Optimization of Automated Dimethylmethylene Blue Assay for Quantification of Pediatric Urine Samples

The metachromatic dye dimethylmethylene blue is used to quantify total glycosaminoglycans in urine. Understanding the interaction of dimethylmethylene blue with glycosaminoglycans is pertinent to optimize the assay procedure depending on the type of sample and interpret the findings meaningfully. The present spectrophotometric study determined the optimum sample-to-dye ratio, primary wavelength for measuring absorbance, after studying the interaction of two different chondroitin sulfate species (unfractionated chondroitin sulfate from bovine trachea vs. chondroitin sulfate oligosaccharide with degree of polymerization of 12, from shark cartilage) with dimethylmethylene blue. Respective dye-glycosaminoglycan complexes of the two chondroitin sulfate species showed significantly different absorbance maxima, while that of the chondroitin sulfate oligosaccharide was closer to absorbance maxima of urine glycosaminoglycans. The chondroitin sulfate oligosaccharide showed relatively less stable absorbance readings at higher concentrations in the reaction volume. Furthermore, the chondroitin sulfate reference materials exhibited differences in the linearity of standard curves and hence parallelism. Based on the findings, the method was semiautomated on Beckman Coulter D✕C 700 biochemistry analyzer using the chondroitin sulfate oligosaccharide as the standard. The urine glycosaminoglycan concentration obtained was slightly lower but reasonably close to that obtained through the External Quality Assurance (EQA) scheme administrated by ERNDIM (European Research Network, Inherited Disorders of Metabolism). The findings of the present study can be used to guide the dimethylmethylene blue assay optimization, redevelopment efforts, and harmonization across laboratories. The chondroitin sulfate oligosaccharide is better than the unfractionated chondroitin sulfate from bovine trachea due to its absorbance maxima closer to urine glycosaminoglycans. On the other hand, unfractionated chondroitin sulfate exhibit poor parallelism leading to falsely lower urine glycosaminoglycan levels.