High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients

Background: Because non-denaturing clectrophoresis and aminopeptidase activity staining often detect noncovalent multienzyme complexes, we adopted procedures to specifically detect the aminopeptidase N (APN) molecule itself in liver disease serum. Methods: Sera or their immunoprecipitate with anti-APN monoclonal antibody were analyzed by sodium dodecyl sultate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional electrophoresis and subsequent Western blotting with rabbit anti-APN serum. Results: In all the patient sera examined, the 140-kDa APN isoform was predominant. In all the sera from 10 patients with cholestatic diseases (8 with extra-hepatic cholestasis and 2 with primary biliary cirrhosis), we observed the 260-kDa isoform that was immunoprecipitated with monoclonal APN antibodies and had a similar isoelectric point to the 140-kDa isoform. However, the 260-kDa isoform was observed faintly in 2 out of 12 patients with other liver diseases, including chronic hepatitis and cirrhosis. Conclusions: We found a novel high-molecular-mass APN isoform (260-kDa) in serum, which is highly likely to be a homodimer of APNs bound covalently and a promising marker of cholestasis. This suggests increased cross-linking reaction between two APN molecules in cholestatic patients. (C) 2003 Elsevier Science B.V. All rights reserved.