Insulin was extracted from pancreases obtained at autopsy from non-diabetic subjects (normal insulin) and from adult-onset diabetic subjects (diabetic insulin). Two pancreases were extracted with I M acetic acid, and the insulin containing fraction of the fluid portion of the extract was separated using Sephadex G 50. This fraction was lyophillised, then incubated with anti-insulin serum (guinea pig) and the insulin-anti-insulin complex separated using Sephadex G 50. Insulin was then dissociated from this complex using I M acetic acid, and separated from other protein with Sephadex G 50. This process yields only substances reacting with anti-insulin serum. Other pancreases were extracted with a mixture containing ethanol, water and hydrochloric acid at pH< 1.5. The fluid portion of this extract was neutralised with ammonia and centrifuged. Insulin was precipitated from the supernate on addition of ethanol and diethyl ether. Catecholamines and cortico-steroids were not precipitated. A mixture of uniformly labeled 14c glucose (2 uc) together with normal or diabetic insulin in a total volume of 2 ml was injected intraperitoneally into each of 10 male Wistar rats. The mixture contained 800-1100 u units of insulin per 2 ml, as determined by immunoassay. After 2 hours the rats were killed and their diaphragms removed. The diaphragmatic glycogen was isolated by digestion with hot 30% potassium hydroxide, followed by precipitation with 66% ethanol. The activity of this glycogen was used as the measure of glucose incorporation into glycogen. Highly significant differences were found between the level of glucose in corporation following diabetic insulins and normal insulins.
Shapcott, Dennis James, "Further Studies on the Abnormal Insulin of Diabetes Mellitus" (1966). University Libraries Digitized Theses 189x-20xx. 64.