Date Full Report Received


Date Abstract Report Received



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Without a current gold standard method to objectively recognize and quantify pain perception in animals, addressing questions about animal welfare is extremely challenging. Furthermore, the lack of scientific evidence makes it very difficult for stakeholders to respond to societal concerns with science-based recommendations for reducing pain caused during castration, tail docking, teeth clipping, animal identification and other procedures. The main goal of this project was to develop and validate a new “gold standard” test for measuring circulating physiological biomarkers of pain in piglets and to compare the results with existing commercial immunoassays.
The primary objective of this proposal was to analytically and clinically validate a novel liquid chromatography-mass spectrometry (LC-MS) method for determining parent and metabolite Substance P (SP), beta-endorphin, calcitonin-gene related peptide (CGRP) and neuropeptide Y (NPY) concentrations in swine plasma. Long known major obstacles to investigating the role of these neuropeptides as a pain biomarker are (1) the inaccuracy of existing immunoassays for measuring plasma concentrations and (2) the correlation of circulating neuropeptide biomarkers with acute and chronic pain following a routine husbandry procedure such as castration. Current enzyme-linked immunosorbent assays (ELISA’s) have not been appropriately validated in pigs and yield inconsistent and non-reproducible results in pain-free individuals. This has prevented establishment of a reference range for circulating neuropeptides in pain-free subjects thus hampering efforts to use these measures as objective pain assessment tools. Furthermore, ELISA and radioimmunoassay (RIA) tests also lack specificity, with significant cross-reactivity reported with neuropeptides and their metabolites. Therefore, many of the physiological effects of neuropeptide metabolites have been mistakenly attributed to the parent peptide hindering our understanding of the specific role of the peptides in pain perception.
This research project resulted in development of a sensitive and specific analytical methodology using LC-MS to measure Substance P, beta-endorphin, and several Substance P metabolites in spiked piglet plasma at the low parts-per-trillion level. The developed
methodology was then applied to un-spiked piglet plasma samples from several sources. The majority of the tested samples had little if any Substance P or its metabolites. A few samples gave detectable amounts of Substance P in concentrations below 5 pg/mL (parts-per-trillion). In contrast, many of the samples demonstrated immunoreactivity in ELISA tests in the 100 pg/mL concentration range. The stark difference between the results measured by the highly specific and sensitive LC-MS and methodologies based on immunoreactivity cast serious doubt on the ability of the ELISA tests to specifically and accurately measure Substance P and other neuropeptides. It is likely that the ELISA tests are detecting interference from other biological molecules in the piglet samples. Although the LC-MS method developed in the project meets the criteria for analytical reproducibility when “spikes” of a known concentration of piglet Substance P are analyzed, the fact that no significant amounts of Substance P were detected (despite repeated efforts) in piglet plasma suggests that this assay will not be useful for assessment of pain in piglets.