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

Abstract

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.