A method for the determination of free, active insulin in the sera of insulin-treated diabetics is described. This involved radioimmunoassay after extraction of free insulin with polyethylene glycol. Recovery tests with cold insulin showed 73 per cent recovery of free insulin and no recovery of bound insulin.The fasting free insulin levels were slightly lower in the patients than in normal persons; exceptions were some patients with complications expected to cause insulin resistance at the peripheral tissue level. Free insulin levels did not correlate with total insulin levels, antibody titers, insulin requirements or conditions of insulin treatment.A very slight increase of insulin was observed after glucose loading in insulin-treated patients, but a marked increase of the free insulin level followed by an exaggerated increase in the total insulin level was observed in a patient with the insulin autoimmune syndrome.The diurnal changes of the free insulin suggested the dynamic states of this fraction and its usefulness for determining control of diabetes with insulin. DIABETES 22: 590-600, August, 1973.Since Berson and Yalow introduced the radioimmunologic method for the determination of plasma insulin, 1 their original method and modifications have been widely used and the method's reliability generally accepted. The plasma insulin level in patients previously treated with insulin, however, cannot be determined by the original method or most of its modifications, due to the presence of human anti-insulin antibody in the plasma which combines with the added radioinsulin and interferes with the reaction between radioinsulin, endogenous insulin and guinea pig antiinsulin antibody.By Grodsky and Forsham's method in which the plasma insulin is extracted with acid ethanol prior to the immunoassay, 2 the presence of human antibody causes no interference in the radioimmunoassay. However, the obtained insulin level represents total insulins, including antibody-bound inactive insulin. For the study of insulin dynamics in patients treated with insulin, the determination of free, active insulin is required. In this report, a simple method for the determination of free insulin in serum containing insulin antibodies is described. MATERIALS AND METHODSExtraction of free insulin. To 1 ml. of plasma chilled in an ice water bath, 1 ml. of cold 25 per cent (w/w) polyethylene glycol solution was added with an automatic syringe, and the mixture was immediately agitated with a vortical mixer for one minute and centrifuged at 3,000 r.p.m. for forty-five minutes in a refrigerated centrifuge. The supernatant was used for the radioimmunoassay.Extraction of total insulin. A 1 ml. quantity of plasma and 0.2 ml. of 1.0 N HC1 were mixed and, after one hour at room temperature, 1.4 ml. of 25 per cent polyethylene glycol solution was added. After mixing with a vortical mixer, 0.2 ml. of 1.0 N NaOH was added and the mixture was again agitated with a vortical mixer. After centrifugation at 3,000 r.p.m. for forty-five minutes, the supernatant was...
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