Optimized LC-MS/MS Method for the Detection of ppCCK(21-44): A Surrogate to Monitor Human Cholecystokinin Secretion.

The hormone cholecystokinin (CCK) is secreted postprandially from duodenal enteroendocrine cells and circulates in the low picomolar range. Detection of this digestion and appetite-regulating hormone currently relies on the use of immunoassays, many of which suffer from insufficient sensitivity in the physiological range and cross-reactivity problems with gastrin, which circulates at higher plasma concentrations. As an alternative to existing techniques, a liquid chromatography and mass spectrometry-based method was developed to measure CCK-derived peptides in cell culture supernatants. The method was initially applied to organoid studies and was capable of detecting both CCK8 and an N-terminal peptide fragment (prepro) ppCCK(21-44) in supernatants following stimulation. Extraction optimization was performed using statistical modeling software, enabling a quantitative LC-MS/MS method for ppCCK(21-44) capable of detecting this peptide in the low pM range in human plasma and secretion buffer solutions. Plasma samples from healthy individuals receiving a standardized meal (Ensure) after an overnight fast were analyzed; however, the method only had sensitivity to detect ppCCK(21-44). Secretion studies employing human intestinal organoids and meal studies in healthy volunteers confirmed that ppCCK(21-44) is a suitable surrogate analyte for measuring the release of CCK in vitro and in vivo. ; BBSRC iCASE studentship