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Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans.
Abstract: 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. Conclusions: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions.
Publication Date: 2006-12-14 PubMed ID: 17261277DOI: 10.1016/j.cca.2006.11.025Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper discusses a study which evaluated the reliability of 1,9-dimethylmethylene blue (DMB) dye-binding assays in comparison to agarose gel electrophoresis for quantifying urinary glycosaminoglycans, an important molecule in various biological processes. The results of the study suggested that, despite its simplicity and speed, the DMB test may not be suitable due to interference from other components in mammalian urine.
Methodology and Results
- The study involved the quantification of urinary glycosaminoglycans taken from various species of mammals. This was achieved through the use of three different 1,9-dimethylmethylene blue (DMB) dye-binding assays.
- The results from these assays were then compared with findings obtained using densitometry, a process performed after running the glycosaminoglycans samples through agarose gel electrophoresis and isolating them using ion exchange chromatography.
- It was found that the levels of glycosaminoglycan urinary concentrations in the urine samples varied between 1-20 mg/l, and glycosaminoglycan/creatinine ratios ranged between 2-25×10(-3) across all the mammalian species that were studied.
- The research also revealed a trend of decreasing glycosaminoglycan levels with age in human, cat, and horse species.
Comparison with DMB assay
- Contrastingly, the DMB assays recorded much higher results, with up to 200 mg/l of glycosaminoglycan concentrations in urine and 500×10(-3) glycosaminoglycan/creatinine ratios.
- However, these high measurements were greatly reduced after the samples underwent a 4-hour dialysis, suggesting that other low molecular weight compounds present in urine may have interfered with the DMB tests.
- It was evident from the study that urinary anions like sulfate, phosphate, and citrate reacted with metachromatic dyes such as Toluidine Blue and DMB, further contributing to the interference.
Conclusion
- In summary, the study concluded that while the DMB assay is a quick and straightforward test to carry out, it may not be a suitable method for quantifying urinary glycosaminoglycans in mammals, as other urinary compounds interfere with the test results, leading to inaccuracies.
Cite This Article
APA
de Lima CR, Baccarin RY, Michelacci YM.
(2006).
Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans.
Clin Chim Acta, 378(1-2), 206-215.
https://doi.org/10.1016/j.cca.2006.11.025 Publication
Researcher Affiliations
- Departamento de Bioquímica, Universidade Federal de São Paulo - UNIFESP - São Paulo, SP, Brazil.
MeSH Terms
- Animals
- Creatinine / urine
- Densitometry
- Dogs
- Electrophoresis, Agar Gel
- Glycosaminoglycans / urine
- Goats
- Horses
- Methylene Blue / analogs & derivatives
- Sheep
Citations
This article has been cited 7 times.- Paganini C, Monti L, Costantini R, Besio R, Lecci S, Biggiogera M, Tian K, Schwartz JM, Huber C, Cormier-Daire V, Gibson BG, Pirog KA, Forlino A, Rossi A. Calcium activated nucleotidase 1 (CANT1) is critical for glycosaminoglycan biosynthesis in cartilage and endochondral ossification. Matrix Biol 2019 Aug;81:70-90.
- Min T, Sun J, Yi Y, Wang HX, Hang F, Ai YW, Wang LM. Microanalysis, Pharmacokinetics and Tissue Distribution of Polysaccharide-Protein Complexes from Longan Pulp in Mice. Int J Mol Sci 2015 Oct 15;16(10):24403-16.
- Baccarin RY, Rasera L, Machado TS, Michelacci YM. Relevance of synovial fluid chondroitin sulphate as a biomarker to monitor polo pony joints. Can J Vet Res 2014 Jan;78(1):50-60.
- Coppa GV, Buzzega D, Zampini L, Maccari F, Santoro L, Galeotti F, Galeazzi T, Gabrielli O, Volpi N. Plasmatic and Urinary Glycosaminoglycans Characterization in Mucopolysaccharidosis II Patient Treated with Enzyme-Replacement Therapy with Idursulfase. JIMD Rep 2012;4:79-90.
- Nyren-Erickson EK, Haldar MK, Gu Y, Qian SY, Friesner DL, Mallik S. Fluorescent liposomes for differential interactions with glycosaminoglycans. Anal Chem 2011 Aug 1;83(15):5989-95.
- Alp BF, Akyol I, Adayener C, Senkul T, Gultepe M, Baykal K, Iseri C. The significance of potassium chloride sensitivity test and urinary uronic acid level in the diagnosis of chronic pelvic pain syndrome. Int Urol Nephrol 2009;41(3):483-9.
- Indika NR, Peiris H, Deutz NEP, Perera R. Optimization of Automated Dimethylmethylene Blue Assay for Quantification of Pediatric Urine Samples. Indian J Clin Biochem 2024 Oct;39(4):519-528.
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