Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans

Background: The relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after Agarose gel electrophoresis.

Methods: Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. The results were compared to those obtained by densitometry after Agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography.

Results: Densitometry after Agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25x10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. In comparison, DMB assays gave much higher results - up to 200 mg/l and 500x10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB.

Conclusion: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since Other urinary components interfere with the reactions.