Abstract 365: Stabilization of incretin in human plasma sample
Authors: Yi Jizu , Warunek David , Liu Zhaoxia , Craft David 
Published: June 2012

Incretin peptides, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), possess multiple physiological roles that make them important peptide biomarkers for metabolic disorder. GLP-1 and GIP contribute to approximately 60 -70 % of the total postprandial insulin response in healthy individuals, and potentially have a therapeutic value in treatment of type II diabetes. Glucagon-like peptides (GLP-1, and GLP-2) regulate cell proliferation, differentiation, and apoptosis. GLP-1 also contributes to physiological roles in controlling energy homoestasis and balance through both peripheral signals and brain stem regulations of appetite in the nucleus of the solitary tract (NTS). Circulating GLP-1 acts to inhibit food intake, acute GLP-1 reduces calorie intake, and GLP-1 neuronal circuits of NTS may have anorectic effect in energy homeostasis.

However, the incretin effect is significantly decreased in patients with type 2 diabetes, delaying and reducing insulin release after oral glucose administration. It is well documented that circulating GLP-1 and GIP after their secretion are rapidly digested by the intrinsic blood enzyme dipeptidyl peptidase-IV (DPP-IV). The half-life of these two peptides in in vivo plasma is very short: approximately 2 minutes for intact GLP-1 and 5 minutes for intact GIP.

Ex vivo stabilization of full functions of these peptides is critical for their utility in diagnostics and/or drug development. We directly investigated the stability of these peptides and their metabolites in conventional serum, EDTA plasma, or EDTA plasma with protease inhibitors as stabilizers. Using spiked blood samples, peptides were monitored by time-course MALDI-TOF MS. Mass spectrometry allows detection of the intact peptides for kinetic analysis as well as identification of the cleaved peptides. The results indicate that degradation of GLP-1 fits to a simple Michaelis-Menton Equation (Km = 21.36 nM and Vm = 7.05 nM), while GIP fits an observed first-order reaction, and both having the greatest stability in the inhibited plasma sample. The half-life of GLP-1 peptide was also dependence of the peptide concentration, with lower concentration having less half-life time. Identification of the cleaved peptides indicates that both intrinsic DPP-IV and exo-carboxypeptidase contribute to the digestion of active GLP-1 in ex vivo sample. Further, our study indicates that stabilization of both GLP-1 and GIP was accomplished by including a cocktail of enzyme inhibitors in a blood-collection tube, allowing accurate measurement for biomarker development. We also conducted an evaluation of two commercially available ELISA kits for GLP-1 and GIP demonstrating some kits are sensitive to peptidase degradation while others are not.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2011-365