Specificity and sensitivity of commercially available assays for glucagon and oxyntomodulin measurement in humans

Abstract

Aim: To determine the specificity and sensitivity of assays carried out using commercially available kits for glucagon and/or oxyntomodulin measurements. Methods: Ten different assay kits used for the measurement of either glucagon or oxyntomodulin concentrations were obtained. Solutions of synthetic glucagon (proglucagon (PG) residues 33-61), oxyntomodulin (PG residues 33-69) and glicentin (PG residues 1-69) were prepared and peptide concentrations were verified by quantitative amino acid analysis and a processing-independent in-house RIA. Peptides were added to the matrix (assay buffer) supplied with the kits (concentration range: 1.25-300 pmol/l) and to human plasma and recoveries were determined. Assays yielding meaningful results were analysed for precision and sensitivity by repeated analysis and ability to discriminate low concentrations.

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

“…Peaks 3, 4, and 5 were confirmed as glucagon [21][22][23][24][25][26][27][28][29], glucagon 20-29, and glucagon [19][20][21][22][23][24][25][26][27][28][29] to within 3.8 ppm. These peaks showed lower mass accuracies than the other metabolites, which is likely to be due to their lower abundances limiting mass spectrometer signal.…”

“…Glucagon [19][20][21][22][23][24][25][26][27][28][29] , or miniglucagon, has previously only been identified in liver plasma membranes [6] and pancreatic cells [7] , whereas glucagon [20][21][22][23][24][25][26][27][28][29] , and glucagon [21][22][23][24][25][26][27][28][29] have not been previously reported. Miniglucagon, has its own unique biological activity, which is opposite to that of gluagon , and thus acts as a modulator [8] .…”

“…Further studies are needed to determine whether glucagon [20][21][22][23][24][25][26][27][28][29] bonds [33] . However other studies have suggested that IDE cleaves at Arg 17 -Arg 18 bonds with the formation of glucagon [18][19][20][21][22][23][24][25][26][27][28][29] , which may then be converted into glucagon [19][20][21][22][23][24][25][26][27][28][29] by an unidentified aminopeptidase like enzyme [34] . It is possible that such residual hepatocyte enzymes are present in plasma leading to the formation of glucagon [19][20][21][22][23][24][25][26][27][28][29] , and perhaps lead to the formation of glucagon [20]…”

“…Glucagon [1][2][3][4][5][6][7][8][9][10][11][12][13] and glucagon [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] , and the minor metabolites glucagon [1][2][3][4][5][6][7][8][9][10] , glucagon [14][15][16][17][18][19][20][21][22][23][24][25] and glucagon [23][24]…”

“…immunoassay kits [19] and in 1/3 sandwich ELISA kits [20] recently evaluated. It could also help to explain the 7-fold difference in endogenous glucagon concentrations reported by immunoassays directed against the middle or C-terminal regions of glucagon [21] .…”