DPP-4 activity in plasma was determined using a continuous spectrophotometric assay with the substrate Gly-Pro-pNA (Bachem, Bubendorf, Switzerland), as has been reported previously.9 The assay was based on the ability of DPP-4 to cleave the substrate Gly-Pro-pNA into pNA, resulting in an increase in absorbance at 390 nm, where the absorbance was expressed as milli optical density (mOD). Enzymatic activity was calculated as the slope (in mOD/min) from 4 to 14 minutes. Intra- and inter-assay precisions were 2.28%–7.57% and 3.64%–12.75%, respectively. The lower limit of quantification was 1.43 mOD/min.
Gly pro pna
Manufactured by Bachem
Sourced in Switzerland
Gly-Pro-pNA is a laboratory reagent used as a substrate for the enzymatic detection and measurement of dipeptidyl peptidase IV (DPP-IV) activity. It serves as a chromogenic or fluorogenic substrate, allowing for the quantification of DPP-IV activity through colorimetric or fluorometric methods.
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3 protocols using gly pro pna
1
Spectrophotometric Assay for DPP-4 Activity
2
Quantifying DPP-4 Activity and Metabolic Markers
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To determine DPP-4 activity, we used a continuous spectrophotometric assay with the substrate, Gly-Pro-pNA (Bachem, Bubendorf, Switzerland).16 The intra- and inter-assay precisions were 1.9%–8.3% and 3.5%–5.5%, respectively. The measurements of plasma DPP-4 activity were expressed as the percentage change from baseline DPP-4 activity, which was determined before the first dosing. We quantified the active GLP-1, glucose, glucagon, insulin, and C-peptide levels as previously described.15
The individual pharmacodynamic parameters for each treatment group were calculated with the noncompartmental method. For DPP-4 activity measurements, we calculated the area under the effect–time curve over the dosing interval at steady state (AUECτ,ss) and the maximum (inhibitory) effect on DPP-4 activity at steady state (Emax,ss). These were measured both before (baseline) and after treatment with EVO and EVO + MET. For GLP-1, glucose, glucagon, insulin, and C-peptide measurements, we calculated the areas under the concentration–time curves (AUCs) over the dosing interval. These were measured before (baseline) and after treatment with EVO, MET, and EVO + MET. We used the linear trapezoidal method to calculate the AUECτ,ss and the AUCs. The observed values were used to calculate the Emax,ss.
The individual pharmacodynamic parameters for each treatment group were calculated with the noncompartmental method. For DPP-4 activity measurements, we calculated the area under the effect–time curve over the dosing interval at steady state (AUECτ,ss) and the maximum (inhibitory) effect on DPP-4 activity at steady state (Emax,ss). These were measured both before (baseline) and after treatment with EVO and EVO + MET. For GLP-1, glucose, glucagon, insulin, and C-peptide measurements, we calculated the areas under the concentration–time curves (AUCs) over the dosing interval. These were measured before (baseline) and after treatment with EVO, MET, and EVO + MET. We used the linear trapezoidal method to calculate the AUECτ,ss and the AUCs. The observed values were used to calculate the Emax,ss.
3
Extraction and Characterization of DPP-IV from Rat Intestine
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DPP-IV was extracted from rat intestine segments as previously described. 6 Briefly, intestine segments were homogenized using lysis buffer (PBS containing 100 KIU mL -1 aprotinin and 1% Triton X-100). Then, the obtained samples were centrifuged at 1000g at 4 °C for 10 min to eliminate the cellular debris, then centrifuged twice at 20 000g at 4 °C for 10 min. Supernatants were stored at -80 °C until analysis.
To determine DPP-IV activity, intestinal lysates were incubated with 0.2 mMchromogenic substrate Gly-Pro-pNA (Bachem, Bubendorf, Switzerland) in Tris-HCl buffer at 37 °C. The mixture was measured at 405 nm at 37 °C for 30 min in a microplate reader.
To determine DPP-IV activity, intestinal lysates were incubated with 0.2 mMchromogenic substrate Gly-Pro-pNA (Bachem, Bubendorf, Switzerland) in Tris-HCl buffer at 37 °C. The mixture was measured at 405 nm at 37 °C for 30 min in a microplate reader.
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