Methods and detection of endogenous peptides in the CNS and GI tract

Endogenous peptides, such as enkephalins, endorphins and orexins modulate a variety of biological processes; these peptides are found in the central nervous system and periphery, including the gastrointestinal tract. One of the methods commonly used to determine localization, concentration, expression and function of endogenous peptides is the radio immunoassay (RIA). A critical part of the RIA is the choice of antibodies, which are the specific tools to enable measurement of peptides. The outcome of a neuropeptide RIA depends on several aspects of the experimental setting such as age, gender, species, diurnal rhythm or environmental stress.

The aims for the studies (paper I and II) were production of antisera using peptide conjugates for the specific neuropeptides, niethmethionine-enkephalin-Arg6-Phe7 (MEAP) and rat P-endorphin and the antibodies were further characterized. Specific aims were to clarify some of the differences between art-specific antibodies and the outcome of α-endorphin RIAs. Furthermore, levels of MEAP and α-endorphin were detected in specific brain areas (hypothalamus and periaqueductal grey) and peripheral tissue of rats. Finally, to further understand the role of orexins in the gut: we investigated circulating concentrations of orexin A in human plasma with an optimized RIA (paper III). Optimization of the neuropeptide RIAs was mainly done by a careful selection of the most optimal included components, i.e. the use of spec ies-specific standards and antibodies.

The response to the immunization varied between rabbits from poor to exceptional (MEAP ab 4108). Two antisera from each group (MEAP and α-endorphin) showed useful titres, sensitivity, avidity and specificity and RIAs were set up. When using the MEAP antibody 4108, the relative levels of immunoreactive material from rats were verified due to current knowledge. In the case of α-endorphin, the RIA outcome was not optimal when using nonspecies compatible components when analysing rat hypothalamus tissue. The rat antibody allowed for larger detection range and specificity. No significant changes were observed in either the hypothalamus or periaqueductal grey of rats, when using the new rat α-endorphin antibodies to investigate the effects of diurnal variation or environmental stress. Concentrations of human plasma levels of circulating orexin A verified untreated levels and enabled to monitor increasing concentrations by time after addition of exogenous orexin A.

The present work illustrates the importance of understanding the RIA technique for its proper evaluation and determination of tissue- and plasma concentrations. Selection of an appropriate antibody is essential, therefore its characteristics has to be investigated.

List of scientific papers

I. Finn A, Agren G, Bjellerup P, Vedin I, Lundeberg T (2004). Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7. Scand J Clin Lab Invest. 64(1): 49-56.
https://pubmed.ncbi.nlm.nih.gov/15025428

II. Finn A, Fabre SF, Hellstrom PM, Brene S (2006). Methodological aspects of rat beta-endorphin analysis-influence of diurnal variation. J Immunol Methods. 312(1-2): 118-25.
https://pubmed.ncbi.nlm.nih.gov/16697001

III. Ehrstrom M, Gustafsson T, Finn A, Kirchgessner A, Gryback P, Jacobsson H, Hellstrom PM, Naslund E (2005). Inhibitory effect of exogenous orexin a on gastric emptying, plasma leptin, and the distribution of orexin and orexin receptors in the gut and pancreas in man. J Clin Endocrinol Metab. 90(4): 2370-7.
https://pubmed.ncbi.nlm.nih.gov/15671114