Radioreceptorassay of insulin using human erythrocytes. I. Methodological aspects

It is known that human erythrocytes have specific receptors for insulin. This works describes a radioreceptor assay (RRA) suitable for the determination of the insulin binding to the surface receptors of erythrocytes. The erythrocytes were isolated from heparinated blood collected in the morning after overnight fasting. In the incubation mixture are 4-5 X 10(9) cells/ml, labelled porcine insulin and nonlabeled (cold) insulin in serial concentrations, range 10-10(5) ng/ml. Incubation (150 min. at +15 degrees C) is ended by suspending the incubation mixture in cold buffer. After washing, the tracer insulin bound to erythrocytes is measured on a gamma counter and the percentage of the specific binding is calculated against the total radioactivity determined prior to the washing steps. By this procedure, the maximal binding of 125I-Insulin was 10.55 +/- 0.78% (mean +/- SD) in five normal volunteers and 6.79 +/- 1.77 (mean +/- SD) in five obese nondiabetic patients, having an enhanced insulin response in the oral glucose tolerance test. The slight hemolysis that happened accidentally in some tubes after incubation caused rude degradation of the tracer, the binding being decreased at last by 25%. The method described (according to Gambhir K. K. et al 1977 slightly modified) is specific and thus suitable for estimating the prognosis of obese children, patients with acute and chronic hepatitis and also for the study of the role of erythrocytes in insulin metabolism and possibly in the metabolism of other hormones.