Characterization of circulating insulin in insulin autoimmune syndrome

We examined the forms of circulating insulin in three patients with the insulin autoimmune syndrome by a method combining gel filtration and reverse phase high performance liquid chromatography (RP-HPLC). Insulin bound to circulating antibody was dissociated by molecular sieve chromatography at acid pH. The free insulin peak eluted from a Sephadex G-50 column was subsequently chromatographed on a Bio-Gel P-30 column. In all three patients, insulin coeluted with normal human insulin. However, when the partially purified insulins, obtained by gel filtration, were applied to RP-HPLC, an abnormally migrating insulin was found in two of three patients. The insulins were more hydrophobic than normal human, porcine, or bovine insulin, but were different from each other. A third patient had only a single insulin peak on RP-HPLC which corresponded to normal insulin. In contrast, the insulin from insulin-treated diabetic patients with antibodies to exogenous insulin corresponded to either porcine or bovine and normal human insulin. The antibodies in the circulation of these patients with the autoimmune syndrome were of the immunoglobulin G type and contained kappa and lambda-chains in the same proportions as antibodies in insulin-treated patients. Autoantibodies could not be distinguished from those secondary to exogenous insulin treatment on the basis of displacement of binding by human, beef, or pork insulin. These results suggest that in certain patients with the insulin autoimmune syndrome, there may be a molecular abnormality of circulating insulin. Whether this comprises a cause for the syndrome or is a result of posttranslational processing of insulin remains to be determined.