**3.1.2 Characterization of secreted ADM**

To examine the molecular forms of ADM, extracts of cells cultured on a 100-mm dish and 15 ml of the conditioned medium (1%FBS) were analyzed by reverse-phase high-performance liquid chromatography (HPLC) with a column of TSK ODS 120 A (Tosoh, Tokyo, Japan). A linear gradient of 10% to 60% acetonitrile was run in 0.1% trifluoroacetic acid for 60 minutes and the ir-AM level in each fraction was measured by RIA. The recovery of ir-ADM from this HPLC system was greater than 80%.
