Insulin degradation in vivo: a high-performance liquid chromatographic analysis

The metabolism of insulin in vivo was investigated using an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method. After intravenous injection of A14-[125I]insulin into normals, eight labelled insulin derivatives were found in plasma (peaks 1-8). Two of them (peaks 1 and 7) showed an elution pattern identical with those of reference [125I]monoiodotyrosine and intact A14-[125I]insulin, respectively. Of the other six peaks, five (2-6) eluted before and one (peak 8) after insulin. This pattern was highly reproducible in terms of capacity factors and peak heights. Radioactivity separated by RP-HPLC was further characterized for its trichloroacetic acid precipitability and immunoprecipitability. Fractions corresponding to peaks 4-6 and 8, which showed an immunoprecipitability higher than 50%, were pooled in order to obtain sufficient radioactivity and were found to be insulin separated by Sephadex G-50 chromatography, containing in its structure, after sulphitolysis, intact A-chain and to be partially rebindable to monocyte insulin receptors. These data demonstrate that in blood, products of insulin metabolism circulate which retain a part of the immunological and biological properties of the hormone. These products are clearly separated from one another and from intact insulin by RP-HPLC, suggesting that the appropriate use of this technique may allow a further and more accurate qualitative and quantitative characterization of in vivo insulin metabolism in physiological and pathological conditions.