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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. [ABSTRACT FROM AUTHOR]

Context Accurate glucagon level measurements are necessary for investigation of mechanisms for postprandial hyperglycemia in type 2 diabetes. Objective To 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 Participants Twenty 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 (Δ 0–4W) 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. Results ELISA-based glucagon AUC0–3h was higher than LC-HRMS–based AUC0–3h at baseline and 4 weeks. However, differences in Δ 0–4W-AUC0–3h measured using ELISA and LC-HRMS were not statistically significant. Additionally, Δ 0–4W-AUC0–3h measured using ELISA and LC-HRMS were strongly correlated (r = 0.87, P < 0.001). Conclusions Plasma 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. [ABSTRACT FROM AUTHOR]

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [13C2,15N2]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [13C2,15N2]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [13C2,15N2]xanthine as a substrate, [13C2,15N2]UA as an analytical standard, and [13C3,15N3]UA as an internal standard. The [13C2,15N2]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity ( R2 = 0.998, weighting of 1/ x2) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma. [ABSTRACT FROM AUTHOR]

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2)  = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma., (Copyright © 2016 John Wiley & Sons, Ltd.)

Through the application of TRAP (target-related affinity profiling), we identified a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-dependent and selective with regard to other chemokines. The compounds, however, did not antagonize the binding of (125)I-labeled CCL2 to the CCR2 receptor nor did they block CCR2-mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC(50) = 80 nM) with excellent oral bioavailability in rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.