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

ABSTRACT

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.