*Corresponding author email: jjholst@sund.ku.dk Symbiosis Group Symbiosis www.symbiosisonline.org www.symbiosisonlinepublishing.com No Effect of Aprotinin (Trasylol™) on Degradation of Exogenous and Endogenous Glucagon in Human, Mouse and Rat Plasma Monika J Bak 1,2# , Nicolai Wewer Albrechtsen 1# , Bolette Hartmann 1 , Jens Pedersen 1 , Mikkel Christensen 3 , Tina Vilsboll 3 , Filip Knop 3 , Carolyn F Deacon 1 , Lars O Dragsted 2 and Jens J Holst 1 * 1 NNF Center for Basic Metabolic Research, Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark 2 Departments of Life Science, Faculty of Human Nutrition, University of Copenhagen, DK-2200 Copenhagen, Denmark 3 Department of Internal Medicine, Gentofte Hospital, DK-2900 Hellerup, Denmark # Contributed equally to the preparation of this manuscript Journal of Endocrinology and Diabetes Open Access Research Article With the development of the radioimmunoassay (RIA) technique in 1956 [5], it became possible to measure endogenous glucagon concentrations [6]. At that time, it was speculated that enzymatic degradation of glucagon might occur during storage and handling of the plasma samples, and the addition of the protease inhibitor, aprotinin, to blood samples has since become standard procedure before glucagon measurements [7]. The first glucagon RIAs were developed using the isotope I 131 , which has a short half-life of 8 days, to label the tracer, and Eisentraut et al. observed that addition of aprotinin was essential for prevention of the damaging effect of human plasma on the glucagon tracer (but was not necessary for the protection of unlabeled, endogenous glucagon [8]). Heding [9] and Nonaka and Foa [10], however, both described a significant loss of endogenous glucago฀n levels if samples were not treated with aprotinin. Other proteolytic inhibitors (such as benzamidine) have also been investigated and recommended for reducing glucagon degradation in plasma [11]. In contrast to these early assays, tracers used today are prepared using I 125 , which has a half-life of 60 days, but it has never been systematically examined whether aprotinin is still required in modern-day glucagon assays. The aim of this study was, therefore, to investigate whether aprotinin (Trasylol™) prevents glucagon degradation. We studied possible degradation of exogenous glucagon in rat, mouse and human plasma, as well as endogenous glucagon in blood samples from T2DM patients obtained during fasting and hypoglycemia (stimulating secretion). Materials and Methods Peptides and aprotinin (Trasylol™) Synthetic glucagon 1-29 (No.: H-6790) was obtained from Bachem, Switzerland, and dissolved in phosphate buffer. The concentration of glucagon in prepared solutions was confirmed Introduction Glucagon is the counter-regulatory hormone to insulin regarding their actions on the liver. Glucagon is released from the pancreatic α-cells in response to hypoglycemia and stimulates hepatic glucose output. It is composed of 29 amino acids and is liberated from the precursor molecule, proglucagon, by tissue- specific posttranslational cleavage by prohormone convertase (PC2) in the pancreatic α-cells [1,2]. Neutral endopeptidase 24.11 (NEP) and plasmin activities have been linked to the short half- life (around 5 min) of glucagon in vivo, but evidence points to the kidney as the main site of metabolic clearance [3,4]. Abstract Traditionally, aprotinin (Trasylol™) has been added to plasma samples prior to glucagon analysis. However, the evidence for the need of aprotinin is sparse and based on results obtained when radioimmunoassay (RIA) techniques were still in their infancy. Using RIAs directed against both the C-terminus and a mid-region of glucagon, we challenged the classical view that aprotinin is necessary. Glucagon concentrations in pools of human, mouse and rat plasma (n=30, 25 and 16 of each, respectively) with and without addition of increasing amounts of exogenous glucagon (5, 10, 20, 40 and 60 pM) were similar irrespective of whether or not aprotinin had been added. To investigate whether individual variation occurs in human samples, we measured plasma from 20 patients with gastrointestinal diseases and 20 healthy subjects with or without addition of aprotinin. Again, measured amounts of glucagon, endogenous or added, were not affected by the presence of aprotinin. The effect of aprotinin, present at blood sampling or added later (30 and 60 minutes), on endogenous glucagon values was investigated in T2DM patients (n=5), before and after insulin-induced hypoglycemia. There were no differences between the four treatments. In conclusion, we found no support for use of aprotinin for prevention of glucagon degradation. Keywords: Trasylol™; Aprotinin; Glucagon assay; Radioimmunoassay (RIA); Peptide degradation Received: 19.11.2013, Accepted: 20.01.2014, Published: 22.01.2014 * Corresponding author: Jens Juul Holst, Dept of Biomedical Sciences, the Panum Institute Blegdamsvej 3,bldg 12.2, DK-2200 Copenhagen N, DK, Den- mark, Tel: +45 28757518; E-mail: jjholst@sund.ku.dk