Complete Removal of Glycogen from Tissues by Extraction with Cold Trichloroacetic Acid Solution

Abstract

Much of the experimental work upon the state of glycogen in animal tissues has been interpreted as showing that glycogen exists in tissues in two forms. Early workers (l-4) found that extraction of animal tissues with water or cold trichloroacetic acid solution yielded less glycogen than was obtained by boiling the tissue in an alkaline solution. Various names were used to designate the two fractions. “Easily extractable,” “free,” “lyo-,” and “trichloroacetic acid-extractable” are terms that have been used for the glycogen fraction extracted with water or cold trichloroacetic acid solution; and ‘difficultly extractable,” “fixed,” “bound,” “desmo-,” and “residual” are designations that have been used for the form that required alkaline digestion to extract it from the tissues. Many workers assumed that the difficultly extractable glycogen was bound to protein, but Pfliiger (5) and Meyer (6) found no basis for the assumption of such a binding. Bloom et al. (7) found that in resting, normal, fed rats, the glycogen of the liver extractable with trichloroacetic acid solution comprises 85% of the total glycogen obtained by boiling with 30% KOH solution. The same extraction of the livers of these animals, after they had been fasted for 24 hours, yielded only 7.9% of that obtained by alkaline digestion. These authors found that in the muscle of normal rats the trichloroacetic acidextractable glycogen was 55 y0 of that obtained by alkaline digestion; and 4 hours after subcutaneous epinephrine injection this fraction was 16.4% of the amount obtained by alkaline digestion. Other workers have observed a shifting in the distribution of glycogen between the two fractions as a result of fasting (4, 8lo), epinephrine injection (8, ll-14), insulin administration (12, 14, 15)) thyroxin injection (16)) adrenalectomy (9), hypophysectomy (9), and muscle work (17). In our laboratory it was found that extraction of heart muscle by thorough grinding with 5% trichloroacetic acid solution (Servall Omnimixer once, and the Potter-Elvehjem apparatus three times) yielded an average of 57% of the total glycogen (18). Alkaline digestion of the residue after the four homogenizations with trichloroacetic acid solution, followed by alcoholic precipitation, yielded an additional 43 ‘% of glycogen. The latter was shown to be an essentially pure product by analysis with anthrone reagent and, after hydrolysis, by copper reduction and glucose oxidase techniques (18).