Evaluation of an on‐capillary copper complexation methodology for the investigation of in vitro metabolism of dynorphin A 1–17

Abstract

Dynorphin A 1–17 is an endogenous neuropeptide implicated in a variety of neurological disorders including Alzheimer’s and Parkinson’s diseases and neuropathic pain. Metabolites of this peptide can exhibit their own unique effects in vivo, and it is possible that one of these metabolites is responsible for the neurotoxicity. In this article, the use of CE for the separation of dynorphin A 1–17 from four of its metabolites is described. Buffer additives were investigated to eliminate peptide adsorption to the capillary wall and to improve resolution between closely related metabolites. On-capillary copper complexation was employed and was shown to improve separation efficiency as compared with the separation of native peptides. The method was then applied to in vitro dynorphin metabolism in human plasma as well as rat brain and rat spinal cord slices.

The permeation of dynorphin A 1–6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability

“…In this work and that of others, Dyn A 1–6 has been identified as a major metabolite of the parent peptide Dyn A 1–17 in both the central nervous system (brain and spinal cord) [11, 13, 16] and peripheral tissues (blood and plasma) [6, 7, 13, 21]. Dyn A 1–6 was also identified as a metabolite in the presence of bovine brain microvessel endothelial cells (BBMEC) (supplemental information- Figure S1).…”

“…Dyn A 1–6, the N-terminal fragment of Dyn A 1–17, has been identified as a major metabolite of the parent peptide Dyn A 1–17, by our group and others, in both the central nervous system (brain and spinal cord) [11, 13, 16] and peripheral tissues (blood and plasma) [6, 7, 13, 21]. Therefore, this communication describes the initial studies employed to characterize the BBB permeability of Dyn A 1–6 and the effect of the peptide on cell monolayer integrity, utilizing the in vitro cell culture model of the BBB developed by Audus and Borchardt [1].…”

“…The LC-MS/MS method required minimal sample preparation and no derivatization steps. Improved limits of detection were achieved using LC-MS/MS in comparison to a previously described CE-UV method [13]. Additionally fluorescein was employed as a low molecular weight, membrane-impermeant molecule to probe the effect of Dyn A 1–6 on cell monolayer integrity.…”