Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid

• Published in: Analytical biochemistry Vol. 364; no. 2; pp. 104 - 111
• Main Authors: Lam, Le Hoang, Shimamura, Tomoko, Sakaguchi, Ken, Noguchi, Katsuya, Ishiyama, Munetaka, Fujimura, Yume, Ukeda, Hiroyuki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.05.2007
• Subjects: 3-Hydroxybutyric acid; 3-Hydroxybutyric Acid - analysis; 3-Hydroxybutyric Acid - chemical synthesis; 3-Hydroxybutyric Acid - chemistry; 3HB-GGG; ACE; Angiotensin-Converting Enzyme Inhibitors - chemistry; Angiotensin-Converting Enzyme Inhibitors - classification; Animals; Antihypertension; Biochemistry - methods; Calibration; Captopril - chemistry; Drug Evaluation, Preclinical - methods; Fagopyrum - chemistry; Feasibility Studies; Food Analysis - methods; Formazans - chemistry; Garlic - chemistry; Hippurates - chemistry; Hydrolysis; Hydroxybutyrates; Indicators and Reagents; Inhibitory Concentration 50; Molecular Structure; Oligopeptides - analysis; Oligopeptides - chemical synthesis; Oligopeptides - chemistry; Rabbits; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry; Substrate Specificity; Antihypertension; ACE; 3HB-GGG; 3-Hydroxybutyric acid
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.02.017

Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters—ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 °C for 30 min—the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.