Measuring Sulfated Glycosaminoglycans in Gingival Crevicular Fluid Based on Dimethylmethylene Blue

Abstract

The information about periodontal conditions is necessary for regeneration of periodontal tissues. The measurement of sulfated glycosaminoglycans (S-GAGs) in gingival crevicular fluid (GCF) is important in the search for disease biomarkers. To analyze the amount of S-GAGs in GCF quickly and accurately, we have modified a microplate method previously used for urinary S-GAGs to estimate the content of S-GAGs in GCF without interference of other GCF components. GCF samples were collected from six maxillary anterior teeth and diluted GCF samples were divided into two groups (20 µ:l each), with or without chondroitinase ABC treatment, mixed with a dimethylmethylene blue (DMB) dye solution (180 µl) to detect S-GAG. Absorbance at 540 nm was measured using a microplate reader and the amount of S-GAG was calculated from calibration curves for standard S-GAGs where a linear relationship existed in the concentration range (0-3 µg/100 µl). Other GCF components such as glycoprotein and serum constituents did not influence the measurement of S-GAGs. Although trace amounts of S-GAGs were found in controls with a healthy periodontium (0.05 µg/100 µl ± 0.04, mean±S.E., n=5), considerable amounts of S-GAGs were found in patients with advanced periodontal disease (0.42 ± 0.11, n=6, p<0.05). The accuracy of this simple and rapid method of measuring S-GAGs in GCF makes it useful for screening periodontal conditions.