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

Hyunyee Yoon, Su Hee Cho, Yu Rim Seo, Kyung Sang Yu, Sung Sup Park, Moon Jung Song

Research output: Contribution to journalArticlepeer-review

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 × 104–1.86 × 106 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.

Original languageEnglish
Article number113952
JournalAnalytical Biochemistry
Volume612
DOIs
StatePublished - 1 Jan 2021

Keywords

  • Dipeptidyl peptidase 4 (DPP4)
  • Enzymatic assay
  • Evogliptin (DA-1229) tartrate
  • Human plasma
  • Type 2 diabetes mellitus (T2DM)

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