Measurement of Renin Activity in Human Plasma

Transcription

1 Measurement f Renin Activity in Human Plasma By Peter T. Pickens, M.B., M.R.C.P., F. Merlin Bumpus, Ph.D., A. Murray Llyd, M.B., M.R.A.C.P., Rbert R. Smeby, Ph.D., and Irvine H. Page, M.D. Many methds fr measurement f renin activity have been described since Tigerstedt and Bergman 1 first made an extract f kidney and shwed that when injected it raised bld pressure. Thse methds which rely n the rise f bld pressure fllwing such injectins suffer frm three main disadvantages. First, the magnitude f respnse t a given dse may vary and tachyphylaxis can be elicited. Secnd, relatively large quantities f extracts are required t btain a measurable respnse. Third, renin is an enzyme and nt a pressr substance as thught at first, and this type f assay des nt measure its enzymatic activity as discussed riginally by Plentl and Page. 2 Methds in which renin is allwed t react with substrate in vitr, and in which the resultant angitensin is measured, avid these difficulties. 2 Angitensin des nt prduce tachyphylaxis except in large dses. The sensitivity f renin measurement can be increased by prlnging the time f incubatin. Furthermre, these methds allw cntrl f numerus factrs which affect the rate f frmatin, r destructin, f angitensin such as ph, temperature, substrate cncentratin, presence r absence f inhibitrs r activatrs, and angitensinases. Cntrl f these factrs can be achieved by separating renin as much as pssible frm ther plasma cnstituents, and then adding t it a unifrm Frm the Research Divisin, Cleveland Clinic Fundatin, Cleveland, Ohi. Supprted in part by research Grant H-6835 frm the Natinal Heart Institute and by grants frm the Heart Assciatin f Nrtheastern Ohi, Inc. t Rbert R. Smeby. Accepted fr publicatin May 13, substrate. 2-3 Alternatively, the reactin may prceed under cntrlled cnditins similar t thse in whle plasma, 4 ' 5 with inhibitin f interfering factrs r crrectin fr thse which cannt be inhibited. It is cnvenient t make use f bth types f methds. We have studied sme f the factrs cncerned in measuring renin activity in human plasma. Methds PREPARATION OF RENIN Renin was prepared frm human kidneys by the methd f Haas et al., and was kindly assayed by Dr. Harry Gldblatt. This renin cntained a small amunt f angitensinase with a ph ptimum at 5.5, but at ph 7.5 n angitensin destructin was bserved. RENIN ASSAY Apprximately 20 ml f heparinized bld were cled rapidly in a 50 ml plyethylene tube in ice and all steps f the prcedure prir t incubatin were dne at 0 t 4. Fllwing centrifugatin, the plasma was remved, and its vlume recrded. Fllwing adjustment t ph 6.5 with 1 N HC1, the plasma was dialyzed, with stirring, against 5 liters f a slutin f di-sdium ethylenediamine tetraacetic acid (EDTA, 2.2 g/liter) fr 24 hurs and then against 5 liters f distilled water fr a further 24 hurs using Visking 20/100 dialysis tubing. The vlume f the dialyzed plasma was measured and the heavy precipitate frmed during dialysis remved by centrifugatin. A 0.05 ml sample was then remved fr substrate assay as described belw. T the remaining supernatant was added ne drp f a 1:20 slutin f di-isprpyflurphsphate (DFP) in isprpyl alchl and the slutin adjusted t ph 5.5 with HC1 r NaOH. A sample was remved fr measuring velcity f angitensin frmatin. The remainder (usually abut 10 ml) was measured, transferred t a plyethylene tube and incubated fr fur hurs at 37 C, 1 ml f saline added, the vlume adjusted t 20 ml with distilled water, the ph brught t 5.0 with HC1, and the tube placed in biling water fr 10 min- 438 Circulatin Research, Vl. XV11, Nvember 196}

2 MEASUREMENT OF RENIN ACTIVITY 439 utes. The prtein precipitate was remved by centrifugatin and the supernate adjusted t ph 7.2 with NaOH, and then t ph 6.8 by additin f 0.1!6 acetic acid. If the slutin was cludy it was recentrifuged. An aliqut, usually abut 15 ml, was evaprated t dryness under reduced pressure and the residue prepared fr assay by redisslving in distilled water using 1/20 the riginal vlume. SUBSTRATE ASSAY T 0.05 ml f dialyzed plasma was added 1 ml f 0.01 M EDTA in saline, 0.1 ml f a slutin f human renin cntaining apprximately 1 Gldblatt unit per ml and 0.85 ml saline. After ph adjustment t 7.5 with NaOH, the mixture was incubated fr ne hur at 37 C. The ph was then adjusted t 5.0 with HCl, the tube placed in biling water fr 10 minutes, prtein precipitate remved by centrifugatin and the supernatant assayed. Substrate cncentratin was expressed as nangrams (ng) angitensin prduced. REACTION VELOCITY OF ANGIOTENSIN FORMATION T 0.9 ml f dialyzed plasma was added 0.1 ml f a renin slutin cntaining apprximately 0.01 Gldblatt units (G.U.) per ml, ph was adjusted t 5.5 with HCl if necessary, and the mixture incubated fr fur hurs at 37 C. Then 1 ml f saline was added, the tube placed in biling water fr 10 minutes and the prtein precipitate remved by centrifugatin. Results were expressed as ng f angitensin prduced. ASSAY OF ANGIOTENSIN Angitensin frmed during incubatin was measured by its pressr effect in the rat 7 ' 8 given a gangliplegic agent t increase its sensitivity." Animals weighing between 150 g and 200 g were anesthetized by intraperitneal injectin f 100 mg/kg sdium Amytal (isamylethylbarbituric acid). Atrpine (0.65 mg in 1 ml saline) and pentlinium (5 mg in 1 ml 20% plyvinylpyrrlidne) were given by subcutaneus injectin. The trachea was intubated and bth vagi were cut. The cartid artery was cannulated and cnnected t a mercury manmeter. A fine cannula was placed in ne femral vein fr injectins. Standard slutins f angitensin were made by dilutin, at the time f assay, f a stck slutin f a crude preparatin f natural angitensin equivalent t the fllwing cncentratins f the isleucyl 5 -angitensin n: 33.3 ng/ml, 16.7 ng/ml and 8.3 ng/ml. Injectins f 0.1 r 0.2 ml were given and washed thrugh with saline s that the ttal vlume injected was 0.25 ml. Recently the stck slutin was prepared frm cmmercially available angitensin n (Hypertensin, Ciba) by disslving the cntents f a 2.5 mg angitensin vial in 1 liter f 0.1% bacitracin slutin. Using this slutin 100 ml were diluted t 758 ml with 0.1% bacitracin t give a stck slutin cntaining apprximately 330 ng/ml. This was standardized against a preparatin f knwn activity t determine the cncentratin f bilgically active peptide. This slutin is kept as a stck slutin and is diluted 1:10 with saline at the time f assay. Results ASSAY OF ANGIOTENSIN Bld pressure rise is nt directly prprtinal t the dse f angitensin in the rat assay preparatin, but t the lgarithm f the dse (fig. 1). This linear relatinship makes it pssible t use rather widely spaced standards f 0.83, 1.67, and 3.33 ng and t interplate between them t btain the value f an unknwn. The sensitivity f the animals varies cnsiderably, bth initially m - < 4 Hig.l) O.I ANGIOTENSIN (NANOGRAMS) ANGIOTENSIN (NANOGRAMS) FIGURE 1 Dse-respnse relatinship f angitensin in the rat pressr assay. Circulatin Research, Vl. XVI1, Nvember 196}

3 440 PICKENS, BUMPUS, LLOYD, SMEBY, PAGE (three- t furfld) and during the curse f the day (five- t sixfld). This is illustrated (fig. 2) by results frm several rats, sn after they were prepared, and several hurs later shwing that the linear relatinship is maintained ver a wide range f sensitivity and that the preparatin remains useful fr assay. The lwer limit f sensitivity differs frm animal t animal and figure 3 is a I 14 b. u 6 it u/ A ANGIOTENSIN (Nngrn) FIGURE 2 s Demnstratin f the validity f the rat pressr assay by means f animals f differing sensitivity t angitensin. tracing frm a rat in which it was pssible t measure amunts f angitensin smaller than Ing. It has been fund advantageus t wait at least ne hur after preparatin f the animal, because during the first 30 t 60 minutes sensitivity ften increases. Several injectins f standard (usually fur t six injectins) are given t insure that sensitivity is apprximately cnstant; subsequently at least as many injectins are given f the standard as f unknwn. At least three injectins were given fr each sample t be assayed. The accuracy f the assay has been estimated frm 10 standard slutins cntaining between 10 and 30 ng/ml f angitensin measured as unknwn samples. The percentage errr fr these samples varied frm 1 t 9% with a standard deviatin f ± 5%. ANGIOTENSINASE Whle plasma destrys angitensin, and althugh it may be pssible t measure renin in the presence f angitensinases, 2 we have attempted t inhibit them. Angitensin destructin after fur hurs incubatin culd then nt be detected. Since plasma angitensinase A requires calcium ins, 10 this in was remved by dialysis against EDTA slutin. Hwever, when calcium-free plasma was incubated with small quantities f angitensin fr fur hurs at ph 5.5, lss f angitensin still ccurred (table 1). In whle plasma, Dse (nangrams) Pressr Respnse U U U (mm n drum) (1) Rane -16% t +10* S (2) Rane -13% t + 81 S D.+7* FIGURE 3 Tracing frm a rat assay demnstrating that the respnse t 0.8 ng angitensin can be easily measured. Circulatin Research, Vl. XVI1, Nvember 1965

4 MEASUREMENT OF RENIN ACTIVITY 441 angitensinase activity was greatest at apprximately ph 7, but that angitensinase remaining after remval f calicum ins had a ph ptimum f apprximately 4.5. Di-isprpylflurphsphate (DFP) in a cncentratin f 1 part in 2000, reduced the lss f angitensin s that it was n lnger measurable by ur methd (table 1). Angitensinase inhibitin by DFP was nt reversed by dialysis. Its activity was usually less in fresh plasma than in plasma stred in glass bttles fr several weeks. EDTA was added t the fluid in the bath during the first dialysis rather than at the time f incubatin because it was bserved that in high cncentratin and after repeated dses, it was lethal t rats. If any DFP remained after biling at the end f the incubatin, its cncentratin was s lw, the assay animal was unaffected. Neither EDTA nr DFP, in the cncentratins used, affected the velcity f the reactin f renin with substrate. The angitensinase f kidney renin preparatins was partially inhibited by DFP. RENIN SUBSTRATE Substrate was assayed by incubatin f a small vlume f plasma with an excess f renin. Fr each preparatin f renin, the vlume f a given slutin necessary t prvide an excess was determined by experiment as illustrated in figure 4. The amunt f renin required fr maximum angitensin yield (fig. 4) was multiplied by 10 and this quantity f renin used fr rutine substrate determinatins. The average substrate cn 30 z TABLE 1 Angitensinase Activity in Calcium Free Plasma and Its Inhibitin by DFP Plasma sample ENSI Per cent angitensin remaining after incubatin* ph 5.5 ph 5.5 n DFP DFP added % z RENIN FIGURE 4 Angitensin yield pltted vs. renin level used t shw minimum quantity f renin required t give maximum angitensin yield. Renin in ml f preparatin used. centratin f eight samples was 720 ng/ml befre and 712 ng/ml after dialysis. Therefre, substrate was nt lst during dialysis. Substrate cncentratin in plasma f 29 nrmal subjects ranged frm 510 ng/ml t 1010 ng/ml, mean 729 ng/ml, in 32 patients with hypertensin frm 370 t 1320 ng/ml, mean 668 ng/ml, and in 9 pregnant wmen during the last trimester frm 1930 t 3240 ng/ml, with a mean f 2613 ng/ml. VELOCITY OF ANGIOTENSIN FORMATION AFTER ADDITION OF RENIN Velcity f angitensin frmatin in different samples f plasma, after additin f a cnstant quantity f renin, varies cnsiderably. This variatin is nt due t differences in ttal substrate cncentratin, nr t renin already present in the plasma. When a cnstant quantity f renin (apprximately G.U.) was incubated with a series f dilutins % ph 7.5 n DFP * In each instance 33 ng angitensin were incubated with 10 ml plasma at 37 fr fur hurs. Circulatin Research, Vl. XVII, Nvember

5 442 PICKENS, BUMPUS, LLOYD, SMEBY, PAGE angitensin. The mbility n carbxymethylcellulse paper electrphresis at ph was similar t that f angitensin SUBSTRATE NANOGRAUS/nl FIGURE S Angitensin prduced in tw plasma samples when cnstant amunts f renin were added at differing substrate cncentratins. f tw different plasma samples (fig. 5), the velcity at any substrate cncentratin was apprximately three times greater in ne plasma than in the ther. These bservatins suggest the presence f an inhibitr in the slwer reacting plasma 11 (r an activatr in the faster reacting plasma), r the existence f mre than ne frm f substrate. Hwever, Skeggs et al. 12 have been unable t demnstrate any difference in the rate f reactin f renin with different hg substrates. The results f incubatin f a cnstant quantity f renin with plasma frm nrmal subjects, frm patients with hypertensin, and frm wmen during the last trimester f pregnancy are shwn in figure 6. The angitensin frmed by incubatin f plasma alne has been subtracted, except in three samples taken during pregnancy (pen circles). In the plasma f pregnant wmen angitensin was generated faster. The plasma f the majrity f hypertensive patients had relatively lw rates f angitensin generatin, althugh in fur they were unusually high. EVIDENCE THAT THE SUBSTANCES MEASURED ARE RENIN AND ANGIOTENSIN The substance measured by ur methd was nndialyzable and the effect f ph n the rate f the reactin was similar t that f renin with partially purified substrate. The pressr material measured by the rat assay was absent after dialysis and was frmed during incubatin. Upn injectin int the rat, the characteristics f the pressr respnse were identical t thse prduced by synthetic RECOVERY AND RELIABILITY The recvery f added renin r angitensin and variability have been measured during each step f the methd. In nine experiments a quantity f renin varying frm t 0.1 G.U. f human renin was added t 10 ml f plasma befre, and after, dialysis. After dialysis the recvery varied frm 87 t 108% with a mean f 97%. There was n greater lss with lwer renin cncentratins than with higher. Pssible lss f angitensin during incubatin was determined by adding 33 ng f angitensin t 10 ml f dialyzed plasma and carrying thrugh the cmplete prcedure. The same plasma incubated alne fr fur hurs 9 Z 150- Ui 00- PRODl z 55 z 5 < 50- PLASMA SAMPLE FROM Nrmal Hypertensive Pregnant h 1 t* t. : t u FIGURE 6 m 9 m (tig.6) Amunt f angitensin frmed by a cnstant level f added renin in fur hurs with different plasma samples. The data, except pen circles, have been crrected fr endgenus renin levels. Circulatin Research, Vl. XV11, Nvember 196}

6 MEASUREMENT OF RENIN ACTIVITY 443 with 33 ng f angitensin added immediately befre biling served as a blank. In eight experiments recvery ranged frm 90 t 108%, mean 99%. In anther eight experiments in which 33 ng f angitensin was added t 10 ml f dialyzed plasma, biled at ph 5.0, and evaprated t dryness, recvery varied frm 84 t 96%, mean 89%. Human renin was added t a large vlume f human plasma t prduce a cncentratin f apprximately G.U. per ml. Ten samples, each f 10 ml, were assayed in pairs daily fr five days. The yield f angitensin varied frm 83 t 106 ng, with a mean f 91 ng and the standard errr was ± 8%. There was n greater similarity between samples assayed n the same day than n thers. The lss f added renin and angitensin thrughut the prcess was apprximately 15% and the variability f recvery 85% ± 7%. OTHER FACTORS Incubatin was cnducted at ph 5.5 because it is clse t ptimum fr the reactin f human renin with substrate (fig. 7), and far frm the ptimum fr angitensinase activity f whle plasma. Other factrs that might influence the chice f ph include 1) the activity f the DFP-inhibited angitensinase is less in mre alkaline cnditins; a ui 3 a 3 z 40-i 20- HUMAN KIDNEY RENIN 2) the Michaelis cnstant is lwer at ph 7.5 than at ph 5.5 and, therefre, in nrmal plasma, at ph 7.5 substrate cncentratins are less likely t fall belw the level necessary fr zer rder kinetics t apply than at ph 5.5. Incubatin fr fur hurs was chsen since at this time nrmal plasma generated enugh angitensin t assay readily, and the fall in substrate cncentratin was insufficient t affect significantly the velcity f the reactin. Fr example, the highest renin activity fund in nrmal peripheral venus plasma generated 50 ng f angitensin/10 ml plasma at the end f fur hurs incubatin. The substrate cncentratin was 510 ng/ml, which was reduced by dilutin during dialysis t 465 ng/ml. After incubatin it fell t 460 ng/ml, a decrease f 1.1%. In samples with abnrmally high renin activity, the fall in substrate cncentratin was greater, and the effect n velcity was increased if the initial substrate cncentratin was lw. The highest renin activity we bserved was in plasma f a patient with hepatic cirrhsis and ascites whse plasma generated 585 ng angitensin/10 ml in fur hurs. The substrate cncentratin f this sample was 385 ng/mg, after dialysis 360 ng/ml, and after incubatin 302 ng/ml. This reductin f substrate cncentratin wuld decrease the velcity by apprximately 12% and HUMAN PLASMA (fij.7) I ph FIGURE 7 I PH Curves shwing ph ptima fr crude human kidney renin and fr renin in human plasma. Circulatin Research, Vl. XVII, Nvember 196) 7.5

7 444 PICKENS, BUMPUS, LLOYD, SMEBY, PAGE the effect n ttal yield wuld be apprximately ne-half f this, because the substrate cncentratin fell gradually, thugh nt unifrmly, thrughut the fur hurs' incubatin. The efficiency f dialysis in remving angitensin r ther pressr substances was tested by adding 1 ml f saline cntaining 330 ng f angitensin t 9 ml f plasma. After dialysis fr 24 hurs, 19 ng f angitensin (6%) remained, and after 48 hurs, 6 ng (2%). In nineteen unincubated plasma samples n pressr activity was measurable, althugh several samples had abnrmally high renin cncentratins. Plasma was dialyzed against distilled water rather than saline because f the 20-fld cncentratin prir t assay. Precipitatin f prtein during biling has been fund t be greatest at ph 5.0 and less than 1% remained in slutin. Smetimes the centrifuged specimen was turbid after biling. This turbidity culd usually be remved by centrifugatin after adjusting t ph 6.8. Maximal prtein remval facilitated redisslving the residue after evapratin. Occasinally depressr activity has been bserved and this was readily remved by ether extractin f the slid residue befre disslving in distilled water. KINETICS OF THE RENIN REACTION The kinetics f the renin-renin substrate reactin have been studied previusly by Plentl and Page, 2 and we have studied them again under these particular incubatin cnditins. When renin was incubated with a lw cncentratin f substrate and samples were withdrawn at intervals, the rate f angitensin frmatin decreased as the substrate cncentratin fell, and after three hurs, the rate was n lnger measurable. The rate f the reactin was prprtinal t substrate remaining. Under these incubatin cnditins f lw substrate cncentratins, the reactin is f first rder with respect t substrate. The integrated frm f the first rder equatin is: 2-(lg! k = * loo(m-x) M \ where k = cnstant depending n the enzyme cncentratin. M= initial substrate cncentratin. X = prduct frmed after time t, t = time. n,., 100 (M-X). On pltting lgi ^-TT against t, a straight line shuld be btained if the reactin fllws first rder kinetics. Within the accuracy f the methd the data are cnsistent with first rder kinetics. An attempt has been made t determine whether an equatin such as that f Michaelis and Menten described the relatin between substrate cncentratin and velcity. The equatin is: V v=- 1-- where v = velcity at substrate cncentratin. V = velcity when enzyme is saturated with substrate. s = substrate cncentratin. K m = a cnstant characteristic f the reactin (the Michaelis cnstant). This equatin might describe the results bserved at lw substrate cncentratin, since when s is small, v tends t becme directly prprtinal t it. At high substrate cncentratin when s is large, v tends t becme independent f s. T test the usefulness f this equatin in the intermediate range f substrate cncentratin where s is equal t K, n a series f tubes was prepared cntaining a cnstant cncentratin f renin (apprximately G.U./ml) and substrate cncentratin ranging frm 42 ng/ml t 220 ng/ml. After incubatin fr ne hur at ph 7.5, the reactin was stpped by biling, and the angitensin generated measured (fig. 8). The substrate cncentratin is the mean f that at the beginning and the end f incubatin. The Michaelis cnstant has been calculated, and the pen circles are the values derived frm the Michaelis equatin. Within the accuracy f the methd the experimental values are cn- C'TCulatin Research, Vl. XVII, Nvember 1965

8 MEASUREMENT OF RENIN ACTIVITY 445 sistent with thse predicted by the equatin. The effect f enzyme cncentratin n yield f angitensin has been studied (fig. 9). A plasma sample with high substrate cncentratin, 1060 ng/ml, was chsen in rder that the velcity shuld be little affected by substrate cncentratin. The data fall apprximately n a straight line (the line des nt pass thrugh the rigin because this substrate sample itself cntained pressr material). Under these cnditins velcity is directly prprtinal t enzyme cncentratin. 120n N S Experimentl Redings Thereticl Curve vltu Substrate Nanqrms/ m I Crrected FIGURE 8 Cmparisn f levels f angitensin frmed as determined experimentally with thse calculated frm the Michaelis-Menten equatin. _35-i uj 25- tz a RENIN FIGURE 9 Relatinship between amunt f renin and angitensin (nangrams) frmed in plasma. Numbers n abscissa refer t vlumes f a standardized renin sample. Circulatin Research, Vl. XVII, Nvember 1965 The relatin between enzyme cncentratin and yield f angitensin was als studied at lwer substrate cncentratins. Figure 10 presents data pltted accrding t the methd f Lineweaver and Burk 14 frm an experiment in which tw cncentratins f renin, ne apprximately 10 times greater than the ther, were incubated with a series f dilutins f substrate. One series f tubes was incubated fr 30 minutes and the ther fr 5 hurs, in rder that an easily measurable quantity f angitensin be frmed in bth. In this type f plt the intercept n the hrizntal axis is equal t the negative reciprcal f the Michaelis cnstant (K m ). Within the accuracy f the assay, the values f K m d nt differ and, therefre, the substrate cncentratin giving half maximal velcity is the same fr bth renin cncentratins. Since the readings fall n tw straight lines, the rati f velcities remains cnstant at any substrate cncentratin. We cnclude h FIGURE 10 Lineweaver-Burk plts f the renin-renin substrate reactin at tw levels f renin.

9 446 PICKENS, BUMPUS, LLOYD, SMEBY, PAGE that the substrate cncentratin in nrmal human plasma prvides cnditins adequate fr renin assay. RENIN ACTIVITY IN HUMAN PLASMA Peripheral plasma renin activity has been measured in 31 nrmtensive subjects. The angitensiri frmed in 10 ml f plasma after fur hurs incubatin varied frm 6 t 50 ng with a mean f 26 ng. The highest plasma renin activity was bserved in a patient with cirrhsis and ascites; 585 ng f angitensin were frmed in 10 ml f plasma. Renin activity in renal venus bld btained by catheterizatin f the renal veins, r at peratin, was usually greater (as much as five times greater) than in arterial bld btained cncurrently, but smetimes there was n difference. Discussin Over the years, attempts have been made t find the mst sensitive bilgical preparatin fr measurement f small amunts f angitensin. 15 The bservatin f Page and Taylr 9 that gangliplegics greatly augmented the respnse, prvided a helpful clue and subsequent wrk has usually emplyed this principle in rats, cats and dgs, but especially the frmer because f their size. With varius preparatins prprtinality has been bserved between the height f the pressr respnse and the dse, between the area under the curve and the dse, between the height f the pressr respnse and the square rt f the dse, and between the height f the pressr respnse and the lgarithm f the dse. The latter is valid fr ur preparatin. Many smth muscle preparatins such as guinea pig ileum, rabbit intestine, rat uterus, and rabbit arta have been used fr angitensin assay. The spirally cut rabbit artic strip is very sensitive, but is difficult t prepare and slw in peratin. Pressr assay in the rat is adequately sensitive, and if several readings are btained frm each sample, each cmpared with injectins f standard, it is reasnably accurate. The relatin between substrate cncentratin and velcity in frmatin f angitensin is the subject f cntradictry reprts. Plentl and Page, 2 when using lw substrate levels, btained results which demnstrated that the reactin. f renin with its substrate is f first rder. When the substrate level is high, substrate is nt rate limiting and the reactin is zer rder. Helmer 4 has applied the findings f Plentl and Page generally t the estimatin f renin in human plasma when it was incubated in the presence f sdium chlride. Taquini et al., 10 and Hbler et al., 17 using cnditins very similar t thse f Helmer, reprt that in sme plasma samples the additin f mre substrate makes n measurable difference in reactin rate, suggesting zer rder kinetics. We have demnstrated that plasma samples, when incubated in the absence f any salts and under the cnditins described here, always frmed angitensin at a velcity cmpletely dependent upn renin cncentratin and that further additin f substrate did nt change significantly the rate f frmatin f angitensin. The bservatin that plasma substrate cncentratin was raised during pregnancy cnfirms that f Helmer. 4 In a small number f patients with cirrhsis f liver, plasma cntained less substrate than nrmal. It has been pinted ut by Plentl and Page 2 and later by Brwn et al. 3 that it is necessary t measure reactin velcity. Our methd des nt permit remval f samples at intervals during incubatin but, insfar as velcity falls ff, due t cnsumptin f substrate, an apprximate crrectin can be applied either frm an empirical curve r frm determinatin f K m. Since in ur methd renin cncentratin is lwer and substrate cncentratin is higher than that f Brwn et al., 3 the crrectin is likely t be smaller. In nrmal plasma it is s small (1%) that it can be ignred. In samples with greater renin activity, especially if the initial substrate cncentratin is lw, the crrectin is larger. In nne f thse examined has it been greater than apprximately 6%. We have nt made the crrectin, since the crrected figure wuld have n added clinical significance. Circulatin Research, Vl. XVII, Nvember 1965

10 MEASUREMENT OF RENIN ACTIVITY 447 It has been suggested that it is desirable t assay renin under cnditins similar t thse in whle plasma, withut inactivating angitensinase. is Dialysis alne, by decreasing Ca ++ cncentratin, may partially inactivate angitensinase activity. It may be pssible t measure renin in the presence f angitensinase, 2 but applicatin f the first rder reactin equatin des nt crrect fr lsses due t angitensinase activity. In the methd we have used, plasma angitensinase is inactivated by EDTA and DFP t the degree that its effect is negligible. These inhibitrs prvide a useful alternative t destructin by acidificatin at high salt cncentratin. 3 It is hped that DFP may als prve useful by inactivating sme renal angitensinase, remaining in renin prepared frm human kidney. Variance in angitensin yield with a cnstant amunt f renin added t different samples f nrmal plasma suggests clearly the presence in plasma f an unknwn factr which alters the rate f the renin-renin substrate reactin. Page and Helmer 11 presented evidence that a renin inhibitr is present in bld frm dgs made tachyphylactic with renin. Bucher et al. 5 have als bserved that angitensin frmatin is mre rapid in sme plasma samples than in thers. Hbler et al. 17 and Brunner 19 have fund angitensin frmatin mre rapid in plasma f nephrectmized rats than in nrmal. Our methd, as well as thers, des nt measure the true cncentratin f renin in plasma, but rather "effective renin activity" as it is affected by ther plasma cnstituents, including renin substrate. Summary A methd is described fr estimating plasma renin activity by using renin substrate present in plasma. This methd differs frm ther indirect renin assay methds by (1) incubatin in the absence f ins thus establishing cnditins fr zer rder kinetics fr the reactin between endgeneus renin and substrate and (2) the use f angitensinase inhibitrs di-sdium ethylenediamine tetraacetic Circulatin Research, Vl. XVII, Nvember 1965 acid (EDTA) and d-isprpylflurphsphate (DFP). Recveries f renin added t plasma in levels similar t thse ccurring in plasma are 85% SD ± 1%. The incubatin was dne at ph 5.5 which was shwn t be the ptimum fr human renin reacting with human substrate. By incubating human plasma samples with knwn quantities f human renin, evidence was btained suggesting that factrs ther than enzyme r ttal substrate cncentratins affect the velcity f angitensin frmatin. This variability f reactin rate may be explained by the existence f an inhibitr r activatr in this system r by a variatin in the type f substrate. Acknwledgment We thank Miss Lretta Lac and Miss Frances Prubski fr skillful technical assistance, and express ur gratitude t Dr. P. A. Khairallah and t Dr. H. P. Dustan fr many valuable suggestins and much helpful criticism thrughut the curse f this wrk. References 1. TICERSTEDT, R., AND BERCMAN, P. C: Niere und Kreislauf. Skand. Arch. f. Physil. 8: , PLENTL, A. A., AND PAGE, I. H.: A kinetic analysis f the renin-angitnin pressr system and the standardizatin f the enzymes renin and angitnase. J. Exptl. Med. 78: , BROWN, J. J., DAVIES, D. L., LEVER, A. F., ROBERTSON, J. I. S., AND TREE, M.: The estimatin f renin in human plasma. Bichem. J. 93: , HELMER, O. M., AND JUDSON, W. E.: The quantitative determinatin f renin in the plasma f patients with arterial hypertensin. Circulatin 27: , BOUCHER, R., VEYRAT, R., DECHAMPLAIN, J., AND GENEST, J.: New prcedures fr measurement f human plasma angitensin and renin activity levels. Can. Med. Assc. J. 90: , HAAS, E., LAMFROM, H., AND GOLDBLATT, H.: A simple methd fr the extractin and partial purificatin f renin. Arch. Bichem. Biphys. 48: , PEART, W. S.: New methd f large-scale preparatin f hypertensin with a nte n its assay. Bichem. J. 59: , KHAHIALLAH, P. A., AND PAGE, I. H.: A vaspressr lipid in incubated plasma. Am. J. Physil. 199: , 1960.

11 448 PICKENS, BUMPUS, LLOYD, SMEBY, PAGE 9. PACE, I. H., AND TAYLOR, R. D.: Mechanism f renin tachyphylaxis restratin f respnsiveness by tetraethylammnium in. Science 105: 622, KHAIRALLAH, P. A., BOMPUS, F. M., PAGE, I. H., SMEBY, R. R.: Angitensinase with a high degree f specificity in plasma and red cells. Science 140: , PAGE, I. H., AND HELMER, O. M.: Angitninactivatr, renin and angitnin inhibitr, and the mechanism f angitnin tachyphylaxis in nrmal, hypertensive, and nephrectmized animals. J. Exptl. Med. 71: , SKEGGS, L. T., LENTZ, K. E., HOCHSTRASSER, H., AND KAHN, J. R.: The purificatin and partial characterizatin f several frms f hg renin substrate. J. Exptl. Med. 118: 73-98, SMEBY, R. R., BUMPUS, F. M., KHAIRALLAH, P. A., PAGE, I. H., AND CHERIATHUNDAM, P.: Determinatin f angitensin in bld. Circulatin 28: 807, LINEWEAVER, H., AND BURK, D.: The determinatin f enzyme dissciatin cnstants. J. Am. Chem. Sc. 56: , PAGE, I. H., AND BUMPUS, F. M.: Angitensin. Physil. Rev. 41: , TAQUINI, A. C, BRAUN-MENENDEZ, E., FASCIOLO, J. C, LELOIR, L. F., AND MUNOZ, J. M.: Medicin del hipertensingen. Rev. Sc. Arg. Bil. 19: , HOOBLER, S., SCHROEDER, J., BLAQUIER, P., AND DEMERJIAN, Y.: Further studies n the mechanism whereby nephrectmy augments the pressr respnse t renin. Can. Med. Assc. J. 90: , HELMER, O. M.: Pressr substances and hypertensin. In Hypertensin: Recent Advances. Secnd Hahnemann sympsium n hypertensive disease, ed. by A. M. Brest and J. H. Myer. Philadelphia, Lea and Febiger, 1961, BRUNNER, H.: Angitensinbildung im Serum intakter und nephrektmierter Ratten. Arch. Exptl. Pathl. Pharmacl. 243: , Circulatin Research, Vl. XV11, Nvember 1965