Comparison of Methods to Assay Liver Glycogen Fractions: The Effects of Starvation

Abstract

There are several methods to extract and measure glycogen in animal tissues. Glycogen is extracted with or without homogenization by using cold Perchloric Acid (PCA). Three procedures were compared to determine glycogen fractions in rat liver at different physiological states. The present study was conducted on two groups of rats, one group of five rats were fed standard rodent laboratory food and were marked as controls, and another five rats were starved overnight (15 hour) as cases. The glycogen fractions were extracted and measured by using three methods: classical homogenization, total-glycogen-fractionation and homogenization-free protocols. The data of homogenization methods showed that following 15 hour starvation, total glycogen decreased (36.4±1.9 vs. 27.7±2.5, p=0.01) and the change occurred entirely in Acid Soluble Glycogen (ASG) (32.0±1.1 vs. 22.7±2.5, p=0.01), while Acid Insoluble Glycogen (AIG) did not change significantly (4.9±0.9 vs. 4.6±0.3, p=0.7). Similar results were achieved by using the method of total-glycogen-fractionation. Homogenization-free procedure indicated that ASG and AIG fractions compromise about 2/3 and 1/3 of total glycogen and the changes occurred in both ASG (24.4±2.6 vs. 16.7±0.4, p<0.05) and AIG fraction (8.7±0.8 vs. 7.1±0.3, p=0.05). The findings of 'homogenization assay method' indicate that ASG is the major portion of liver glycogen and is more metabolically active form. The same results were obtained by using 'total-glycogen-fractionation method'. 'Homogenization-free method' gave different results, because AIG has been contaminated with ASG fraction. In both 'homogenization' and 'homogenization-free' methods ASG must be extracted at least twice to prevent contamination of AIG with ASG.