‘Free’ and ‘fixed’ glycogen as physiological entities

¹ Department of Pharmacology, University of Alabama Medical Center, Birmingham, Alabama

An anthrone method is presented for determining both total and glycosidic carbohydrate from the extract of a single tissue sample. Maximal amounts of carbohydrate are extracted with minimal destruction of glycosidic linkages by heating homogenized rat liver for 5 minutes in 2% trichloractic acid (TCA). Following protein precipitation, an aliquot of the supernatant is used for direct anthrone measurement (total carbohydrate) and another is treated with hot 3% KOH for determination of the alkali-stable carbohydrate (glycogen). The amount of ‘fixed’ glycogen, extracted from the TCA precipitate with hot 30% KOH, was found to depend upon the size of the tissue sample and the volume employed during TCA extraction. The larger the amount of tissue in relation to the TCA extraction volume the greater was the quantity fixed. Thorough homogenization and correction for the glycogen-containing filtrate trapped with the precipitate virtually eliminated the fixed fraction. Differences in methods of extraction should not form the basis for a physiological distinction of glycogen as fixed and ‘free’.