes in glycosaminoglycan synthesis by bovine chondrocytes.