Preanalytical impact on the accuracy of measurements of glucagon, GLP-1 and GIP in clinical trials

Abstract

Background

Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans.

Methods

Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at −80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA).

Results

Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin.

Conclusions

The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials.

Keywords:

• Preanalytics
• glucagon
• GLP-1
• GIP
• diabetes

Acknowledgement

The illustrations in supplementary figure 1 was created with Biorender.com. The data has been presented at the American Diabetes Association conference in 2022.

Author contribution

CR, SASK and NJWA conceived the study idea. CR wrote the first draft of the manuscript. SASK, MMR, NJJ and NH performed, and CR assisted in conducting the experimental study days. CR, BH and JJH measured glucagon, GLP-1 and GIP. All authors revised and accepted the final version of the manuscript.

Disclosure statement

Nicolai J. Wewer Albrechtsen has received research support and speaker fees from Mercodia, Novo Nordisk, Merck, MSD, and Boehringer Ingelheim.

Data availability statement

Data may be shared upon reasonable request and following approval by the Danish Data Protection Agency.

Additional information

Funding

Associate Prof. Nicolai J. Wewer Albrechtsen is supported by NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001), EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude (1052-00003B). NNF Center for Protein Research is supported financially by the NNF (grant agreement NNF14CC0001) and MSc Sasha A S Kjeldsen is supported by Aase og Ejnar Danielsens Fond (21-10-0287) and A. P. Møller Fonden (2021-00683).