Abstract

The metabolic fate of adrenocorticotropic hormone (ACTH) fragment 4–10 (4–10) was evaluated following incorporation of a nonradioactive ¹²⁷I-tag and with selective detection of I⁺ at m/z 127 by inductively coupled plasma mass spectrometry (ICP-MS). ¹²⁷I has all the advantages of radioactive ¹²⁵I as a metabolite tracer and, together with its detection in the femtogram range, has led to a successful metabolite profiling of ¹²⁷I-ACTH (4–10) in vitro. The observed metabolic stability of this peptide in tissue preparations from human was plasma > kidney S9 > liver microsomes > liver cytosol, liver S9. Metabolic turnover of ¹²⁷I-ACTH (4–10) was not NADPH-dependent and, together with inhibition by protease inhibitor cocktail and EDTA, is consistent with metabolism exclusively by proteases. Our preliminary studies using chemical inhibitors suggested the involvement of metalloprotease, serine peptidase, and aminopeptidase in ¹²⁷I-ACTH (4–10) metabolism. The liver is the primary site of metabolic clearance of ¹²⁷I-ACTH (4–10), with kidney S9 taking four times longer to produce a metabolite profile comparable to that produced by liver S9. A total of six metabolites retaining the ¹²⁷I-tag was detected by ICP-MS, and their structures were elucidated using a LTQ/Orbitrap. ¹²⁷I-ACTH (4–10) underwent both N- and C-terminal proteolysis to produce ¹²⁷I-Phe as the major metabolite. The ¹²⁷I-tag had minimal effect on the metabolic turnover and site of proteolysis of ACTH (4–10), which, together with ICP-MS providing essentially equimolar responses, suggests that the use of a ¹²⁷I-tag may have general utility as an alternative to radioiodination to investigate the metabolism of peptide therapeutics