Assessment and optimization of kinetic methods for angiotensin-converting enzyme in plasma

Abstract

In the kinetic angiotensin-converting enzyme (ACE) method, a practical and optimal buffer is 80 mmol/L borate buffer at pH 8.2 (37 °C). A lag phase is detected in the reaction, and a 5-min incubation of substrate and plasma is suggested before the kinetic measurement. The substrate, N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG), concentration is maximized at 1.0 mmol/L and the measurement wavelength is at 345 nm to ensure linearity of measurement. The proposed procedure uses a 1:9 plasma-to-reagent volume ratio. The linear range of the assay extends to ∼170 U/L, representing a 25% substrate hydrolysis. The FAPGG absorptivity is determined by measuring the difference in absorbance between 1.0 mmol/L FAPGG and the product solutions. The wavelength fidelity is checked by noting the expected absorbance value of the FAPGG solution, and a 1.0-nm deviation from 345 nm alters the absorbance by 15.5%. The precision of ACE assays at ∼60 and 100 U/L is 3.5% and 2.4% within batch and 2.9% and 2.6% between batch, respectively. The reference interval (2.5th to 97.5th percentiles) is 41-139 U/L, and there is no difference between values for men and women.