A nonradioactive approach to investigate the metabolism of therapeutic peptides by tagging with127I and using inductively-coupled plasma mass spectrometry analysiss

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 Iat 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 therapeuticsThe 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 Iat 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