Competitive Inhibitor Binding Assay (IBA) of Captopril and Other Inhibitors of Angiotensin Converting Enzyme

Abstract

We developed a novel assay method for the determination of captopril and enalapril serum concentrations. This method is based on a new principle of enzyme assay, inhibitor binding assay (IBA), recently introduced by us for the measurement of serum angiotensin converting enzyme (ACE). The assay principle is based on binding of ¹²⁵I-labelled ACE inhibitor (351A, p-hydroxy-benzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) to ACE. A competitive IBA was established using captopril as a standard and human serum ACE as a binder. For the assay of captive captopril, 0.1 ml of venous blood was taken into 9.9 ml of ice-cold buffer. Following separation of cells, the supernatant was stored at –20°C until assayed. Tubes containing added serum ACE as a binder, labelled inhibitor, and unknown samples, were incubated for 1 hour at 37°C. Following separation of bound label the content of captopril was read from a standard curve. Results in healthy subjects showed peak blood concentrations ranging from 220 to 1300 ng/ml within 30–60 minutes after a single oral dose of 50 mg captopril, and virtual disappearence of active drug from the blood within 3 hours. About 50 % of the measurable active captopril was lost during storage of diluted plasma for one month at –20°C The assay method is simple and sensitve, and measures active drug only. The present new assay principle may prove convenient and feasible for the assay of captopril and other ACE inhibitors in biological fluids.