Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction.

OBJECTIVE: Acid digestion of animal tissues yields two fractions of glycogen, acid soluble (ASG) and insoluble (AIG). The current study was performed to improve the assay method for glycogen fractions in rat liver in different physiological states. MATERIALS AND METHODS: All steps of the assay were manipulated and optimized to measure the content of ASG and AIG in fed and starved rat liver. RESULTS: In postmortem liver tissue, total glycogen was decreased slowly at 4°C and rapidly at 25°C but was well stabilized at -20°C and -70°C. At room temperature, ASG underwent autolysis at the rate of 1.3% and decreased by half at 35 min, while AIG increased slightly. The yield of the recovery of ASG during four successive extractions depends on the tissue concentration, and at the ratio of 50 mg tissue per 2 mL perchloric acid (PCA) was about 93.2%, 6.3%, 0.3% and 0.05% respectively. The increase in the time and extent of homogenization of the tissue with cold PCA and using ultrasonication had not any significant effect on the extraction yield of ASG. The time of centrifugation of the tissue extract could be reduced from 15 to 7.5 minutes with no significant decrease in the recovery of ASG. On extraction with ethanol, the yield of recovery of ASG reached the maximal level of 97.5% at a final ethanol concentration of 60%. The recovery of ASG was not improved in the presence of KCl. During 24 starvation, total glycogen depleted completely and the change occurred entirely in ASG, while AIG did not change significantly. CONCLUSIONS: The CV% was less than 5% for the optimized assays of glycogen fractions. ASG is the main and metabolically active portion of glycogen in rat liver.

A new protocol for separation of acid soluble and insoluble fractions from total glycogen and simultaneous measurements.

OBJECTIVE: The glycogen is extracted routinely from animal tissues with cold perchloric acid (PCA). Acid soluble glycogen (ASG) is extracted, while the insoluble fraction (AIG) is liberated using hot alkaline. The current study was performed to separate and measure ASG, AIG and total glycogen in the same sample simultaneously. MATERIALS AND METHODS: The protocol has the four phases of tissue digestion, extraction, separation of fractions and measurement. The liver tissue was weighed and digested with four volumes of 30% KOH and heated in boiling water bath for 10 min. Total glycogen was extracted with ethanol at a final concentration of 55%. The suspension of total glycogen was separated into the two fractions of acid soluble and insoluble by adding of 30 µL PCA (70%) followed by a short and mild centrifugation. Total glycogen, ASG and AIG have derived from the same sample and analyzed for glucose. RESULTS: Analysis of different weights of the liver tissue using the current procedure shows that the fractions of glycogen are measured accurately. The CV% was less than 5% for inter- and intra-assays of total glycogen and ASG. The CV% was more than 5% for inter-assays of AIG, but it lessened in intra-assays. During 24 h starvation, total glycogen depleted completely (71.4 ± 8.3 mg/g wet vs. 4.4 ± 1.2, p ≤ 0.004) and the change occurred entirely in ASG (66.9 ± 7.8 vs. 1.9 ± 1.1, p ≤ 0.004), while AIG did not change significantly (4.4 ± 1.3 vs. 2.2 ± 0.9, p ≤ 0.08). CONCLUSIONS: The values of ASG, AIG and total glycogen obtained by the current protocol are the same as the classical homogenization method but the procedure is more easy and precise. ASG is the main and metabolically active portion of glycogen in rat liver.

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.