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Optimization and validation of a fluorogenic dipeptidyl peptidase 4 enzymatic assay in human plasma

Authors
Yoon, HyunyeeCho, Su HeeSeo, Yu RimYu, Kyung-SangPark, Sung SupSong, Moon Jung
Issue Date
1-1월-2021
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Keywords
Dipeptidyl peptidase 4 (DPP4); Enzymatic assay; Evogliptin (DA-1229) tartrate; Human plasma; Type 2 diabetes mellitus (T2DM)
Citation
ANALYTICAL BIOCHEMISTRY, v.612
Indexed
SCIE
SCOPUS
Journal Title
ANALYTICAL BIOCHEMISTRY
Volume
612
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/50167
DOI
10.1016/j.ab.2020.113952
ISSN
0003-2697
Abstract
During the development of a specific dipeptidyl peptidase 4 (DPP4) inhibitor to treat type 2 diabetes, a fluorogenic kinetic analysis for DPP4 enzymatic activity using Gly-Pro-Aminomethylcoumarin (AMC) as a substrate was optimized and validated for recombinant DPP4 and human plasma samples. The sensitivity, calibration curve, detection range, accuracy, precision, recovery efficiency, Km constant, short/long-term stability, and stability after freezing-thawing cycles were analyzed. DPP4 enzymatic activity (mU/min) was measured as the initial velocity (Vo) of the enzymatic reaction over time. The sensitivity of the Vo value was 14,488 mU/min for recombinant DPP4 and 17,995 mU/min for human plasma samples. The dynamic ranges of the calibration curve were linear and reliable between 1.11 x 10(4)-1.86 x 10(6) mU/min of the mean Vo value and in the DPP4 concentration range of 23.4-3,000 ng/mL. The assay's accuracy and precision met acceptance criteria for all samples. Plasma DPP4 was stable under various storage temperatures, even after three freeze-thaw cycles. Our optimized, validated bioanalytic method for measuring DPP4 activity in plasma samples was successfully employed to evaluate the effect of evogliptin (DA-1229) tartrate, which irreversibly and dose-dependently inhibits DPP4 enzymatic activity, without the dilution effect of human plasma samples and irrespective of the co-treated metformin.
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