Accurate quantification of plasma glucagon levels in humans is necessary for understanding the physiological and pathological importance of glucagon. Although several immunoassays for glucagon are available, they provide inconsistent glucagon values owing to cross-reactivity of the antibodies with peptides other than glucagon. To overcome this limitation, we developed a novel method to measure glucagon levels by a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS) assay via parallel reaction monitoring (PRM) without immunoaffinity enrichment. Using stable isotope-labeled glucagon as an internal standard and 200 μL of plasma, the lower limit of quantification was 0.5 pM. This method was applied to measure plasma glucagon levels during the oral glucose tolerance test (OGTT) and meal tolerance test (MTT) in healthy volunteers, and its results were compared with those of sandwich enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). During the OGTT, this method showed significant suppression of plasma glucagon levels, and similar patterns were observed with sandwich ELISA and RIA. In contrast, during the MTT, plasma glucagon levels were slightly elevated according to the LC-MS/MS and sandwich ELISA results and were reduced according to RIA results. Our newly developed LC-MS/MS method overcomes a lack of specificity among currently available immunoassays for glucagon and may contribute to a better understanding of the importance of glucagon. Graphical abstract Flowchart for the extraction and quantification of glucagon in human plasma, and plasma glucagon responses in healthy volunteers quantified by the present LC-MS/MS, sandwich ELISA, and RIA during OGTT and MTT.
ContextAccurate glucagon level measurements are necessary for investigation of mechanisms for postprandial hyperglycemia in type 2 diabetes.ObjectiveTo evaluate the accuracy of postprandial glucagon level measurements using a sandwich ELISA vs a recently established liquid chromatography-high resolution mass spectrometry (LC-HRMS) method in type 2 diabetes mellitus.Design and ParticipantsTwenty patients with type 2 diabetes treated with insulin underwent a meal test before and after administration of the dipeptidyl peptidase-4 inhibitor anagliptin for 4 weeks. Blood samples were taken serially after the meal, and glucagon levels were measured using both ELISA and LC-HRMS. We compared the change from baseline to 4 weeks (Δ0–4W) using the area under the curve for plasma glucagon during the meal test [area under the curve (AUC)0–3h] measured using ELISA and LC-HRMS.ResultsELISA-based glucagon AUC0–3h was higher than LC-HRMS–based AUC0–3h at baseline and 4 weeks. However, differences in Δ0–4W-AUC0–3h measured using ELISA and LC-HRMS were not statistically significant. Additionally, Δ0–4W-AUC0–3h measured using ELISA and LC-HRMS were strongly correlated (r = 0.87, P < 0.001).ConclusionsPlasma glucagon levels during a meal test in patients with type 2 diabetes measured using ELISA were consistently higher than those measured using LC-HRMS. However, given that the changes in glucagon levels measured using ELISA before and after dipeptidyl peptidase-4 inhibitor therapy were similar to those based on LC-HRMS, this ELISA seems to be useful for evaluating the effect of the drug interventions on postprandial glucagon levels.
Glucagon is detected in plasma even after total pancreatectomy, and it is debated whether this glucagon is derived from the gastrointestinal tract. Here, we applied sandwich enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography–high‐resolution mass spectrometry to measure plasma glucagon levels in one patient after partial pancreatectomy (one‐seventh of the pancreas remaining) and three patients after total pancreatectomy. Sandwich ELISA detected higher glucagon levels in pancreatectomy patients than in healthy individuals. In contrast, liquid chromatography–high‐resolution mass spectrometry showed that plasma glucagon levels in pancreatectomy patients were below the lower limit of quantification. Plasma glucagon measured by sandwich ELISA showed a striking correlation with plasma glicentin, suggesting cross‐reaction with this gastrointestinal glucagon‐related peptide. These results indicated that pancreatectomized patients falsely showed pseudo‐hyperglucagonemia when measured by glucagon sandwich ELISA.
Glucagon has an important role in glucose homeostasis. Recently, a new plasma glucagon assay based on liquid chromatography-high resolution mass spectrometry was developed. We evaluated the influence of a dipeptidyl peptidase-4 inhibitor (anagliptin) on plasma glucagon levels in Japanese patients with type 2 diabetes by using this new assay. Methods: Twenty-four patients with type 2 diabetes were enrolled in a prospective, singlecenter, randomized, open-label study and were randomly allocated to 4 weeks of treatment with metformin (1000 mg/day) or anagliptin (200 mg/day). A liquid test meal labeled with sodium [ 13 C] acetate was ingested before and after the treatment period. Samples of blood and expired air were collected over 3 h. Plasma levels of glucose, glucagon, C-peptide, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were measured, and gastric emptying was also evaluated. Results: Twenty-two patients completed the study (metformin group: n = 10; anagliptin group: n = 12). Glycemic control showed similar improvement in both groups. In the anagliptin group, there was a slight decrease of the incremental area under the plasma concentration versus time curve for glucagon after the test meal (P = 0.048). In addition, the plasma level of active GLP-1 and GIP was increased, and plasma C-peptide was also increased versus baseline. Neither anagliptin nor metformin delayed gastric emptying. Conclusions: In patients with type 2 diabetes maintained endogenous insulin secretion, anagliptin increased the plasma level of active GLP-1 and GIP in association with a slight stimulation of insulin secretion and slight inhibition of glucagon secretion, but did not delay gastric emptying.
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