No Effect of Aprotinin (Trasylol) on Degradation of Exogenous and Endogenous Glucagon in Human, Mouse and Rat Plasma

Jens Juul Holst

doi:10.15226/2374-6890/1/1/00101

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.

Highlights

Glucagon is the counter-regulatory hormone to insulin regarding their actions on the liver
The first glucagon RIAs were developed using the isotope I131, 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
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

Summary

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 tissuespecific posttranslational cleavage by prohormone convertase (PC2) in the pancreatic α-cells [1,2]. Other proteolytic inhibitors (such as benzamidine) have been investigated and recommended for reducing glucagon degradation in plasma [11]. In contrast to these early assays, tracers used today are prepared using I125, 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

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