To investigate the stability of glucagon-like peptide 1 (GLP-1) and glucagon in plasma under short- and long-term storage conditions. Pooled human plasma (n=20), to which a dipeptidyl peptidase 4 (DPP4) inhibitor and aprotinin were added, was spiked with synthetic GLP-1 (intact, 7–36NH2 as well as the primary metabolite, GLP-1 9–36NH2) or glucagon. Peptide recoveries were measured in samples kept for 1 and 3 h at room temperature or on ice, treated with various enzyme inhibitors, after up to three thawing–refreezing cycles, and after storage at −20 and −80 °C for up to 1 year. Recoveries were unaffected by freezing cycles or if plasma was stored on ice for up to 3 h, but were impaired when samples stood at RT for more than 1 h. Recovery of intact GLP-1 increased by addition of a DPP4 inhibitor (no ice), but was not further improved by neutral endopeptidase 24.11 inhibitor or an inhibitor cocktail. GLP-1, but not glucagon, was stable for at least 1 year. Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared with immediate analysis, regardless of storage time. Plasma handling procedures can significantly influence results of subsequent hormone analysis. Our data support addition of DPP4 inhibitor for GLP-1 measurement as well as cooling on ice of both GLP-1 and glucagon. Freeze–thaw cycles did not significantly affect stability of GLP-1 or glucagon. Long-term storage may affect glucagon levels regardless of storage temperature and results should be interpreted with caution.
Nicolai J Wewer Albrechtsen, Monika J Bak, Bolette Hartmann, Louise Wulff Christensen, Rune E Kuhre, Carolyn F Deacon, and Jens J Holst
Justyna Modrzynska, Christine F Klein, Kasper Iversen, Henning Bundgaard, Bolette Hartmann, Maike Moss, Nikolaj Rittig, Niels Moeller, Jens J Holst, and Nicolai J Wewer Albrechtsen
Objective: Glucagon and glucagon-like peptide-1 (GLP-1) originate from the common precursor, proglucagon, and their plasma concentrations have been reported to be increased during inflammatory conditions. Increased blood glucose levels are frequently observed in septic patients, and therefore we hypothesized that glucagon, but not GLP-1, is increased in individuals with inflammation.
Design: Prospective longitudinal cohort study.
Materials and Methods: We measured glucagon and GLP-1 in plasma sampled consecutively in three cohorts consisting of patients with infective endocarditis (n=16), urosepsis (n=28) and post-operative inflammation following percutaneous aortic valve implantation or thoracic endovascular aortic repair (n=5). Correlations between C-Reactive Protein (CRP), a marker of systemic inflammation, and glucagon and GLP-1 concentrations were investigated. Additionally, glucagon and GLP-1 concentrations were measured after a bolus infusion of lipopolysaccharide (LPS, 1ng/kg) in nine healthy young males.
Results: Glucagon and CRP were positively and significantly correlated (r=0.27; P=0.0003), whereas no significant association between GLP-1 and CRP was found (r=0.08, P=0.30). LPS infusion resulted in acute systemic inflammation reflected by increased temperature, pulse, tumor necrosis factor-α (TNFα), interleukin-6 (IL-6) and concomitantly increased concentrations of glucagon (P<0.05) but not GLP-1.
Conclusions :Systemic inflammation caused by bacterial infections or developed as a non-infected condition is associated with increased plasma concentration of glucagon, but not GLP-1. Hyperglucagonemia may contribute to the impaired glucose control in patients with systemic inflammatory diseases.