Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies.
This article describes a simple and selective procedure used for direct measurement of sulfated glycosaminoglycans (GAGs) in biological samples and its application to the determination of GAGs during tissue regeneration and myogenic differentiation. We describe a modified procedure of previous GAG assays that has improved specificity, reproducibility, and sensitivity. The assay is based on the ability of sulfated GAGs to bind the cationic dye 1,9-dimethylmethylene blue. We describe conditions that allow isolation of the GAG-dye complex. This complex was dissociated; the optical density measurement of the dissociated dye permitted quantification of GAGs in biological samples. Applied to the study of myogenic cell differentiation in vitro, muscle repair, and skin ulceration, this method revealed significant modifications in the patterns of expression of different sulfated GAGs in these tissues. In particular, application of the method after nitrous acid treatment revealed that heparan sulfate and chondroitin sulfate ratio changed during muscle regeneration process.
Barbosa
Isabelle
I
Laboratoire de Recherche sur la Croissance, la Réparation et la Régénération Tissulaires (CRRET), CNRS FRE-2412, Université Paris 12-Val de Marne, 61 Avenue du Général de Gaulle, 94010 Créteil cedex, France.
Garcia
Stéphanie
S
Barbier-Chassefière
Véronique
V
Caruelle
Jean-Pierre
JP
Martelly
Isabelle
I
Papy-García
Dulce
D
D000818:Animals; D002138:Calibration; D002454:Cell Differentiation; D002460:Cell Line; D006025:Glycosaminoglycans / Q000032:analysis*; D006497:Heparan Sulfate; D008297:Male; D008751:Methylene Blue / Q000031:analogs & derivatives*; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012038:Regeneration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000378:metabolism* / Q000473:pathology
D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D008751:Methylene Blue; C435946:dimethylmethylene blue; C013786:A73025