Reverse phase high performance liquid chromatographic analysis of circulating insulin in the insulin autoimmune...

Some patients with the insulin autoimmune syndrome have circulating insulin that is heterogeneous. We used reverse phase high performance liquid chromatographic analysis to identify the forms of plasma insulin in patients with this syndrome and compared the results with those in patients with insulin-treated diabetes and patients with hyperinsulinism. Under acidic conditions, free insulin dissociated from insulin antibodies eluted from Bio-Gel P-30 columns as a single peak. When such insulin fractions were applied to reverse phase high performance liquid chromatography, a major insulin peak emerged with the same retention time as standard human insulin in all six patients with the syndrome. In addition, a minor insulin peak was consistently found at relatively high acetonitrile concentrations. However, this hydrophobic insulin also was found in two of four insulin-treated diabetic patients and in one of two hyperinsulinemic patients who did not have insulin antibodies. Preliminary characterization of the variant insulin revealed that it has a molecular size between those of proinsulin and insulin and retains the immunoreactivity of insulin, but not C-peptide. It may be an aggregate of insulin molecule or proinsulin intermediates. Since the variant insulin was not found only in patients with the insulin autoimmune syndrome, it seems unlikely that an altered endogenously produced insulin induces the generation of autoantibodies to insulin in this syndrome.