HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF INSULIN SYNTHESIS IN BIOLOGICAL-SYSTEMS

This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [H-3]leucine into insulin in biological systems. The first step of the procedure w as size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH2PO4-methanol (9:1, v/v). By this step the bu lk of both protein and radioactivity was separated from tritiated insu lin. The second step, which employs reversed-phase chromatography on a n octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simpl e and reproducible method was employed to test insulin synthesis in cu ltured human retinoblastoma Y79 cells.