Optimized determination of angiotensin I-converting enzyme activity with hippuryl-L-histidyl-L-leucine as substrate.
The determination of the angiotensin I-converting enzyme activity (ACE, kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) is necessary to control the course and the treatment of sarcoidosis, as well as to monitor the therapeutic use of enzyme inhibitors such as captopril in hypertension or congestive heart failure. Numerous synthetic substrates are known with which to measure the enzyme activity. A discontinuous method using hippuryl-L-histidyl-L-leucine was tested and improved. The cleavage product, hippurate, reacts with cyanuric chloride to give a yellow complex which can be measured at 405 nm using a spectral line photometer. Enzyme activity, kinetic constants and activation energy are dependent on the chloride ion concentration. Optimal test concentrations are 1.1 mol/l potassium chloride and 3.0 mmol/l hippuryl-L-histidyl-L-leucine at pH 8.3. Higher substrate concentrations effect an inhibition of the enzyme reaction. A Michaelis constant of 0.9 mmol/l was found with serum as enzyme source. An activation energy of 57 kJ/mol was obtained from the relation between the logarithm of velocity of enzyme reaction and reciprocal value of absolute temperature. Furthermore, a linear dependence on chloride ion concentration was observed. The histogram of the enzyme activities in sera from 146 healthy volunteers shows a non-gaussian distribution. The reference interval at 25 degrees C is characterized by a median of 24 units/l with the 2.5th and the 97.5th percentiles at 13 units/l and 42 units/l, respectively. The corresponding values at 37 degrees C are 27 units/l and 86 units/l with a median of 48 units/l. No significant sex and age dependence could be found. A potent ACE inhibitor such as captopril leads to a rapid decrease of the enzyme activity within 60 min after oral administration. In the following hours, the enzyme activity slowly increases.
Schnaith
E
E
Institut für Klinische Chemie und Laboratoriumsdiagnostik, Heinrich-Heine-Universität, Düsseldorf, Germany.
Beyrau
R
R
Bückner
B
B
Klein
R M
RM
Rick
W
W
D002863:Chromogenic Compounds / Q000378:metabolism*; D006801:Humans; D006973:Hypertension / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D012016:Reference Values; D012507:Sarcoidosis / Q000201:enzymology; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods
D002863:Chromogenic Compounds; D009842:Oligopeptides; C010980:hippuryl-histidyl-leucine; D007703:Peptidyl-Dipeptidase A