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 substrate s are known with which to measure the enzyme activity. A discontinuous method using hippuryl-L-histidyl-L-leucine was tested and improved. T he 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 concentr ations are 1.1 mol/l potassium chloride and 3.0 mmol/l hippuryl-L-hist idyl-L-leucine at pH 8.3. Higher substrate concentrations effect an in hibition of the enzyme reaction. A Michaelis constant of 0.9 mmol/l wa s found with serum as enzyme source. An activation energy of 57 kJ/mol was obtained from the relation between the logarithm of velocity of e nzyme reaction and reciprocal value of absolute temperature. Furthermo re, a linear dependence on chloride ion concentration was observed. Th e histogram of the enzyme activities in sera from 146 healthy voluntee rs shows a non-gaussian distribution. The reference interval at 25 deg rees C is characterized by a median of 24 units/l with the 2.5th and t he 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 wit h a median of 48 units/l No significant sex and age dependence could b e found. A potent ACE inhibitor such as captopril leads to a rapid dec rease of the enzyme activity within 60 min after oral administration. In the following hours, the enzyme activity slowly increases.