In vitro metabolic stability of obestatin: Kinetics and identification of cleavage products

The in vitro metabolic stability testing on synthetic obestatin peptides from two different species (human hOb and mouse mOb) using HPLC analysis is described. A reversed-phase C18 column of 300 Å pore size was used, with a gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting peptide metabolic products, while UV (DAD) and fluorescence served quantitative purposes. Differences in the metabolic degradation kinetics of hOb and mOb were found in plasma, liver and kidney homogenate, with half-lives ranging between 12.6 and 138.0 min. Proteolytic hydrolysis at the N-terminal Phe residue and cleavage at Pro4–Phe5 were found to be two major metabolic pathways, accounting for more than 50% of the metabolic degradation. Several other labile peptide bonds were located. The influence of a standard protease inhibitor cocktail was investigated, as well as the metabolism of iodinated human obestatin in liver homogenate. Our results indicate that the major instability of obestatin peptides, as currently used in biomedical investigations, should be taken into account in the interpretation of the obtained results.