A
Sensitive
Fluorimetric
A s s a y
A n g i o t e n s i n - c o n v e r t i n g
for
S e r u m
E n z y m e
JOAN FRIEDLAND, P H . D . , AND EMANUEL SILVERSTEIN, M.D.,
PH.D.
Laboratory of Molecular Biology, Department of Medicine and Graduate Program in Biochemistry .STAT Down.state Medical Center, Brooklyn, New York 11203 ABSTRACT
T H E OBSERVATION that serum angiotensinconverting enzyme is elevated in many patients with sarcoidosis22,29"™ has focused attention on the role of this enzyme in disease and suggested the need for a clinically applicable assay using unpurified serum or plasma. Angiotensin-converting enzyme 31 is a halide-requiring dipeptidase that catalyzes the cleavage of the carboxyl end of the decapeptide angiotensin I to form the pressor octapeptide angiotensin II and the dipeptide L-histidyl-L-leucine 3 ' 13 :
The substrate, angiotensin 1, is itself formed by proteolytic cleavage from the serum protein precursor angiotensinogen catalyzed by the enzyme renin, which is present in juxtaglomerular cells of the kidney and released in a controlled manner from them. Angiotensin-converting en-
Received August 28, 1975; received revised manuscript October 20, 1975; accepted lor publication October 20, 1975. Address reprint requests to Dr. Silverstein.
* The following abbreviations are used: Fl, fluorescence intensity; standard amino acid abbreviations are used: e.g., his-leu, L-histidyl-t.-leucine.
asp-arg-val-tyr-ile-his-pro-phe-his-leu* —> angiotensin I asp-arg-val-tyr-ile-his-pro-phe + his-leu angiotensin II
416
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Friedland, Joan, and Silverstein, Emanuel: A sensitive, fluorimetric assay for serum angiotensin-converting enzyme. Am J Clin Pathol 66: 4 1 6 - 4 2 4 , 1976. A simple, rapid, highly sensitive and reproducible assay for angiotensin-converting enzyme in untreated serum is described. It is based on the conversion of the substrate analog, hippuryl-L-histidyl-L-leucine (5 HIM in 0.1 M K phosphate, />H 8.3-0.3 M NaCl) to hippurate and L-histidyl-Lleucine, which is quantified spectrofluorimetrically (X excitation = 360 nm; X fluorescence = 500 nm) by formation of a fluorescent adduct with ophthaldialdehyde. T h e chloride requirement and inhibition and activation patterns correspond to those for angiotensin-converting enzyme. T h e K„, for hippuryl-L-histidyl-L-leucine was 1.33 niM. The mean value of serum angiotensin-converting enzyme for 58 normal human subjects (mean age, 32 years; range 19-57) was 32.2 ± 1.30 (SE), with a standard deviation of 9.87 nmol/min/ml serum. The assay is useful for the diagnosis and possible management of sarcoidosis and may have other applications in the future. (Key words: Serum angiotensin-converting enzyme; Sarcoidosis, serum angiotensin-converting enzyme in; Angiotensin I; Angiotensin II; Hippuryl-L-histidyl-L-leucine; L-histidyl-t.-leucine; Fluorescent assay; oPhthaldialdehyde.)
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SERUM ANGIOTENSIN-CONVERTING ENZYME
based on formation of the fluorescent adduct of o-phthaldialdehyde and the histidyl moiety of the L-histidyl-L-leucine product 6,15,27,28 formed from the hippurylL-histidyl-L-leucine substrate. It is applicable to untreated sera, simple, rapid, and highly sensitive, requiring as little as 1 /u.1 or less of serum, and has been applied to the study of large numbers of sera in sarcoidosis. 29 Materials and Methods Materials Hippuryl-L-histidyl-L-leucine was purchased from Bachem Fine Chemicals, Marina del Rey, CA, L-histidyl-L-leucine and o-phthaldialdehyde from the Sigma Chemical Co., and purified methanol from the Baker Chemical Co. Inorganic chemicals were reagent-grade. Serum Samples Blood was obtained from healthy Blood Bank donors (unless otherwise noted) by arm venipuncture. T h e blood was allowed to clot in a new clean test tube (Vacutainer) for about an hour at room temperature, centrifuged for 10 minutes at 900 X g, and the serum carefully removed with a Pasteur pipette and stored at —76 to - 8 6 C. Reagents Distilled and deionized water was used throughout. Hippuryl-L-histidyl-L-leucine (25 mM; ^ H 8.3; molecular weight 466) was prepared by dissolving 46.6 mg hippuryl-L-histidyl-L-leucine in 4 ml of 25 mM NaOH. Phospho-saline buffer (pH 8.3) was prepared by dissolving 87.09 g (0.5 mole) K 2 H P 0 4 and 87.67 g (1.5 mole) NaCl in 900 ml H 2 0 , adjusting the pH to 8.3 with 1 N H O , adding water to a total volume of 1 1 and readjusting the pH if
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zyme is thus an important element in the renin-angiotensin system of blood pressure and aldosterone control, 10 as well as in neural action. 9 In addition to plasma, angiotensin-converting enzyme is present in various organs, particularly in lung, 7 the organ that appears to be responsible for much of the rapid conversion in vivo of angiotensin I to angiotensin II. 26 Angiotensin-converting enzyme has been assayed with angiotensin I as substrate biologically by contractile 1 ' 2,4,16 ' 18,33 or blood pressure 24 response, radiometrically by measuring the release in histidyl-leucine of radioactivity in the terminal leucine in angiotensin I,17 spectrofluorimetrically, 6,27 spectrophotometrically by ninhydrin reaction 11 and by separation of product and precursor by countercurrent distribution. 32 Simpler substrate analogs have been assayed by similar methods, with the exception of biological activity, since no such activity is g e n e r a t e d with the analogs 8-12'19'27.34 Partially purified human plasma or serum angiotensin-converting enzyme has been determined radiometrically with [ 14 C]leu-5-ile-angiotensin I 21 as substrate and fluorimetrically with angiotensin I or benzyloxycarbonyl-phe-his-leu or benzyloxycarbonyl-pro-phe-his-leu,27 while purified or unpurified human plasma was assayed with angiotensin I substrate biologically5,14 or radioimmunologically. 14 In our experience the spectrophotometric assay utilizing hippuryl-L-histidyl-Lleucine substrate 8 has not been universally applicable clinically, since significantly high levels of lipids interfered with the extraction and subsequent solubilization of the product hippuric acid. This assay also required fastidious technic and was relatively insensitive. T h e spectrofluorimetric method described in this paper is a modification of a method previously described that was unsuitable for assay of unpurified serum angiotensin-converting enzyme, and is
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FRIEDLAND AND SILVERSTEIN
important control for the slow breakdown of hippuryl-L-histidine-L-leucine in NaOH. In order to keep the blank low the development of fluorescence should be started as soon as possible after the addition of NaOH. Fluorescent Product Development One hundred microliters of o-phthaldialdehyde reagent were added to each tube, which was mixed with a vortex mixer. Exactly 10 min later the reaction was terminated by the addition of 200 jid of 3 M HC1 and the tube again thoroughly mixed. T h e HC1 addition was accompanied by precipitation of a presumptive proteino-phthaldialdehyde complex, which was removed by centrifugation for 10 min at 1,000 X g. T h e fluorescence was read in a 1-cm rectangular fluorescence cuvette in a Perkin-Elmer MPF-4 fluorimeter with 8 mm entrance and exit slitst and a 7.5 = v energy output between 30 and 90 min after addition of HC1, during which time it was stable. The excitation wavelength was 360 nm and the emission fluorescence wavelength was 500 nm. Calculations
Enzymatic activity in nmoles L-histidylL-leucine/min/ml serum was calculated by two methods. The first is applicable to ratios of less than 2 of corrected assay/ Enzyme Incubation standard fluorescence. The second method must be used for ratios greater than 2, Ten microliters of serum were added to and is also applicable to ratios of less than 2. 240 fjii of substrate-buffer solution at 37 C Method 1. Angiotensin-converting enin a 12 X 75-mm test tube tightly covered zymatic activity, nmol his-leu*/min/ml with Parafilm, mixed with a vortex mixer, serum = and incubated for 15 min at 37 C. Duplicate reaction mixtures were stopped by addition of 1.45 ml of 0.28 M NaOH and t Optimum exit and entrance slit settings will mixing with a vortex mixer. A substrate vary with the lamp output and thefluorimeterused. buffer blank was similarly treated at the For the Perkin-Elmer MPF-4 spectrofluorimeter a useprocedure is to set the exit slit at 8 mm and same time except that enzyme was added ful adjust the entrance slit so that the standard reads immediately after NaOH, and was an approximately 50 FI units.
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necessary. This buffer keeps well at room temperature but precipitates at 5 C. Substrate-buffer solution was prepared by mixing buffer, substrate and water at a ratio of 1:1:2.8 (final concentration in the assay mixture: 0.1 M K phosphate, pH 8.3, 0.3 M NaCl; 5 mM hippuryl-L-histidyl-Lleucine). Two per cent o-phthaldialdehyde (200 mg/cl purified methanol) was prepared immediately prior to addition of enzyme to a series of assay mixtures. A product standard consisted of 0.2 ml 0.516 mM L-histidyl-L-leucine (13.86 mg/dl H 2 0 ) , 1 ml buffer, and 3.6 ml H 2 0 ; 0.24 ml of standard solution contained 5.16 nmol L-histidyl-L-leucine. Dilutions of the standard were used for establishing the standard curve. Serum was added to the standard after addition of 0.28 N NaOH to eliminate any possible error from enzymatic hydrolysis of L-histidyl-L-leucine. Since the variation among standards containing different sera was less than 5%, usually three of the series being assayed were added to the standard and the mean value of fluorescence intensity obtained was used to correlate fluorescence with the content of the reaction product, L-histidyl-L-leucine. T h e fluorescence of a blank containing water and serum was subtracted from that of the standard and serum.
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5.166 nmol his-leu (standard) 100§ ml - 1 serum (FI* assay - FI assay blank) 15 min (FI standard - FI standard blank)
contained L-histidine-L-leucine and buffer and was handled as described for the enzyme assays, including a zero-time control. Hydrolysis was accompanied by a decrease in fluorescence from that obtained with the initial concentration as determined in the zero-time control. T h e quantity hydrolyzed was determined by multiplying the fraction hydrolyzed (decrease in Fl/initial FI) by the initial concentration. No correction was made for the fluorescence of L-histidine since it was only about 8% of the fluorescence of L-histidyl-L-leucine.
Hydrolysis of L-histidyl-L-leucine
Initial Assay Development
Results
Initially the fluorimetric method of Hydrolysis of the product L-histidylCheung and Cushman 6 was used to assay L-leucine was determined fluorimetrically by adding serum to an assay mixture that angiotensin-converting enzyme in serum, but was unsuccessful. Essentially no § The activity in 10 fx\ was converted to activity fluorescence was obtained, apparently because o-phthaldialdehyde also reacts with per ml by multiplying by 100.
i
1
1
1
300
Frc. 1. Standard curve of L-histidyl-L-leucine quantity versus fluorescence intensity. The units of Lhistidyl-L-leucine are given directly in terms of velocity/ml serum in the standard assay as described in Materials and Methods. The actual L-histidyl-Lleucine concentration in nmol/ml may be obtained by multiplying by 0.6.
200
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L-HISTIDYL-L-LEUCINE, nmols x min - 1 x m l " 1 serum
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Method 2. A standard curve of (standard — blank) fluorescence versus L-histidyl-Lleucine was prepared by using integral multiples of the routine standard up to 7 under the same condition as for the routine standard (Fig. 1). T h e quantity of Lhistidyl-L-leucine in nmol/.01 ml serum in each standard was converted to nmol/ min/ml of serum by multiplying by 100/15 min. Enzymatic activity was read from the standard curve using the fluorescence obtained in the assay corrected for fluorimeter variation by multiplying by fluorescence of standard when the curve was made/fluorescence of standard at the time of assay.
FRIEDLAND AND SILVERSTEIN
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A.J.C.P.—Vol.
66
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FIG. 2. The velocity of formation of L-histidyl-L-leucine catalyzed by serum angiotensin-converting enzyme as a function of the concentration of the substrate, hippuryl-L-histidyl-L-leucine. The serum used had an activity of 30 nmol/min/ml serum. Standard assay conditions were used (see Materials and Methods). the abundant serum protein, leaving insufficient reagent to react with the Lhistidyl-L-leucine product formed from hippuryl-L-histidyl-L-leucine. By in-
creasing the o-phthaldialdehyde concentration from 0.2 to 2% there was sufficient reagent to form the fluorescent Lhistidyl-L-leucine adduct. Under these conditions the fluorescence intensity of L-histidyl-L-leucine in buffer in the presUJ z ence of serum was 80% of that obtained in o the absence of serum. Even at a serum conUJ tent of 10 /id instead of 50 /Ltl a concentra_l y^ I in tion of o-phthaldialdehyde greater than -J o 0.2% was required, since the fluorescence ' E -I c intensity with 0.2% o-phthaldialdehyde was >only 62% of that with 2% o-phthaldialdeo H hyde. These results indicated the importo tance of controlling for the effect of X serum per se on the fluorescence intensity. This early assay procedure, (half the 10 15 20 volume of Cushman and Cheung 6 (0.125 MINUTES ml total reaction volume, 0.05 ml serum, FIG. 3. Linearity with respect to time of the forma- 2% o-phthaldialdehyde) had two drawtion of L-histidyl-L-leucine from hippuryl-L-histidylL-leucine catalyzed by serum angiotensin-converting backs. First, the assay was not linear even enzyme (activity, 30 nmol/min/ml serum). Standard in the upper normal range due to great assay conditions were used (see Materials and deviation from the linear portion of the Methods).
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.05
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FIG. 5. Linearity with respect lo serum concentration of the velocity of i.-histidyl-i.-leucine formation catalyzed by serum angiotensin-conL-Hi stidyl-L-Leucine Peptidase verting enzyme. The reaction mixture contained M NaCl, 0.1 K phosphate,/>H 8.3. 5 mm hippurylThe activity of the L-histidyl-L-leucine 0.5 L-histidyl-L-leucine, and enzyme as shown in llie hydrolyzing activity was measured in figure in 0.25 ml. Standards and blanks were preseveral sera under the conditions of the pared for each serum concentration to control any influence of serum on fluorescence intensity. Serum fluorimetric assay (50 /xl sera in 125 /i-1 quantities smaller than 10 \x\ were obtained by using 0.1 M K phosphate - 0.3 M NaCl,//H 8.3), serum diluted 1/10 with physiologic saline solution. using as substrate 0.2 ITIM L-histidyl-Lleucine, a concentration typically formed in the angiotensin-converting enzyme reaction mixture. This measurement indicated 1 1 i i o that an underestimation of as much as 15% ' 30 /- might be introduced in the assay of angiotensin-converting enzyme in this system. T h e velocity of the L-histidyl-L-leucine c peptidase reaction was determined at varying L-histidyl-L-leucine concentra^ i G 20 tions. T h e K,„ for L-histidyl-L-leucine of the peptidase was estimated from \h — 1/S plots 23 to be 0.16 mM. This is similar to y^ the value of 0.2 IIIM for the K,„ of porcine 20 ~ lung L-histidyl-L-leucine peptidase. Reduction of the volume of serum used to _i 10 (A and increase in total reaction volume to 250 fjil would reduce the concentration of the L-histidyl-L-leucine product tenfold. i T h e angiotensin-converting enzymatic 15 10 MINUTES activity of most human sera (32 nmol/ FIG. 4. Linearity with respect to time oi~ the forma- min/ml serum; 0.02 HIM L-histidyl-L-leution of L-histidyl-L-leucine catalyzed by a high cine formed in 15 min) would result in a activity of serum angiotensin-converting enzyme from a patient with sarcoidosis. The standard assay L-histidyl-L-leucine concentration well described in Materials and Methods was used. below the K,„ for L-histidyl-L-leucine of Calculations were done by a standard curve (Method 2) sincefluorescenceintensity is not linear for most its peptidase activity. From the rates of of these points. hydrolysis of 0.02 ITIM L-histidyl-L-leucine
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curve of fluorescence intensity with Lhistidyl-L-leucine generated under these conditions, and, to a lesser extent, to significant deviation from initial rate conditions for sera with elevated activities. Second, human serum was found to contain a peptidase that hydrolyzes L-histidylL-leucine. Since we found the o-phthaldialdehyde-L-histidine addition product to have a quantum yield of about 8% that of the o-phthaldialdehyde-L-histidyl-L-leucine addition product, the activity measured would be lower than the true activity by almost the entire amount of the Lhistidyl-L-leucine hydrolyzed.
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FRIEDLAND AND SILVERSTEIN
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Table 1. Inhibition and Activation of Serum Angiotensin-converting Enzyme by Various Compounds*
CuSO, o-Phenanthroline Dithiothreiiol CdS04t CdS04t EDTAt EDTAt EDTAt\ CoCI2t / EDTAt\ CoCl2t / EDTAt\ CoCI2t J
1 x 10"3 1 x 10-33 1 x 10" 5 x 10"5 5 x 10~45 5 x 10"4 5 x 1(T4 5 x 10"4 1 x 105 x 10"44 5 x 10"5 5 x 10"4 1 x 10"
Serum Angiotensinconverting Enzyme Activity, % of Control 6 2 3 12 2 29.7 0.05 0.1
activity of 34 nmol/min/ml, which is in the normal range. T o avoid spuriously high blanks due to base-catalyzed hydrolysis, addition of NaOH to the blank, followed by enzyme, was made at the same time that NaOH was added to terminate the enzymatic reaction of the reacted samples. Kinetics
The effect of substrate concentration on reaction velocity is given in Figure 2. Maximum activity was found at 5 ITIM hippuryl-L-histidyl-L-leucine and inhibi104 tion at 15 niM. T h e K,„ determined from the \lv - 1/S plot 23 was 1.33 m\i. 128 A 5-niM concentration of hippuryl-Lhistidyl-L-leucine was selected as close to * Standard assay conditions were used as described in Materials and optimum for the standard assay. This conMethods. tf I'reincubated with enzyme for 15 min. at 42 C prior to assay at centration of substrate was also used for the 25,~f higher concentration than the concentration in lite reaction rabbit lung enzyme system in which, mixture that is shown. similarly, the K„, was 2.6 mM and inhibi8 in 250-/U.1 assays using 10 /A of sera from tion was present above 10 mM substrate. Initial rate conditions were present and a several patients, it was estimated that less linear release of product with time would than 2.5% of the L-histidyl-L-leucine be expected since assay of serum of the formed would be likely to be hydrolyzed highest activity (240 nmol/min/ml serum) in the new assay system. Therefore, essenresulted in utilization of less than 3% of tially no correction would be necessary for the substrate during the 15 min assay. T h e hydrolysis of L-histidyl-L-leucine. formation of the product, L-histidyl-Lleucine, was linear with time both for serum Apparent Hydrolysis of Hippwyl-L-histidylof normal activity (Fig. 3) and for serum L-leucine in NaOH of markedly elevated activity from a paThe fluorescence intensity of the zero- tient with sarcoidosis (Fig. 4). The velocity time blank containing NaOH and enzyme of the reaction was directly proportional increased linearly with the time elapsed to the concentration of serum enzyme between NaOH addition and fluorescence added, indicating that the assay measures development with o-phthaldialdehyde, the amount of enzyme present and is valid suggesting a slow base-catalyzed hydrolysis (Fig. 5). The enzyme was inactive in the of hippuryl-L-histidyl-L-leucine to hip- absence of chloride. The action of inhibipurate and L-histidyl-L-leucine. When tors and activators (Table 1) conformed to for angiotensin-converting large quantities of enzyme were used, this that expected 8 enzyme. effect was small compared with the total fluorescence developed. However, the inNormal Values crease in fluorescence intensity of the blank in 30 min was equivalent to 1.3 T h e mean serum angiotensin-convertnmol/min/ml and would result in a value ing enzyme in 58 healthy blood bank 4% below the true one for an enzymatic donors (51 male, 19-57 years of age,
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Compound
Concentration (M)
A.J.C.P. —Vol. 66
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SERUM ANGIOTENSIN-CONVERTING ENZYME
mean age, 31.6 years; 7 female, 2 1 - 4 4 years old, mean age, 33.3 years) was 32.2 ± 1.30 (standard error of the mean) ± 9.87 (standard deviation) nmol/min/ml serum. The values of two individuals were between 2 and 3 standard deviations greater than the mean, and that of one was greater than 3 standard deviations. No value was more than 2 standard deviations below the mean. Discussion
herein is readily adaptable to automation, in contrast to the spectrophotometric method, 8 which would present more difficulties. Note Added in Proof: Serum angiotensinconverting enzyme has recently been found to be elevated in most patients with Gaucher's disease (Lieberman J: The specificity and nature of serum angiotensin-converting enzyme (ACE) elevations in sarcoidosis. Ann NY Acad Sci, in press; Silverstein E, Friedland J: Elevated serum and spleen angiotensin convening enzyme (ACE) and serum lysozyme (1.) in chronic Gaucher's disease (GD). Clin Res 24: 478A, 1976). Acknowledgments. Michael Kitt assisted in the initial phase of this work. Charlotte Setton assisted in obtaining blood samples. Dr. D. W. Cushman provided helpful references and a gift of hippurylL-histidyl-L-leucine.
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The present fluorescent assay for serum angiotensin-converting enzymatic activity has several advantages in comparison with the spectrophotometric method. 8 ' 22 It is applicable to sera drawn at any time and is extremely sensitive. The present assay could be run on as little as 1 fil of serum by lengthening incubation time if necessary or increasing the sensitivity range of the fluorimeter if possible on the instrument available. Further increase in sensitivity could be obtained by a decrease in the size of the incubation mixture and use of microcuvettes. T h e method is thus on a micro level, and almost any size of sample can be assayed. This is important where sample size is limited, as in pediatrics, 25 where multiple determinations are required, and for micro sampling studies. In contrast, the spectrophotometric assay requires 100 fil of serum and a 30-minute incubation time in the normal range. The present fluorescent assay can be run on large numbers of sera at a time, and is rapid and relatively simple to perform. T h e angiotensin-converting enzyme assay herein described appears to be useful for diagnosis and management of patients with sarcoidosis, who may have extremely elevated levels.22'29 Serum enzyme elevation has also recently been reported to occur in the neonatal idiopathic respiratory distress syndrome, 25 and other clinical uses may become apparent in the future. T h e fluorescent method described
423
References 1. Andersen JB: Converting enzyme activity in liver damage. Acta Pathol Microbiol Scand 71:1-7, 1967 2. Bakhle YS: Conversion of angiotensin I to angiotensin II by cell-free extracts of dog lung. Nature (Lond) 220:919-921, 1968 3. Bakhle YS: Converting enzyme in vitro measurement and properties. Handbook Exp Pharm 37:41-80, 1974 4. Barrett JD, Sambhi MP: Simultaneous assay of angiotensin I and II and determination of converting enzyme activity. J Pharmacol Exp Ther 170:326-333, 1969 5. Boucher R, Kurihara H, Grise C, et al: Section III. Conversion of angiotensin I. Measurement of plasma angiotensin I convening enzyme activity. Circ Res 26-27 suppl: I 83-91, 1970 6. Cheung HS, Cushman DW: Inhibition of homogenous angiotensin converting enzyme of rabbit lung by synthetic venom peptides of Bothrops jararaca. Biochim Biophys Acta 293: 451-463, 1973 7. Cushman DW, Cheung HS: Concentration of angiotensin-converting enzyme in tissues of the rat. Biochim Biophys Acta 250:261265, 1971 8. Cushman DW, Cheung HS: Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem Pharmacol 20:1637-1648, 1971 9. Daul CB, Heath RG, Carey RE: Angiotensinforming enzyme in human brain. Neuropharmacology 14:75-80, 1975 10. Davis JO: The control of renin release. Am ] Med 55:333-350. 1973 11. Dorer FE, Skeggs LT, Kahn JR. et al: Angiotensin converting enzyme. Method of assay and partial purification. Analyt Biochem 33:102113,1970
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24. 25.
26. 27.
28.
29. 30.
31. 32. 33.
34.
enzyme dissociation constants. J Am Chem Soc 56:658-666, 1934 Loyke HF: Converting enzyme in rat serum. Proc Soc Exp Biol Med 134:248-251, 1970 Matlioli L, Zakheim RM, Mullis K, et al: Angiotesin-I-converting enzyme activity in idiopathic respiratory distress syndrome of the newborn infant and in experimental alveolar hypoxia in mice. J Pediatr 87:97-101, 1975 NG KKF, Vane JR: Fate of angiotensin I in the circulation. Nature (Lond) 218:144- 150, 1968 Piquilloud Y, Reinharz A, Roth M: Studies on angiotensin converting enzyme with different substrates. Biochim Biophys Acta 206:136142, 1970 Shore PA, Burkhalter A, Cohn VH Jr.: A method for the fluoronietric assay of histamine in tissues. J Pharmacol Exp Ther 127: 182-186, 1959 Silverstein E, Friedland J, Lyons H, et al: Serum angiotensin converting enzyme activity in sarcoidosis. Clin Res 23:352A, 1975 Silverstein E, Friedland J, Lyons H, et al: Elevated angiotensin converting enzyme activity in non-necrotizing granulomatous lymph nodes in sarcoidosis. Clin Res 23: 352A, 1975 Skeggs LT, Marsh WH, Kahn JR, et al: The existence of two forms of hypertensin. ) Exp Med 99:275-282, 1954 Skeggs LT, Kahn JR, Shumway NP: The purification of hypertensin II. J Exp Med 103: 301-307, 1956 Ueda E, Akutsu H, Kokubu T, el al: Partial purification and properties of angiotensin I converting enzyme from rabbit plasma. Jap Circ J 35:801-806, 1971 Yang HYT, Erdos EG, Levin Y: Characterization of a dipeptide hydrolase (kininase II: angiotensin I converting enzyme) J Pharmacol Exp Ther 177:291-300, 1971
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12. Eliseeva YE, Orekhovich VN: Isolation of carboxycathepsin and examination of its specificity. Dokl Akad Nauk, SSSR, (English trans) 153:1434-1436, 1964 13. Erdos EG: Angiotensin I converting enzyme. Circ Res 36:247-255, 1975 14. Fit/. A, Boyd GW, Peart WS: Converting enzyme activity in human plasma. Circ Res 28:246253, 1971 15. Gregcrnian Rl: Identification of histidylleuciiie and other histidyl peptides as normal constituents of human urine. Biochem Med 1: 151-167, 1967 16. Helmer OM: Differentiation between two forms of angiotonin by means of spirally cut strips of rabbit aorta. Am J Physiol 188:571-577, 1957 17. Huggins CG, Thainpi NS: A simple method for the determination of angiotensin I converting enzyme. Life Sci 7: part II 633639, 1968 18. Huggins CG, Corcoran R], Gordon JS, et al: Kinetics of the plasma and lung angiotensin I converting enzymes. Circ Res 26-27 suppl: I 93-101, 1970 19. Igic R, Erdos EG, Yeh, HSJ, Sorrells K, Nakajima T: Angiotensin I converting enzyme of the lung. Circ Res 30-31 suppl:ll 51, 1972 20. Lee H|, Larue JN, Wilson IB: Angiotensinconverting enzyme from guinea pig and hog lung. Biochim Biophys Acta 250:549-557, 1971 21. Lee H|, Larue JN, Wilson IB: Human plasma converting enzyme. Arch Biochem Biophys 142:548-551, 1971 22. Leiberman J: A new confirmatory test for sarcoidosis. Serum angiotensin converting en/.vme. Effect of steroids and chronic lung disease. Am Rev Resp Dis 109:743, 1974 23. Lineweaver H, Burk D: The determination of
A.J.C.P.—Vol.66
Sensitive
Fluorimetric
A s s a y
A n g i o t e n s i n - c o n v e r t i n g
for
S e r u m
E n z y m e
JOAN FRIEDLAND, P H . D . , AND EMANUEL SILVERSTEIN, M.D.,
PH.D.
Laboratory of Molecular Biology, Department of Medicine and Graduate Program in Biochemistry .STAT Down.state Medical Center, Brooklyn, New York 11203 ABSTRACT
T H E OBSERVATION that serum angiotensinconverting enzyme is elevated in many patients with sarcoidosis22,29"™ has focused attention on the role of this enzyme in disease and suggested the need for a clinically applicable assay using unpurified serum or plasma. Angiotensin-converting enzyme 31 is a halide-requiring dipeptidase that catalyzes the cleavage of the carboxyl end of the decapeptide angiotensin I to form the pressor octapeptide angiotensin II and the dipeptide L-histidyl-L-leucine 3 ' 13 :
The substrate, angiotensin 1, is itself formed by proteolytic cleavage from the serum protein precursor angiotensinogen catalyzed by the enzyme renin, which is present in juxtaglomerular cells of the kidney and released in a controlled manner from them. Angiotensin-converting en-
Received August 28, 1975; received revised manuscript October 20, 1975; accepted lor publication October 20, 1975. Address reprint requests to Dr. Silverstein.
* The following abbreviations are used: Fl, fluorescence intensity; standard amino acid abbreviations are used: e.g., his-leu, L-histidyl-t.-leucine.
asp-arg-val-tyr-ile-his-pro-phe-his-leu* —> angiotensin I asp-arg-val-tyr-ile-his-pro-phe + his-leu angiotensin II
416
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Friedland, Joan, and Silverstein, Emanuel: A sensitive, fluorimetric assay for serum angiotensin-converting enzyme. Am J Clin Pathol 66: 4 1 6 - 4 2 4 , 1976. A simple, rapid, highly sensitive and reproducible assay for angiotensin-converting enzyme in untreated serum is described. It is based on the conversion of the substrate analog, hippuryl-L-histidyl-L-leucine (5 HIM in 0.1 M K phosphate, />H 8.3-0.3 M NaCl) to hippurate and L-histidyl-Lleucine, which is quantified spectrofluorimetrically (X excitation = 360 nm; X fluorescence = 500 nm) by formation of a fluorescent adduct with ophthaldialdehyde. T h e chloride requirement and inhibition and activation patterns correspond to those for angiotensin-converting enzyme. T h e K„, for hippuryl-L-histidyl-L-leucine was 1.33 niM. The mean value of serum angiotensin-converting enzyme for 58 normal human subjects (mean age, 32 years; range 19-57) was 32.2 ± 1.30 (SE), with a standard deviation of 9.87 nmol/min/ml serum. The assay is useful for the diagnosis and possible management of sarcoidosis and may have other applications in the future. (Key words: Serum angiotensin-converting enzyme; Sarcoidosis, serum angiotensin-converting enzyme in; Angiotensin I; Angiotensin II; Hippuryl-L-histidyl-L-leucine; L-histidyl-t.-leucine; Fluorescent assay; oPhthaldialdehyde.)
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based on formation of the fluorescent adduct of o-phthaldialdehyde and the histidyl moiety of the L-histidyl-L-leucine product 6,15,27,28 formed from the hippurylL-histidyl-L-leucine substrate. It is applicable to untreated sera, simple, rapid, and highly sensitive, requiring as little as 1 /u.1 or less of serum, and has been applied to the study of large numbers of sera in sarcoidosis. 29 Materials and Methods Materials Hippuryl-L-histidyl-L-leucine was purchased from Bachem Fine Chemicals, Marina del Rey, CA, L-histidyl-L-leucine and o-phthaldialdehyde from the Sigma Chemical Co., and purified methanol from the Baker Chemical Co. Inorganic chemicals were reagent-grade. Serum Samples Blood was obtained from healthy Blood Bank donors (unless otherwise noted) by arm venipuncture. T h e blood was allowed to clot in a new clean test tube (Vacutainer) for about an hour at room temperature, centrifuged for 10 minutes at 900 X g, and the serum carefully removed with a Pasteur pipette and stored at —76 to - 8 6 C. Reagents Distilled and deionized water was used throughout. Hippuryl-L-histidyl-L-leucine (25 mM; ^ H 8.3; molecular weight 466) was prepared by dissolving 46.6 mg hippuryl-L-histidyl-L-leucine in 4 ml of 25 mM NaOH. Phospho-saline buffer (pH 8.3) was prepared by dissolving 87.09 g (0.5 mole) K 2 H P 0 4 and 87.67 g (1.5 mole) NaCl in 900 ml H 2 0 , adjusting the pH to 8.3 with 1 N H O , adding water to a total volume of 1 1 and readjusting the pH if
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zyme is thus an important element in the renin-angiotensin system of blood pressure and aldosterone control, 10 as well as in neural action. 9 In addition to plasma, angiotensin-converting enzyme is present in various organs, particularly in lung, 7 the organ that appears to be responsible for much of the rapid conversion in vivo of angiotensin I to angiotensin II. 26 Angiotensin-converting enzyme has been assayed with angiotensin I as substrate biologically by contractile 1 ' 2,4,16 ' 18,33 or blood pressure 24 response, radiometrically by measuring the release in histidyl-leucine of radioactivity in the terminal leucine in angiotensin I,17 spectrofluorimetrically, 6,27 spectrophotometrically by ninhydrin reaction 11 and by separation of product and precursor by countercurrent distribution. 32 Simpler substrate analogs have been assayed by similar methods, with the exception of biological activity, since no such activity is g e n e r a t e d with the analogs 8-12'19'27.34 Partially purified human plasma or serum angiotensin-converting enzyme has been determined radiometrically with [ 14 C]leu-5-ile-angiotensin I 21 as substrate and fluorimetrically with angiotensin I or benzyloxycarbonyl-phe-his-leu or benzyloxycarbonyl-pro-phe-his-leu,27 while purified or unpurified human plasma was assayed with angiotensin I substrate biologically5,14 or radioimmunologically. 14 In our experience the spectrophotometric assay utilizing hippuryl-L-histidyl-Lleucine substrate 8 has not been universally applicable clinically, since significantly high levels of lipids interfered with the extraction and subsequent solubilization of the product hippuric acid. This assay also required fastidious technic and was relatively insensitive. T h e spectrofluorimetric method described in this paper is a modification of a method previously described that was unsuitable for assay of unpurified serum angiotensin-converting enzyme, and is
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important control for the slow breakdown of hippuryl-L-histidine-L-leucine in NaOH. In order to keep the blank low the development of fluorescence should be started as soon as possible after the addition of NaOH. Fluorescent Product Development One hundred microliters of o-phthaldialdehyde reagent were added to each tube, which was mixed with a vortex mixer. Exactly 10 min later the reaction was terminated by the addition of 200 jid of 3 M HC1 and the tube again thoroughly mixed. T h e HC1 addition was accompanied by precipitation of a presumptive proteino-phthaldialdehyde complex, which was removed by centrifugation for 10 min at 1,000 X g. T h e fluorescence was read in a 1-cm rectangular fluorescence cuvette in a Perkin-Elmer MPF-4 fluorimeter with 8 mm entrance and exit slitst and a 7.5 = v energy output between 30 and 90 min after addition of HC1, during which time it was stable. The excitation wavelength was 360 nm and the emission fluorescence wavelength was 500 nm. Calculations
Enzymatic activity in nmoles L-histidylL-leucine/min/ml serum was calculated by two methods. The first is applicable to ratios of less than 2 of corrected assay/ Enzyme Incubation standard fluorescence. The second method must be used for ratios greater than 2, Ten microliters of serum were added to and is also applicable to ratios of less than 2. 240 fjii of substrate-buffer solution at 37 C Method 1. Angiotensin-converting enin a 12 X 75-mm test tube tightly covered zymatic activity, nmol his-leu*/min/ml with Parafilm, mixed with a vortex mixer, serum = and incubated for 15 min at 37 C. Duplicate reaction mixtures were stopped by addition of 1.45 ml of 0.28 M NaOH and t Optimum exit and entrance slit settings will mixing with a vortex mixer. A substrate vary with the lamp output and thefluorimeterused. buffer blank was similarly treated at the For the Perkin-Elmer MPF-4 spectrofluorimeter a useprocedure is to set the exit slit at 8 mm and same time except that enzyme was added ful adjust the entrance slit so that the standard reads immediately after NaOH, and was an approximately 50 FI units.
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necessary. This buffer keeps well at room temperature but precipitates at 5 C. Substrate-buffer solution was prepared by mixing buffer, substrate and water at a ratio of 1:1:2.8 (final concentration in the assay mixture: 0.1 M K phosphate, pH 8.3, 0.3 M NaCl; 5 mM hippuryl-L-histidyl-Lleucine). Two per cent o-phthaldialdehyde (200 mg/cl purified methanol) was prepared immediately prior to addition of enzyme to a series of assay mixtures. A product standard consisted of 0.2 ml 0.516 mM L-histidyl-L-leucine (13.86 mg/dl H 2 0 ) , 1 ml buffer, and 3.6 ml H 2 0 ; 0.24 ml of standard solution contained 5.16 nmol L-histidyl-L-leucine. Dilutions of the standard were used for establishing the standard curve. Serum was added to the standard after addition of 0.28 N NaOH to eliminate any possible error from enzymatic hydrolysis of L-histidyl-L-leucine. Since the variation among standards containing different sera was less than 5%, usually three of the series being assayed were added to the standard and the mean value of fluorescence intensity obtained was used to correlate fluorescence with the content of the reaction product, L-histidyl-L-leucine. T h e fluorescence of a blank containing water and serum was subtracted from that of the standard and serum.
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5.166 nmol his-leu (standard) 100§ ml - 1 serum (FI* assay - FI assay blank) 15 min (FI standard - FI standard blank)
contained L-histidine-L-leucine and buffer and was handled as described for the enzyme assays, including a zero-time control. Hydrolysis was accompanied by a decrease in fluorescence from that obtained with the initial concentration as determined in the zero-time control. T h e quantity hydrolyzed was determined by multiplying the fraction hydrolyzed (decrease in Fl/initial FI) by the initial concentration. No correction was made for the fluorescence of L-histidine since it was only about 8% of the fluorescence of L-histidyl-L-leucine.
Hydrolysis of L-histidyl-L-leucine
Initial Assay Development
Results
Initially the fluorimetric method of Hydrolysis of the product L-histidylCheung and Cushman 6 was used to assay L-leucine was determined fluorimetrically by adding serum to an assay mixture that angiotensin-converting enzyme in serum, but was unsuccessful. Essentially no § The activity in 10 fx\ was converted to activity fluorescence was obtained, apparently because o-phthaldialdehyde also reacts with per ml by multiplying by 100.
i
1
1
1
300
Frc. 1. Standard curve of L-histidyl-L-leucine quantity versus fluorescence intensity. The units of Lhistidyl-L-leucine are given directly in terms of velocity/ml serum in the standard assay as described in Materials and Methods. The actual L-histidyl-Lleucine concentration in nmol/ml may be obtained by multiplying by 0.6.
200
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Method 2. A standard curve of (standard — blank) fluorescence versus L-histidyl-Lleucine was prepared by using integral multiples of the routine standard up to 7 under the same condition as for the routine standard (Fig. 1). T h e quantity of Lhistidyl-L-leucine in nmol/.01 ml serum in each standard was converted to nmol/ min/ml of serum by multiplying by 100/15 min. Enzymatic activity was read from the standard curve using the fluorescence obtained in the assay corrected for fluorimeter variation by multiplying by fluorescence of standard when the curve was made/fluorescence of standard at the time of assay.
FRIEDLAND AND SILVERSTEIN
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A.J.C.P.—Vol.
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FIG. 2. The velocity of formation of L-histidyl-L-leucine catalyzed by serum angiotensin-converting enzyme as a function of the concentration of the substrate, hippuryl-L-histidyl-L-leucine. The serum used had an activity of 30 nmol/min/ml serum. Standard assay conditions were used (see Materials and Methods). the abundant serum protein, leaving insufficient reagent to react with the Lhistidyl-L-leucine product formed from hippuryl-L-histidyl-L-leucine. By in-
creasing the o-phthaldialdehyde concentration from 0.2 to 2% there was sufficient reagent to form the fluorescent Lhistidyl-L-leucine adduct. Under these conditions the fluorescence intensity of L-histidyl-L-leucine in buffer in the presUJ z ence of serum was 80% of that obtained in o the absence of serum. Even at a serum conUJ tent of 10 /id instead of 50 /Ltl a concentra_l y^ I in tion of o-phthaldialdehyde greater than -J o 0.2% was required, since the fluorescence ' E -I c intensity with 0.2% o-phthaldialdehyde was >only 62% of that with 2% o-phthaldialdeo H hyde. These results indicated the importo tance of controlling for the effect of X serum per se on the fluorescence intensity. This early assay procedure, (half the 10 15 20 volume of Cushman and Cheung 6 (0.125 MINUTES ml total reaction volume, 0.05 ml serum, FIG. 3. Linearity with respect to time of the forma- 2% o-phthaldialdehyde) had two drawtion of L-histidyl-L-leucine from hippuryl-L-histidylL-leucine catalyzed by serum angiotensin-converting backs. First, the assay was not linear even enzyme (activity, 30 nmol/min/ml serum). Standard in the upper normal range due to great assay conditions were used (see Materials and deviation from the linear portion of the Methods).
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.05
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FIG. 5. Linearity with respect lo serum concentration of the velocity of i.-histidyl-i.-leucine formation catalyzed by serum angiotensin-conL-Hi stidyl-L-Leucine Peptidase verting enzyme. The reaction mixture contained M NaCl, 0.1 K phosphate,/>H 8.3. 5 mm hippurylThe activity of the L-histidyl-L-leucine 0.5 L-histidyl-L-leucine, and enzyme as shown in llie hydrolyzing activity was measured in figure in 0.25 ml. Standards and blanks were preseveral sera under the conditions of the pared for each serum concentration to control any influence of serum on fluorescence intensity. Serum fluorimetric assay (50 /xl sera in 125 /i-1 quantities smaller than 10 \x\ were obtained by using 0.1 M K phosphate - 0.3 M NaCl,//H 8.3), serum diluted 1/10 with physiologic saline solution. using as substrate 0.2 ITIM L-histidyl-Lleucine, a concentration typically formed in the angiotensin-converting enzyme reaction mixture. This measurement indicated 1 1 i i o that an underestimation of as much as 15% ' 30 /- might be introduced in the assay of angiotensin-converting enzyme in this system. T h e velocity of the L-histidyl-L-leucine c peptidase reaction was determined at varying L-histidyl-L-leucine concentra^ i G 20 tions. T h e K,„ for L-histidyl-L-leucine of the peptidase was estimated from \h — 1/S plots 23 to be 0.16 mM. This is similar to y^ the value of 0.2 IIIM for the K,„ of porcine 20 ~ lung L-histidyl-L-leucine peptidase. Reduction of the volume of serum used to _i 10 (A and increase in total reaction volume to 250 fjil would reduce the concentration of the L-histidyl-L-leucine product tenfold. i T h e angiotensin-converting enzymatic 15 10 MINUTES activity of most human sera (32 nmol/ FIG. 4. Linearity with respect to time oi~ the forma- min/ml serum; 0.02 HIM L-histidyl-L-leution of L-histidyl-L-leucine catalyzed by a high cine formed in 15 min) would result in a activity of serum angiotensin-converting enzyme from a patient with sarcoidosis. The standard assay L-histidyl-L-leucine concentration well described in Materials and Methods was used. below the K,„ for L-histidyl-L-leucine of Calculations were done by a standard curve (Method 2) sincefluorescenceintensity is not linear for most its peptidase activity. From the rates of of these points. hydrolysis of 0.02 ITIM L-histidyl-L-leucine
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curve of fluorescence intensity with Lhistidyl-L-leucine generated under these conditions, and, to a lesser extent, to significant deviation from initial rate conditions for sera with elevated activities. Second, human serum was found to contain a peptidase that hydrolyzes L-histidylL-leucine. Since we found the o-phthaldialdehyde-L-histidine addition product to have a quantum yield of about 8% that of the o-phthaldialdehyde-L-histidyl-L-leucine addition product, the activity measured would be lower than the true activity by almost the entire amount of the Lhistidyl-L-leucine hydrolyzed.
421
DYL Is/I
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FRIEDLAND AND SILVERSTEIN
422
Table 1. Inhibition and Activation of Serum Angiotensin-converting Enzyme by Various Compounds*
CuSO, o-Phenanthroline Dithiothreiiol CdS04t CdS04t EDTAt EDTAt EDTAt\ CoCI2t / EDTAt\ CoCl2t / EDTAt\ CoCI2t J
1 x 10"3 1 x 10-33 1 x 10" 5 x 10"5 5 x 10~45 5 x 10"4 5 x 1(T4 5 x 10"4 1 x 105 x 10"44 5 x 10"5 5 x 10"4 1 x 10"
Serum Angiotensinconverting Enzyme Activity, % of Control 6 2 3 12 2 29.7 0.05 0.1
activity of 34 nmol/min/ml, which is in the normal range. T o avoid spuriously high blanks due to base-catalyzed hydrolysis, addition of NaOH to the blank, followed by enzyme, was made at the same time that NaOH was added to terminate the enzymatic reaction of the reacted samples. Kinetics
The effect of substrate concentration on reaction velocity is given in Figure 2. Maximum activity was found at 5 ITIM hippuryl-L-histidyl-L-leucine and inhibi104 tion at 15 niM. T h e K,„ determined from the \lv - 1/S plot 23 was 1.33 m\i. 128 A 5-niM concentration of hippuryl-Lhistidyl-L-leucine was selected as close to * Standard assay conditions were used as described in Materials and optimum for the standard assay. This conMethods. tf I'reincubated with enzyme for 15 min. at 42 C prior to assay at centration of substrate was also used for the 25,~f higher concentration than the concentration in lite reaction rabbit lung enzyme system in which, mixture that is shown. similarly, the K„, was 2.6 mM and inhibi8 in 250-/U.1 assays using 10 /A of sera from tion was present above 10 mM substrate. Initial rate conditions were present and a several patients, it was estimated that less linear release of product with time would than 2.5% of the L-histidyl-L-leucine be expected since assay of serum of the formed would be likely to be hydrolyzed highest activity (240 nmol/min/ml serum) in the new assay system. Therefore, essenresulted in utilization of less than 3% of tially no correction would be necessary for the substrate during the 15 min assay. T h e hydrolysis of L-histidyl-L-leucine. formation of the product, L-histidyl-Lleucine, was linear with time both for serum Apparent Hydrolysis of Hippwyl-L-histidylof normal activity (Fig. 3) and for serum L-leucine in NaOH of markedly elevated activity from a paThe fluorescence intensity of the zero- tient with sarcoidosis (Fig. 4). The velocity time blank containing NaOH and enzyme of the reaction was directly proportional increased linearly with the time elapsed to the concentration of serum enzyme between NaOH addition and fluorescence added, indicating that the assay measures development with o-phthaldialdehyde, the amount of enzyme present and is valid suggesting a slow base-catalyzed hydrolysis (Fig. 5). The enzyme was inactive in the of hippuryl-L-histidyl-L-leucine to hip- absence of chloride. The action of inhibipurate and L-histidyl-L-leucine. When tors and activators (Table 1) conformed to for angiotensin-converting large quantities of enzyme were used, this that expected 8 enzyme. effect was small compared with the total fluorescence developed. However, the inNormal Values crease in fluorescence intensity of the blank in 30 min was equivalent to 1.3 T h e mean serum angiotensin-convertnmol/min/ml and would result in a value ing enzyme in 58 healthy blood bank 4% below the true one for an enzymatic donors (51 male, 19-57 years of age,
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Compound
Concentration (M)
A.J.C.P. —Vol. 66
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SERUM ANGIOTENSIN-CONVERTING ENZYME
mean age, 31.6 years; 7 female, 2 1 - 4 4 years old, mean age, 33.3 years) was 32.2 ± 1.30 (standard error of the mean) ± 9.87 (standard deviation) nmol/min/ml serum. The values of two individuals were between 2 and 3 standard deviations greater than the mean, and that of one was greater than 3 standard deviations. No value was more than 2 standard deviations below the mean. Discussion
herein is readily adaptable to automation, in contrast to the spectrophotometric method, 8 which would present more difficulties. Note Added in Proof: Serum angiotensinconverting enzyme has recently been found to be elevated in most patients with Gaucher's disease (Lieberman J: The specificity and nature of serum angiotensin-converting enzyme (ACE) elevations in sarcoidosis. Ann NY Acad Sci, in press; Silverstein E, Friedland J: Elevated serum and spleen angiotensin convening enzyme (ACE) and serum lysozyme (1.) in chronic Gaucher's disease (GD). Clin Res 24: 478A, 1976). Acknowledgments. Michael Kitt assisted in the initial phase of this work. Charlotte Setton assisted in obtaining blood samples. Dr. D. W. Cushman provided helpful references and a gift of hippurylL-histidyl-L-leucine.
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The present fluorescent assay for serum angiotensin-converting enzymatic activity has several advantages in comparison with the spectrophotometric method. 8 ' 22 It is applicable to sera drawn at any time and is extremely sensitive. The present assay could be run on as little as 1 fil of serum by lengthening incubation time if necessary or increasing the sensitivity range of the fluorimeter if possible on the instrument available. Further increase in sensitivity could be obtained by a decrease in the size of the incubation mixture and use of microcuvettes. T h e method is thus on a micro level, and almost any size of sample can be assayed. This is important where sample size is limited, as in pediatrics, 25 where multiple determinations are required, and for micro sampling studies. In contrast, the spectrophotometric assay requires 100 fil of serum and a 30-minute incubation time in the normal range. The present fluorescent assay can be run on large numbers of sera at a time, and is rapid and relatively simple to perform. T h e angiotensin-converting enzyme assay herein described appears to be useful for diagnosis and management of patients with sarcoidosis, who may have extremely elevated levels.22'29 Serum enzyme elevation has also recently been reported to occur in the neonatal idiopathic respiratory distress syndrome, 25 and other clinical uses may become apparent in the future. T h e fluorescent method described
423
References 1. Andersen JB: Converting enzyme activity in liver damage. Acta Pathol Microbiol Scand 71:1-7, 1967 2. Bakhle YS: Conversion of angiotensin I to angiotensin II by cell-free extracts of dog lung. Nature (Lond) 220:919-921, 1968 3. Bakhle YS: Converting enzyme in vitro measurement and properties. Handbook Exp Pharm 37:41-80, 1974 4. Barrett JD, Sambhi MP: Simultaneous assay of angiotensin I and II and determination of converting enzyme activity. J Pharmacol Exp Ther 170:326-333, 1969 5. Boucher R, Kurihara H, Grise C, et al: Section III. Conversion of angiotensin I. Measurement of plasma angiotensin I convening enzyme activity. Circ Res 26-27 suppl: I 83-91, 1970 6. Cheung HS, Cushman DW: Inhibition of homogenous angiotensin converting enzyme of rabbit lung by synthetic venom peptides of Bothrops jararaca. Biochim Biophys Acta 293: 451-463, 1973 7. Cushman DW, Cheung HS: Concentration of angiotensin-converting enzyme in tissues of the rat. Biochim Biophys Acta 250:261265, 1971 8. Cushman DW, Cheung HS: Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem Pharmacol 20:1637-1648, 1971 9. Daul CB, Heath RG, Carey RE: Angiotensinforming enzyme in human brain. Neuropharmacology 14:75-80, 1975 10. Davis JO: The control of renin release. Am ] Med 55:333-350. 1973 11. Dorer FE, Skeggs LT, Kahn JR. et al: Angiotensin converting enzyme. Method of assay and partial purification. Analyt Biochem 33:102113,1970
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24. 25.
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29. 30.
31. 32. 33.
34.
enzyme dissociation constants. J Am Chem Soc 56:658-666, 1934 Loyke HF: Converting enzyme in rat serum. Proc Soc Exp Biol Med 134:248-251, 1970 Matlioli L, Zakheim RM, Mullis K, et al: Angiotesin-I-converting enzyme activity in idiopathic respiratory distress syndrome of the newborn infant and in experimental alveolar hypoxia in mice. J Pediatr 87:97-101, 1975 NG KKF, Vane JR: Fate of angiotensin I in the circulation. Nature (Lond) 218:144- 150, 1968 Piquilloud Y, Reinharz A, Roth M: Studies on angiotensin converting enzyme with different substrates. Biochim Biophys Acta 206:136142, 1970 Shore PA, Burkhalter A, Cohn VH Jr.: A method for the fluoronietric assay of histamine in tissues. J Pharmacol Exp Ther 127: 182-186, 1959 Silverstein E, Friedland J, Lyons H, et al: Serum angiotensin converting enzyme activity in sarcoidosis. Clin Res 23:352A, 1975 Silverstein E, Friedland J, Lyons H, et al: Elevated angiotensin converting enzyme activity in non-necrotizing granulomatous lymph nodes in sarcoidosis. Clin Res 23: 352A, 1975 Skeggs LT, Marsh WH, Kahn JR, et al: The existence of two forms of hypertensin. ) Exp Med 99:275-282, 1954 Skeggs LT, Kahn JR, Shumway NP: The purification of hypertensin II. J Exp Med 103: 301-307, 1956 Ueda E, Akutsu H, Kokubu T, el al: Partial purification and properties of angiotensin I converting enzyme from rabbit plasma. Jap Circ J 35:801-806, 1971 Yang HYT, Erdos EG, Levin Y: Characterization of a dipeptide hydrolase (kininase II: angiotensin I converting enzyme) J Pharmacol Exp Ther 177:291-300, 1971
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12. Eliseeva YE, Orekhovich VN: Isolation of carboxycathepsin and examination of its specificity. Dokl Akad Nauk, SSSR, (English trans) 153:1434-1436, 1964 13. Erdos EG: Angiotensin I converting enzyme. Circ Res 36:247-255, 1975 14. Fit/. A, Boyd GW, Peart WS: Converting enzyme activity in human plasma. Circ Res 28:246253, 1971 15. Gregcrnian Rl: Identification of histidylleuciiie and other histidyl peptides as normal constituents of human urine. Biochem Med 1: 151-167, 1967 16. Helmer OM: Differentiation between two forms of angiotonin by means of spirally cut strips of rabbit aorta. Am J Physiol 188:571-577, 1957 17. Huggins CG, Thainpi NS: A simple method for the determination of angiotensin I converting enzyme. Life Sci 7: part II 633639, 1968 18. Huggins CG, Corcoran R], Gordon JS, et al: Kinetics of the plasma and lung angiotensin I converting enzymes. Circ Res 26-27 suppl: I 93-101, 1970 19. Igic R, Erdos EG, Yeh, HSJ, Sorrells K, Nakajima T: Angiotensin I converting enzyme of the lung. Circ Res 30-31 suppl:ll 51, 1972 20. Lee H|, Larue JN, Wilson IB: Angiotensinconverting enzyme from guinea pig and hog lung. Biochim Biophys Acta 250:549-557, 1971 21. Lee H|, Larue JN, Wilson IB: Human plasma converting enzyme. Arch Biochem Biophys 142:548-551, 1971 22. Leiberman J: A new confirmatory test for sarcoidosis. Serum angiotensin converting en/.vme. Effect of steroids and chronic lung disease. Am Rev Resp Dis 109:743, 1974 23. Lineweaver H, Burk D: The determination of
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