Endocrinol. Japon. 1985, 32 (6), 803-809 An Improved Method for Measuring Angiotensin I Converting Enzyme Activity Using A Highly Sensitive Angiotensin II Radioimmunoassay HIROSHI HIDAKA*, Susumu SAWADA, RIHEI SATO AND HIROSHI OKA The 1st. Department of Internal Medicine, Faculty of Medicine, University of Tokyo. 7-3-1 Hongo Bunkyo-ku, Tokyo *Shizuoka Prefectural University, Hamamatsu Abstract A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmuno- assay. To protect against angiotensin II destruction, bestatin, an inhibitor of angiotensinases, was added to the reaction mixture. Pepstatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radio- immunoassay for angiotensin II. Angiotensin I converting enzyme (ACE) Das and Soffer, 1975; Soffer, 1976). To (E.C.3.4.15.1, kininase II) reacts with the measure ACE activity, a spectrophotometric inactive peptide, angiotensin I (AI), liberat- method (Cushman and Cheung, 1971) or its ing the carboxy terminal dipeptide, Histidyl- modification (Lieberman, 1975) using syn- Leucine, and the vasoactive octapeptide, thetic substrate (Hippuryl-Histidyl-Leucine: angiotensin II (AII) (recently reviewed by Hip-His-Leu) is generally employed. How- ever, the sensitivity of this method is not Received April 1, 1985 sufficient to assess low levels of activity. Abbreviations AI: Angiotensin I AII: Angio- Another method used in the determination tensin II ATase: Angiotensinase ACE: Angio- tensin I Converting Enzyme of this enzyme activity has been described. Endocrinol. Japon. 804 HIDAKA et al. December 1985 Hollemans et al. (1969) and Poulsen et al. antiserum obtained was more than 1: 100,000 with (1971) reported a radioimmunoassay method an initial binding of 50% (B/F=1). The cross for All converted from AI. A drawback in reactivities to angiotensin analogue and various this latter method is that All may be des- reagents in ACE assay have been reported by Hidaka et al. (1982). troyed by serum angiotensinases (ATase) and Other reagents were of analytical grade and that generation of AI by plasma renin ac- were obtained from commercial sources. tivity may occur during the incubation of the reaction mixture. In this paper, we report Assay for ACE activity (using AI as a substrate): a highly sensitive method for measuring ACE The assay for ACE was carried out as follows: activity using AI as a substrate, in which, The serum sample was diluted four times with AI, the intrinsic substrate for ACE, is con- 0.05M HEPES buffer, pH 7.4, containing 90mM verted to All in the reaction. Generated NaCl. 0.1ml of diluted serum (1:4), 0.1ml of All is determined by All radioimmunoassay, pepstatin (1mM), 0.1ml bestatin (0.1mM), 0.1 ml of Al (60ng/ml) and 0.1ml of HEPES buffer without All destruction by angiotensiases. (0.05 M, pH 7.4 containing 90mM NaCl) were mixed and the reaction mixture (0.5ml) was in- cubated for 20min at 37•Ž. The reaction was Materials and Methods stopped by adding MK-521 (1ƒÊM, containing 1mM EDTA) and cooling in an ice bath. The Hippuryl-Histidyl-Leucine(Hip-His-Leu),brady- All generated was determined by direct radio- kinin-potentiating peptide (BPP), angiotensin immunoassay (RIA). ACE activity was calculated as follows; I (AI), angiotensin II (AII) , and bestatin were purchased from Protein Research Foundation, All eq pg/ml/min Osaka, Japan. N-Carboxy-dipeptide-lysine analog =Generated All (37•Ž)- Blank All (4•Ž) (MK-521: (S)-1[N2-(1-carboxy-3-phenylpropyl)-L- lysyl]-L-proline dihydrate, Patchett , et al., 1980) Inhibitory effect of pepstatin on plasma renin ac- was supplied by Merck Sharp and Dohme Re- tivity: search Laboratories. Pepstatin A was donated 0.2ml of the plasma sample (EDTA=2Na by Dr. T. Aoyagi, Institute of Microbial Chem- 1mg/ml), 0.1ml of 0.1M Tris-acetate buffer (pH istry, Tokyo, Japan. 125I AI and 125I All were 7.4, 30mM EDTA), 0.01ml of 12mM PMSF purchased from New England Nuclear Co., (phenylmethanesulfonyl fluoride), and 0.1ml of Boston, USA. HEPES (N-2-hydroxyethylpiperazin- pepstatin (various concentratins prepared) were N"-2-ethanosulfonic acid) was purchased from mixed and the reaction mixture (0.41ml) was Calbiochem Co., California, USA. Antiserum incubated for 60min at 37•Ž. The AI generated to All was produced in rabbits by the use of was measured by RIA (Hidaka et al., 1980). thyroglobulin-conjugate, recently reported in Hidaka et al. (1982). Our method is essentially Inhibitory effects of MK-521 and EDTA on serum based on that of Goodfriend et al. (1964). AII- ACE: conjugate was prepared by the method as follows; Diluted serum (1:4) was mixed with 0.1ml Briefly, 10mg of Porcine thyroglobulin and 20 of MK-521 or BPP (1ƒÊM) and 0.1ml of EDTA mg of All were dissolved in 0.5ml of water. (1mM), respectively (each inhibitor having been To this mixture, 0.25ml of water containg 100 diluted to various concentrations). ACE activity mg of 1-cyclohexyl-3(2-morpho-linoethyl)-carbodi- of the serum was determined by the method imide-metho-P-toluenesulfonate was added as a originally described by Cushman (1971). coupling agent. The copolymer was dialized against distilled water for 24 hrs., lyophilized and Direct RIA for angiotensin II: then emulsified into complete Freund's adjuvant Direct RIA for All was made in accordance for immunization. 0.1mg mixture (1: 1) of with the AI measurement method described pre- Freund's adjuant and AII-conjugate was injected viously by Hidaka et al. (1982). Briefly, 0.7ml into the toe-pads and leg muscles of rabbits. of 0.1M Tris-buffer (pH 7.4, 30mM EDTA, Boosting was performed monthly by intraperitoneal 0.025% BSA), 0.1ml of All antiserum (1:100,000 administration over six months. The best titer of tiert), 0.1ml of 125I All (app. 10,000cpt/tube), Vol.32, No.6 AN IMPROVED METHOD FOR MEASURING ACE ACTIVITY 805 0.1ml of -synthetic All (0-400pg/tube), were action mixture (0.4ml) was incubated for 20min mixed and the reaction mixture (1ml) was in- at 37•Ž. The inhibitory effect of bestain on cubated for 16-20 hr at 4•Ž. The bound and ATase was determined by All recovery, as de- free antisera were separated by adding a dextran- scribed above. coated charcoal suspension at 4•Ž. Bound radio- activity was counted with a gamma counter, ANSAR, ABBOT Lab. There was 0.5% cross- Results and Discussion reactivity with AI and no cross-reactivity with best4tin, pepstatin, or MK-521. The sensitivity Binding rate of All antiserum to 0pg of the assay had a lower limit of 6pg/ml and All in the standard curve was 75% in a the coefficient of variation was 11.5% (n=20). final dilution of 1:100,000. The interference Measurement of serum angiotensinase (ATase) of AI and the proteolytic enzyme inhibitors activity: with employed All antiserum was examined Diluted serum (1:4) 0.2ml, All (1000, 400, (Hidaka et al., 1982). The cross reactivity 200pg/0.1ml) 0.1ml and pepstatin (1mM), were (50% binding) was as much as 0.5% to AI mixed and the reaction mixture (0.4ml) was in- and none to bestatin, peptatin, or MK-521. cubated for 20, 40, 60 min at 37•Ž. After the The mean recovery of synthetic All (400, incubation was completely stopped by adding 1ƒÊM MK-521, containing 1mM EDTA, direct 200, 100, 50, 25, and 12.5pg) added to RIA for All was carried out. ATase activity five plasma samples was 100.6% average was calculated by All recovery which was ex- (109.1-91.3% ranged). pressed by the ratio of the remaining amount to The renin inhibitor, pepstatin, was added the added All after serum ATase had destroyed to the reaction mixture. By using pepstatin All during the incubation. in the assay system we were able to ensure the quantity of substrate AI, since the pep- Inhibitory effect of bestatin on serum ATase: Diluted serum (1:2) 0.2ml, All (1000pg/0.1 statin stopped the intrinsic production of ml), pepstatin (1ml), and bestatin 0.1ml (prepared AI. The inhibitory effect of pepstatin on various concentrations) were mixed and the re- plasma renin was examined; The 50% in- Fig. 1. Angiotensin II de- composed by angiotensin- ase. Added angiotensin II concentrations are desig- nated as follows:-0- 200 pg/tube, -•œ- 400pg/tube, -△-1000pg/tube . Sample dilution is 4 fold. Insert bar graph demonstrates decomposition by angio- tensinase in various sam- ples (n=10). Sample dilu- tion is 2 fold. Reaction mixture was incubated 60 min at 37•Ž.