Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions

Copyright © 2014 John Wiley & Sons, Ltd. RATIONALE Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS Glucagon < inf > 3-29 < /inf > and [pGlu] < sup > 3 < /sup > glucagon < inf > 3-29 < /inf > were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu] < sup > 3 < /sup > glucagon < inf > 3-29 < /inf > as well as glucagon < inf > 19-29 < /inf > , or 'miniglucagon', along with the novel metabolites glucagon < inf > 20-29 < /inf > and glucagon < inf > 21-29 < /inf > . The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated.