The Determination of Glycogen in Tissue.

Abstract

The method in general use for the determination of glycogen in tissue 1 involves the use of strong KOH to effect a separation of glycogen from proteins. Other methods 2 for treatment of tissue have not proved satisfactory.

A suitable method, however, has been found by treating boiling water extracts of tissue (liver only has been employed thus far) with trichloracetic acid. A filtrate from this mixture gives quantitative values for tissue glycogen. The use of KOH is rendered unnecessary and the procedure saves considerable time, particularly when amounts of tissue of more than a gram are involved.

Procedure: Tissue is weighed and plunged into boiling water for about 2 minutes when it is ground thoroughly and re-boiled. The mixture is cooled and diluted to a known volume and after mixing it is centrifuged. The creamy supernatant fluid is removed (the bulk of the proteins are insoluble) and an aliquot part added to a definite volume of trichloracetic acid (we have employed an equal volume), allowed to stand 15 minutes and filtered. Trichloracetic acid of strengths varying from 2 to 5% has been found satisfactory but with 2% acid it has been necessary to refilter. A second filtration has in this case always given satisfactory solutions. To an aliquot part of the filtrate 3 volumes of alcohol and a pinch of N. Cl are added, and the mixture is allowed to stand, preferably overnight. The deposit obtained after centrifuging is a clean white precipitate and the alcoholic fluid is perfectly clear. The deposit is washed twice with 80% alcohol (containing NaCl), once with 95% alcohol, once with absolute alcohol and once with ether. The precipitate is dissolved in water and hydrolyzed. Three methods, Folin-Wu, 3 Folin-Wu, 4 and Hagedorn and Jensen 5 have given identical values for the glucose in the hydrolysates.