Indika Neluwa-Liyanage R, Peiris Hemantha, Deutz Nicolaas E P, Perera Rasika
Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka.
Center for Translational Research in Aging & Longevity, Texas A&M University, College Station, TX USA.
Indian J Clin Biochem. 2024 Oct;39(4):519-528. doi: 10.1007/s12291-023-01173-w. Epub 2023 Dec 18.
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.
异染染料二甲基亚甲蓝用于定量尿液中的总糖胺聚糖。了解二甲基亚甲蓝与糖胺聚糖的相互作用，对于根据样品类型优化检测程序并对结果进行有意义的解释至关重要。本分光光度研究在研究了两种不同硫酸软骨素（来自牛气管的未分级硫酸软骨素与来自鲨鱼软骨的聚合度为12的硫酸软骨素寡糖）与二甲基亚甲蓝的相互作用后，确定了最佳的样品与染料比例、测量吸光度的主波长。两种硫酸软骨素的各自染料 - 糖胺聚糖复合物显示出明显不同的最大吸光度，而硫酸软骨素寡糖的最大吸光度更接近尿液糖胺聚糖的最大吸光度。在反应体积中较高浓度下，硫酸软骨素寡糖的吸光度读数相对不太稳定。此外，硫酸软骨素参考物质在标准曲线的线性以及因此的平行性方面表现出差异。基于这些发现，该方法在贝克曼库尔特DxC 700生化分析仪上以硫酸软骨素寡糖为标准进行了半自动操作。获得的尿液糖胺聚糖浓度略低，但与通过欧洲代谢性遗传疾病研究网络（ERNDIM）管理的外部质量保证（EQA）计划获得的浓度相当接近。本研究结果可用于指导二甲基亚甲蓝检测的优化、重新开发工作以及跨实验室的协调。硫酸软骨素寡糖优于来自牛气管的未分级硫酸软骨素，因为其最大吸光度更接近尿液糖胺聚糖。另一方面，未分级硫酸软骨素的平行性较差，导致尿液糖胺聚糖水平错误地降低。
