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

H. Shokri-Afra, A. Ostovar-Ravari, M. Rasouli

Department of Clinical Biochemistry and Immunogenetic Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran. mehdi.rasouli@yahoo.com

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• Metabolism

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.

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