Metabolic profiling of opioid peptides in canine pituitary and selected brain regions using HPLC with a radioreceptor assay detector

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Abstract
A combination of gradient reversed-phase high performance liquid chromatography (RP/HPLC) with a radioreceptor assay detector that uses two ligands is used to obtain effectively the metabolic profile of endogenous receptoractive opioid peptides in the canine pituitary and in seven selected brain regions including the hypothalamus, caudate nucleus, mid-brain, amygdala, thalamus, pons-medulla, and the hippocampus. Gradient RP/HPLC separates a mixture of endogenous peptides over a wide range of hydrophobicities. A novel opioid preparation from canine limbic system synaptosomes is utilized in a radioreceptorassay screen; tritiated etorphine (ET) or D-2ala, D-5leu-leucine enkephalin (DADL) is used as the competitively displaced ligand. This receptor-rich preparation contains several receptor types, and thus serves well as a screen with the required low level of specificity. Subsequent analysis with other detectors of high specificity (MS, RIA) will follow this screen in other studies. Etorphine interacts with several of the opioid peptide-preferring receptors, whereas DADL is more specific towards the delta receptor that preferentially binds the smaller pentapeptides of the enkephalin family. The highest amount of peptide receptor activity found in this study is in the pituitary tissue, a smaller amount in the hypothalamus and caudate nucleus, and still lower amounts in the other five brain tissue extracts. This variation in peptide concentration most probably reflects three separate factors that operate in this biologic system: differential tissue-specific processing patterns of the large peptide precursors; distribution of the three opioid peptide systems; and the receptor preparation and the radioligand used in the assay. The structures of the receptoractive compounds in each RP/HPLC peak await mass spectrometric confirmation.