Free Covalent Aggregates of Therapeutic Insulin in Blood of Insulin-Dependent Diabetics

Abstract

Immunoreactive insulin (IRI) in the circulation of diabetics using insulin includes a 12,000-Mr covalent aggregate of insulin. In this study, both free and bound IRI were measured in nine type I diabetics who were treated sequentially for 3 wk with constant doses of conventional beef-pork, biosynthetic human, and beefpork insulins (phases 1, 2, and 3, respectively). The aggregate accounted for 19 ± 5.7, 38 ± 7.7, and 26 ± 7% of bound IRI, and 19 ± 8.3, 35 ± 7.3, and 31 ± 5.0% of free IRI during phases 1, 2, and 3, respectively. Taken as concentrations (μU/ml), the absolute amounts of aggregate in the bound fraction were significantly (P < .001) greater than those that were free, whereas the ratios of aggregate to monomer within each pool were similar (P > .1). The relative and absolute amounts of the insulin aggregate during each of the three treatment phases (compared by analysis of variance) were indistinguishable (P > 0.5). However, the aggregate was overestimated by a factor of ∼2 when measurements were made on the basis of an insulin- monomer standard. We conclude that both the bound and free fractions of IRI contain insulin aggregate. Overreactivity of the aggregate in the radioimmunoassay contributes to the so-called hyperinsulinism of type I diabetes. As with insulin monomer, most of the 12,000-Mr aggregate is bound to antibodies. Because some of the aggregate is free, its biologic consequences must be assessed.