Studies on the State of Insulin in Blood: Materials and Methods for the Estimation of “Free” and “Bound” Insulin-Like Activity in Serum*

Abstract

New techniques have been introduced by the authors and collaborators in the course of their studies on the state, transport and regulation of insulin in man. The discovery that a portion of insulin in blood circulates in a complex form extractable by cationic exchange resins (Nacycle) led to the development of a simple technique for the separation of this portion of insulin from whole serum. The observation that insulin in its complex form is devoid of insulin activity, when assayed in vitro under specified conditions, resulted in the finding that factors present in adipose tissue extracts could dissociate the blood insulin complex (es). Methods have been developed therefore for the extraction, storage, preservation and “standardization” of these factors from adipose tissue. Studies on the ratio of “free” and “bound” insulin-like activity in serum suggest that this ratio does not represent a purely physicochemical equilibrium between “bound” and “free” insulin. This ratio depends upon the metabolic state of the individual donor. It has been found by the authors and collaborators that a rise of blood glucose “triggers” a mechanism which results in the in vivo dissociation of the “bound” form of insulin in blood. A system has been developed for the estimation of “free” and “bound” insulin-like activity in serum. These techniques are presented in detail in the present report. An outline is also included of the rat diaphragm assay (assay of Vallance- Owen and Hurlock) used for the estimation of the insulin-like activity in our studies.