| The kinetics and mechanisms of human insulin disposition in the airways were studied in the isolated perfused rat lung (IPRL), specifically with respect to absorption mechanisms, metabolism kinetics and the influence of insulin self-association on each of these pathways.; Insulin dosing solutions (0.1 mL) at various concentrations, with or without different biochemical modifiers, were administered into the airways of the IPRL. Insulin absorption profiles determined by validated ELISA were fitted to a kinetic model to generate rate constants for absorption (ka) and non-absorptive loss (ke), describing the parallel processes of insulin absorption and elimination in the airways.; Monophasic, apparent first-order absorption profiles were observed following 1.5 and 3.0 IU/mL instillation and the values for ka were insignificantly different. Moreover, insulin absorption was significantly reduced in the presence of atrial natriuretic peptide, a polypeptide suppressing passive absorption of FITC-labeled dextran in the IPRL. These data strongly suggested that insulin was absorbed predominantly via passive diffusion.; Quantitative centrifugal filtration followed by zinc terpyridine titration was newly developed to assess the hexameric content of insulin in dosing solutions, with a support from the data via CD spectroscopy. Insulin hexamer content increased from 16 to 94% with increasing insulin concentration from 0.025 to 1.0 mg/mL. As insulin concentration and thus, hexamer content increased, the extent of insulin absorption in the IPRL from these solutions decreased with decreasing values for both ka (0.21 → 0.05 hr-1) and ke (2.04 → 0.90 hr-1) generated by the kinetic model analysis. This suggested that the kinetics of insulin absorption and metabolism depended on the degree of self-association.; Insulin absorption was significantly increased in the presence of bacitracin and L-leucine p-nitroanilide, suggesting the involvement of aminopeptidase N (APN) in insulin's metabolic loss in the airways. To further identify the metabolites, insulin was incubated in the airways of a non-perfused isolated rat lung, and bronchoalveolar lavage fluid was analyzed using LC/MS. Only one major metabolite, des-PheB1-Ins, was detected, which was identical to a product following insulin incubation with APN. These data suggested that APN was the primary enzyme responsible for insulin metabolism in the airways of the IPRL. |
