The tendency of insulin in high concentrations to selfassociate and the widespread presence of insulindegrading enzymes suggest that fragments and/or aggregates of insulin may circulate in normal and insulin-dependent diabetic (IDOM) individuals. To examine this possibility, we have analyzed, by sensitive physicochemical methods, immunoreactive insulin (IRI) taken from the blood of 9 healthy volunteers and 12 insulin-dependent diabetic patients. IRI from the blood of the normal volunteers was composed of 6000 (91.0 ± 1.4%) and 9000 (9.0 ± 1.4%) molecular weight (mol wt) material. By 10% polyacrylamide disc gel electrophoresis (PAGE) and reverse-phase, high-performance liquid chromatography (HPLC), the 6000 mol wt material was indistinguishable from human insulin standards and insulin fragments were not found. C-peptide reactivity in the 9000 mol wt material confirmed the expected presence of proinsulin and intermediates of proinsulin conversion.
IRI harvested from the blood of 12 C-peptide-negative IDDMs, using a variety of insulin preparations, also separated into 6000 (80.5 ± 3.9%) and 9000–12,000 (19.5 ± 3.9%) mol wt material. By HPLC, 6000 mol wt IRI was either pork insulin (in volunteers using pure pork insulin) or a mixture of beef (∼90%), pork (∼10%) and deamidated beef (trace) insulin in those using a beef-pork mixture. However, the 9000–12,000 mol wt material had characteristics entirely distinct from proinsulin of either human or animal origin: (1) C-peptide reactivity was undetectable using any of three sensitive radioimmunoassay systems, (2) on PAGE it migrated more rapidly than proinsulin-like material, and (3) in contrast to proinsulin, it was unaffected by proteolytic degradation.
We conclude that a covalent aggregate of therapeutic insulin circulates in diabetic patients who use a variety of insulin preparations. Insulin aggregates may account for a relatively large amount of the total IRI in IDDMs, and these aggregates may promote the formation of anti-insulin antibodies and affect the action of insulin.
