The RAPID Method for Blood Processing Yields New Insight in Plasma Concentrations and Molecular Forms of Circulating Gut Peptides

The correct identification of circulating molecular forms and measurement of peptide levels in blood entails that the endocrine peptide being studied is stable and recovered in good yields during blood processing. However, it is not clear whether this is achieved in studies using standard blood processing. Therefore, we compared peptide concentration and form of 12 I-125-labeled peptides using the standard procedure (EDTA-blood on ice) and a new method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls, and Dilution (RAPID). During standard processing there was at least 80% loss for calcitonin-gene-related peptide and cholecystokinin-58 (CCK-58) and more than 35% loss for amylin, insulin, peptide YY forms (PYY(1-36) and PYY(3-36)), and somatostatin-28. In contrast, the RAPID method significantly improved the recovery for 11 of 12 peptides (P < 0.05) and eliminated the breakdown of endocrine peptides occurring after standard processing as reflected in radically changed molecular forms for CCK-58, gastrin-releasing peptide, somatostatin-28, and ghrelin. For endogenous ghrelin, this led to an acyl/total ghrelin ratio of 1: 5 instead of 1: 19 by the standard method. These results show that the RAPID method enables accurate assessment of circulating gut peptide concentrations and forms such as CCK-58, acylated ghrelin, and somatostatin-28. Therefore, the RAPID method represents an efficacious means to detect circulating variations in peptide concentrations and form relevant to the understanding of physiological function of endocrine peptides. (Endocrinology 150: 5113-5118, 2009)