Simple Spectrophotometric Quantification of Urinary Excretion of Glycosaminoglycan Sulfates (1)
The basic dye 1,9-dimethylmethylene blue (DMB), first studied by Taylor and Jeifree, has been used to assay sulfated GAGS in cartilage tissue cultures. Maximum absorbance of the peak resulting from the reaction between DMB and C-4-S is at 535 nm. The dye alone has two peaks of absorbance, at 597 and 648 nm. When the ug of dye was reacted with the C-4-S standard. The color was stable longer when smaller quantities were assayed under the same conditions. The absorbance of the complex found with DMB was linearly correlated with the GAG content, up to 12 ug on each polysaccharide.
Within-run precision was calculated from 20 replicate analysis of three 24-h urine collections with GAG contents of 2.4, 62.1, and 175.3 mg. The CV was 2.5%, 1.6%, and 2.1%, respectively. Between-run precision, determined om data for 10 replicate analyses of six urine collections containing increasing amounts of GAGS (3.1 to 199.7 mg), ranged from 1.8% to 4.5%. When various amounts of GAGS were added to 24-h urine specimens, between 71% and 107% of the added GAGS were accounted for.
The mean (± SD) urinary GAG excretion by 80 healthy subjects (both sexes) was 12.8 ± 5.0 mg/24 h (range: 3.0-30.3 mg/24 h) by the DMB method and 4.9 ± 2.0 mg/24 h (range: 1.1-11.8 mg/24 h) by the borate-carbazole procedure.
To determine urinary sulfated GAGS, a precipitation step minimizes any interference from salts and negatively charged compounds (e.g., glycoproteins, nucleic acids).
Assay procedures: Urinary GAGS were isolated by precipitation with cetylpyridinium chloride (CPC). The pellet was resuspended in deionized water (final volume, 5 mL). Use as blank 0.5 mL of de-ionized water. Use as standards, 4, 8, and 12 pg ofC-4-S in a final volume of 0.5 mL. The samples are 0.05-0.5 mL of resuspended urinary GAGS at a final volume of 0.5 mL (see above). Add to each 2.5 mL of the reagent dye solution. After 5 mm, read the absorbance at 535 nm, using a plastic disposable cuvette.