Characterization and improvement of phenol-sulfuric acid microassay for glucose-based glycogen.

OBJECTIVES Phenol-sulfuric acid reagent is used to measure the concentration of glyco-polymers and -conjugates. There are several uncertainties on glycogen measurement in the tissues. We aimed to improve phenol-sulfuric reagent for microassay of glucose based-glycogen in small tube or microplate. MATERIALS AND METHODS The condition of the reaction was optimized and scaled down for both small tube and microplate application. RESULTS The color intensity was found to be a function of all components of the assay mixture, that is, the amount of sugar and phenol together with the volume of total water and acid. The absorbance increased in the range of 4-10 mg of phenol and reached the plateau between 10-16 mg per 1 mL of acid. The color intensity was a linear function of total water volume (sugar-water- phenol). The sensitivity increased eight times as total water volume was changed from 50 up to 400 µL. The curve for acid volume peaked at about 1 mL. The optimal assay condition was determined to be 13 mg of phenol (200 µL 6.5%), 400-425 µL of total water volume (100 µL of sugar, 100 µL water) for 1 mL of acid. The initial spontaneous high temperature is essential the reaction to proceed and any handling gives inconsistent results and decreases the precision and sensitivity of the method. The values were scaled down by a factor of 0.5 for tube application and reading in cuvet or microplate and by 0.2 or 0.15 for microplate application. CONCLUSIONS The results indicated that phenol-sulfuric acid reagent could be scaled down to 1.0, 0.5 and 0.20, 0.15 mL of sulfuric acid for microassay of glucose based-glycogen.