Development of a new assay for measurement of total angiotensin-converting enzyme after inhibition.
1. We set out to develop a simple, rapid, highly sensitive and reproducible assay to quantify total angiotensin-converting enzyme (ACE) in serum and tissues after ACE inhibition. 2. Total ACE was measured by dissociating ACE inhibitor from the enzyme using zinc chelation (EDTA). The enzyme reconstituted with zinc ion (ZnSO4) was then measured by enzymatic fluorimetric assay. Angiotensin-converting enzyme inhibition was produced in vitro by incubation of enalaprilat or perindoprilat with human serum or cell membranes from rat heart. To achieve the highest recovery rate, we studied concentrations of EDTA from 1 to 300 mmol/L with a pH range from 4 to 13, where the incubation times of EDTA were between 1 and 12 h. After washing off ACE inhibitor and excess EDTA, the resulting concentrates and pellets were then resuspended with Tris buffer containing ZnSO4 to restore ACE activity. 3. The optimal assay conditions to dissociate the drug from the enzyme were 300 mmol/L EDTA in Tris buffer at pH 11 for 12 h with cell membranes and 23 mmol/L EDTA at the same pH incubated for 4 h with serum. The recovery of ACE activity was 81.7 +/- 15.8 and 97.3 +/- 2.9% in tissues and serum, respectively. Intra- and interassay variability coefficients were 5.6 and 12.8% in tissue, respectively, 3.2 and 13.0% in serum, respectively. The method allows quantification of ACE in 100 microliters or less serum and 20 mg or less tissue. 4. The total ACE concentration after ACE inhibition can be determined by this assay, which is suitable for micro-sampling studies of both tissue and serum ACE levels.
Yang
Y
Y
Hypertension Laboratory, Prince Henry Hospital, University of New South Wales, Australia. Y.Yang@unsw.edu.au
Macdonald
G J
GJ
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D001681:Biological Assay; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D015773:Enalaprilat / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008297:Male; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000806:Angiotensin-Converting Enzyme Inhibitors; D002614:Chelating Agents; D007211:Indoles; C053500:perindoprilat; D004492:Edetic Acid; D007703:Peptidyl-Dipeptidase A; D015773:Enalaprilat