Subject Standardization, Acclimatization, and Sample Processing Affect Gut Hormone Levels and Appetite in Humans

Background & Aims: Gut hormones represent attractive therapeutic targets for the treatment of obesity and type 2 diabetes. However, controversy surrounds the effects that adiposity, dietary manipulations, and bariatric surgery have on their circulating concentrations. We sought to determine whether these discrepancies are due to methodologic differences. Methods: Ten normal-weight males participated in a 4-way crossover study investigating whether fasting appetite scores, plasma aryl-ghrelin, active glucagon-like peptide-1 (GLP-1), and peptide YY3-36 (PYY3-36) levels are altered by study-induced stress, prior food consumption, and sample processing. Results: Study visit order affected anxiety, plasma cortisol, and temporal profiles of appetite and plasma PYY3-36, with increased anxiety and cortisol concentrations on the first study day. Plasma cortisol area under the curve (AUC) correlated positively with plasma PYY3-36 AUC. Despite a 14-hour fast, baseline hunger, PYY3-36 concentrations, temporal appetite profiles, PYY3-36 AUC, and active GLP-1 were affected by the previous evening's meal. Sample processing studies revealed that sample acidification and esterase inhibition are required when measuring acylghrelin and dipeptidyl-peptidase IV inhibitor addition for active GLP-1. However, plasma PYY3-36 concentrations were unaffected by addition of dipeptidyl-peptidase TV. Conclusions: Accurate assessment of appetite, feeding behavior, and gut hormone concentrations requires standardization of prior food consumption and subject acclimatization to the study protocol. Moreover, because of the labile nature of aryl-ghrelin and active GLP-1, specialized sample processing needs to be undertaken.