Radio-Immunoassay of Angiotensin II

2l.n.ll

NADTO-IMMIJNOASSAY OT' AI\TGTOTAIVSTN TÏ

E, G. TI:IL,MSHURST, M.B. rB.S., M.R.A.C.P.,

Department of Meôicine,

University of Acl.eJ.alcle

A thesls subrdtteð to the Universfty of .4.ðeLaide for tho ilegree of

DOCTOA OF MEDÏCÏNE

.April", 'l !11 STATEMH\IT

Th:is thesis contains no materLal which has been accepterl for the awarð of any other d"egree or d-ipl.oma 1n any Uníversity and. to the best of my knowled.ge and belief containe no materi-al prevíously publíshed. or written by another person, except when d.ue reference Ís nacle in the text.

E. G. T{TIMSHT]RST

April - 1971 AClOVOWÏ,EDGEMMÍTS

This work was carried. out d"uring tenure of the positÍon of Temporary Lecturer in the Department of Meåicine, University of

Ad.e1aid.e. ï am ind.ebted. to Professor B"S, Hetzel, formerly

Michell Prof essor of Med.icÍne in the Universíty of Ad.e1aid-e, anô Prof'essor A.G, Ttlangel, present Miehell Profassor of Med.icino for the opportunity to work on thÍs prajeet. My supervisor,

Dro R.D" Sordon, proviåed. constant encouragement and ad-více d.uring the course of the work and I am partioularly grateful for hís critícal appraisal of the manuserÍpt. fhe invaluable technical assistance of Mrs. H.A. Zeunert is gratefully acknowleðged as is the assistance of Mr" W. Nolan in the preparation of the figures. The work described- in Chapter / was earrùed out in collaboration r¡¡ith Dr. G.H. Mcfntosh of the C.S.f ,R.0" DívÍsùon of Nutritional Siochemistry, Adelaid.e, who performeå all the surgical procedures describeð.

Finally, ï would like to thank the Members of the Honorary

Med"Íeal Staff of The Queen Elízabeth Hospital for perrrission to stud"y patients ad.mÍtteô under thelr care. TASLE OF CONTAVîS

CHÀPrm. 1 ¡ INTRODUCTION '1

.I " REI\TTN-ANGTOTENSTN SYSTEM 2, 2. AADïoïMMUN0ASSAY TECHNIQUE ¿¿.

CI{APTER- I? : ANGIOTMISÏN IADIOTM/ft]NOASSAY METHOD 4.1 .

1, .ÀNTIBODY PRODUCTTON 4e"

2, TODINATTON l+9.

3" STPARAîION OF BOU}ID FROM FREE PEPÎIDE ËD

4. ASSAT PROGEDTIRE ?Õ.

5. SPECÏFTCTTY OF THA ASSAY 57"

6. SnNSTTTVTIY OF THE ASSAY 59.

7. RTPRODUCTBTLTTY OF THE ASSAY 59"

B. ASSAY OF ANGTOTEIVSTN TT TN PLASMA SAMPLES 6t.

9" MEA.SUREMEI\ïf 0F RIIVTN ACrrVIrT, RENIN O0NCUVrRArrCIN ÀND RM..IÏN SIIBSTRATE BY BTOASSAY 6l+

CnAPTm. 3 t -NORMAT RANçE OT' PI,ASMA ANGTOTET{STN IT .AM RESPONSE TO PHYSTOTOGTCT,L STIMITTT 68.

1 NORMAL RANGE OF PIASMA ANGIOTM.ISTN TT 69.

2 RESPONSE OF PLASMA ANGTOTHVSTN TT TO PHYSTOLOGTOAL STTMTII,T 72,

3" CORRTIATTON BE[lfrEEIV PTASMA ANGIOTEIVSIN TT CONCENTAATICIN "7G. AND PT,ASMA RS|NTN ACTTVITY I )o cHAPrm. _¿ : PLASIVIA ANGTO ÐISIN II tHrffiS TN PATH0LOGICAL coNpIgtONÊ 77.

1 CONGESTTVE CANDTAC FATLURE 78.

2. CÏRRHOSÏS OF THE L]I/T,R 80"

NTPHROTÏC SYNDROME 81 " CIIÁPTEN. L (continued.) l+. PRIII{ARY ALDoSTERONTSM 81 " 5. ITYPERTUVSIoN 82,

6, ANEPI.IRTc SI]BJEurs 86.

CIIAPTUR 5 z APPLICAîTON OF ANGTOTEXVSTN TT RAÐTOThMUNOASSAY TO MEASTTRMIMTT OF PLASMA RTTÍTN ACTTVTTY 87.

1 . euYmAL BB.

2. RET{At VU{OUS PLASMA RA.IIN ACTTVITT RATTOS 91 " CHAPTER 6 z RENTN-ANETOTEI\TSTN SYSTNd IN PRFêNANCY 96"

1 ÏNTRODUCTTON 97.

¿6 PLASMA ANGTOTENSTN TT CONCETfTRATIONS TN NORMÀT PRæNANCY 101 .

3" PLASMA ANGIOIST{STN TI CONCN\TTRATTONS IN PRT-ECI,AMP TC TOXAmÍTA 106,

CHAPTER. 7 SHEEP STÛDTES 1 08.

1O INTRODI'OTTON 109.

2. CANNUTATTON OF RHVAÍ, TYMFHATTCS 111 .

3, PLASMA AND REIVAT Lfn/PH REMN CONCElflrRAfTON, RENIN ACTrVIrr, AND ANÊrorEîfSIN II CoNCENIR.ATI0N TN NoRMAL SHEM 112,

¿þ" RMITN AND ANGTOTM{STN TT TEVETS TN SHEEP FOTT,OWTNG SODTUM DæLETTON 117.

5. tr'FECT OF MM.CURTC CHLORTDE TNFUSTON ON PT,Á,SMA AND üTMPH RET{TI\ AND ANGTOTÉ'I\ISTN TT 119.

6 MFTCT OF MM.CURTC CHTORIDE TNFUSION ON RENAT LYIUPH FtOlJV RATE AND RH{AL HISÎOLOGY 123.

7. EF'TTCT OF MER.CURTC CHTORTDE TNFTISTON ON URTNARY RENTN CONCENTN.å,TION 125.

8. DISCUSSTON 126. CHAP:TM. B : DISCUSSION 129.

CHAPTTR. 9 SUMMARY 7l+2,

BTBITOGRA.P¡ÍY 145. 'î

cHAPrm, I

ÏNTRODUTTTON

1" REI\IÏN*ANGTOTENSTN STSTMú (¿) Håstoråeal

(¿i) Estimatiçn r¡f Reni"n teineentra*ion or Aotåvít;y in Bl"rood. and" P1asma

(¿¿¿) S'¿bet pate Measi:¡:emen'b (i") Es*imatf.on of Angtotensånaee .Aetivåty

(") 3.Lood Á.ngiateneÍ.n Esti.matåon

(lr¿) Såte af Proåuetåon c¡f Renån

& ) Loeatåon Ín th.e Kidney ç rrRen$.r¡s (b ) ån Extrarenal- îi$sues

(v:i:l) Regulatlon of 3.ctivíty of the Renin-AragS"otensi.r: S¡rs'ûem

a Sod.åunn 3al-anee FLuåd BaL.anee e S¡nmpathetíc Nen¡ous Systenr ð H¡poxÍ"a e 0ostrogeris f Fotassåtrur g Feeðbaek Control *:f Renín Seoretåon h IntegratÍon of Con*ro1 MeeÏ:a¡:åsms

2 rJ"DÏOTMMI.INOASSAY

(i) Ge¡renal

(it) Angi otensín Rad.ioÍrnmunoessay í,\'r ' . .1.! "ì l:,', :'r i¡: ,l !ìtr¡ r): t¡i,l

æ" .{ ¡: vl\ .

THÄPTE!ì. 1

ÏNTRODUCTÏON

"1o FENåN_-SN-eJ9ÏENISIN jiYSrEIt,I (i") H{ggpBIgA!

lhe existenee of the reni-n*angiotensin system we,s fÍrst

suspeeted following the work of Tigerstedt anð. Bergman (lggg), who

ðemonstrated. a prolonged. rÌse in arterÍ,al blood. pressure í,n anaesthetiøed- rabbÍts following the ånjeatå.on r¡f extra*t"s of rabbib 9- kÍ.d.ney. The renal pressor substance was founå in sa1åne extraets c¡f fresh rabbi"t kÍd"ney or of the rlny resí,ðue obtaineð. after trea,tment

of rabbåt kå,ð.ney wi"th aleohol, The authors eoi"ned the :reiTie "reni'n'* for the aettve substanee, whíeh was obd;aåned. from ee¡rtex but not

'Èo any .pppree$"ab1e extent freinr med.ulla of the kid.ney" lhe main

propertS"es of the substance weres i"t was non-d.ia1yøab1e, stable a&

5600 Uut d.estroyed. by boilå.ng, solu-bL.e in water, glyeeråne anå i,n d"ålute sa.lt sol-uti.ons, but Ínsol,ubLe i.n a,eetorre anð ån aLcohol.

The blcod. pressure rise was not abe¡lished by seetåon of th,e cervi..eal spinal. eorð zror by d.estruction of the spinal eorå, Ínei.ieating that the effeet of the pressor agent was irråepend.ent nf the nervÕus system.

The elassie paper of Gnl.d.blatt gt_gL UgSt*) åes,er.!bed. the pnoåuatåon cf sl,¿stair:e,l h¡rpertensíern in d"r-,gs by renal artery

oonstr:letion wÍth a elip" tonstråotåon of one renal antery nr*d- tn

a slåght te moôerate råse ån blo,etl ïrressu.re wåth a tenrîeney t<: return towarås nor"rnal after a peråeid. of tÍme, whereas bil-ateral renal isehaemi"a camrnonly proåuced. very hígh levels of btood. pressure" 3"

lhe authors felt that the h¡rperter:síon prodrieed by only moðerateS-y selrere renal artery eonstrietion, with no signs of sígnifieant.renal"

funetional åmpairnent, resembled" benign nephrosclerosfs in man,

whereas Bevere eonstrietion fronn the beginning Ied. to marked" el-evatåon

of blood pressuree severe d"Ísturbanee of renal funetion, and. uraemia,

tire latter pieture resenblÍng malågnant nephroseleroeís in man. The prod"uotion of e:rryerimental h¡pertension with a resemblanee to

elå.nåoa1 varj"cd;ìee of h¡ipertensive d-ísease in man stÍ.nruLated. a large amount of work aÍmed. at defining the role of the kådneys ån thís situation,

The work of fÍgerstedt and Bergman was eonfirmed ån 1!JB by Píekerång anð. PrÍnznetal- and. by Lanåt.s Ê!_gl" piekeríng ar:d

PrÍnzmetal showed. that a prolonged" rise in bl"ood. pressure may be procLueed. 1n the anaesthetizeiL rabbit by intravenous Ínjeetion of extraets prepareel from fresh kid.ney of several species" They eon- fårmeð that the aetåve substanee, I'renin'r, Tras present i-n the cortex but not ån the medulla of the kidney and that it had ehemÍeal properti.es suggestive of a protein strueture. fhey showed also. that the substance eould- be assayed" biologically by eomparison of the bloocl pressure response prod.uoeð ån unanaes'thetizeð. rabbíüs by the extraet with that preåueed uy a 6t*nðard preparati"on of reRÍn. Land.is e1*.q1. (lg:g)o workÍ-ng with rebbits, found that ttunheated kidney extraeterî had a variabl,e effeet on blsod pressure but that heating ta 55*56@Ç for 20 nd"nutes and. fåltration prod.uced. a¿r extraet whieh eonsisten.tLy elevated. bl"ood. pressurso fhe substance roÈponsÍble foi^ the press6,r ì+. aotivity of renal extraets was destroyed by heating ta 65CIe, ð.ið' nqt pass thrcugh a dialysis membrane, anò was precipitateð with ammonium sulphate. Golðblatt (lgSg) anð" Yfi-Ison anð Piekerir:g (tg:g) ðeseribe¿ the experlmental, proðr.retion Ín animals, by renal artery eonstrietion, of the fibrinoÍð arteri-olar necrosis eharaeteristíe of malignant h¡rpertensÍon, in the same ð"istribution as in man exeept for the sparing of the kidney. The sparing cf the kj-d-ney prornpteå the suggestion (CofatfaN*, 1938) tnat the height of the arterÍal pressr¡re ïras ån important faetor ån the proð.ueti.on of the arterÍolar lesions" Tn 1939, Page, anð Braun'-Men{ndez è:t g1- Ín¿epen¿ently showeå

.that renin had. the properties of an enøJnile, splitting a plasma eonstÍtuent into a smalLer moleeule now known as angiotensin" Page founå that injeetÍon of purifåeð renin ån Ringerr s solution ðid not proðuee vasoeonstrietåon in experínental animals, but that ineubation with I'renín-activator" in plasma restoreð the vasoconstrÍetor aetivity. lhÍs tJpe of observatíon leð to the esneept of renj'n aeting on a plasma substrate to form an aetive vasoeonstrietor substanee. The exåstenee of two forms of angiotensLn vqas åemonsi;rated. form, angíotensÍ,n Ï, by skeggs sg*gå (.rg:h). They showeå that the first was formeö. by the enz¡rmatic aetåon of renin on a plasna substrate anð that this was rapiÕly eonverteå to angiotensin TI by the aetåern of a

seeonð enuJ¡mê present in plasnra'

Tkre bÍoehemåca1 d.efinåtåon of the renin*angiotensín systen Ï was taken further by Lentz .g3--Cl þgfA). ?hey showeå that angiotensin contained one mole of leueine anð one mole eif histåðine in ad-d.Ítion ta 5. the a.mÍno acå.ð.s of ang5..r:tens5.n IIu and. that, conversion ef arrgåotevrsin Ï by eonvertirrg ena¡rme invol.ved hyðrt:J-ys5.s of the phcny1aL.anåne-hi.stt.ååne bonÕ to fonn angi.stensin TT and. histlåy3.. Leucine,

lhe amino aeåd sequenee.fc,r horse arrgíotensin Ï1. was

t¡æ-åso*his*pro-phe. l}:e amåno aeÍð. sequencre of angiotensín formed. by the action of rabbåt renÍn en ox substrate was reporteü at about

.the same tåme (Peart;, '1916g El1,ir:tt ¿rrrd Ps¡art, 195'7h the ainínc aeid" seqrÌenoe was shown to i:ec asp*e,Itg.-va1.*tyr-va1"-hís*prr:-phe-l:i.s* leuo Tt was thought tha* a1.1 the a¡rij-no aeå.ðs had. tlie L-eonfiguratåon ar¿¡l- that the N-tern-¡"ina.L resÍ,rlue wå.s åspå,rtÍ.e aeið, not asparag$,,ne, The synthesås of å.rrgiotensån was ach:ì"eveå shortS"y afterward.s by

Bumpu.s a}. ('tgSl) and by Sehwyzer a&. ret e$- ßgSA) " Drle to the above work, the eoneept of the renån-a,r:gíotensin systen had" d.evelopeû as follows:

SUSSTBATE

RENÏN J ANçTOTH'{SIN T (Decapeptiðe)

I üOifum.rÏNe EI\TZYMI J ANGTCITEIrÍSIN TT (Oetapeptåðe) ANETOfM{SffiÁ.SES tI ÏNATTÏTE FRAGMEIVfS 6

(ri) ESTTMATTON OF REINTN CONOE}ITRATTON OR ACÎIVITY TN BTOOD a{p P!AE_m{ Direet ehemieal" estÍmation of renin in plasma i-s not presently feasibLe anå awaÍts the rlevelopment and charaeterization of a pure renín preparatÍon. The currently avaÍlable method.s d.epend. on meas'úrement of the angiotensån generated d.uring the Íneubati"on of plasma samples ín vitro, The generated angiotensin is measured. by bi-oassay, which is based, on either the pressor aetÍon or the aetÍon in eausing eontraetion of smetc'üh musel-e" Most methqrd,s d.o not gi"re a preeíse meÐ,surement of the amount of enz¡rme present ín plasma since there are many faetors whieh affeet the rate of enz¡me-substrate reaetÍon anô thus the anount of angiotensÍn generated. by pJ"asma when i"ncubated. in vitro, The laek of a uni.versal renin stan¿ara preparation has meant that absalute values for renin aetivÍty or rôneentratlon obtalned. by groups using d"ifferent method.s cannot be eompared.. However, it Ís stil1 possíble to cornpare patterns of ehange in reËponse to vartous stímuU".

The assay teehnÍques d.Í.f'fer in tha.t some measur"e the amount of angiotensin generateð after a specific duration of ineubation, whereas others employ enzJnne kinetie techniques anð use eåther the i"nitial or maxímal veloeity erf angtotensån formatíon å,s a rneasure of the renin eontent of the plasrna samples.

The end, prod.uet of the Ín vitro ånor"ibation of plasrua samples is mainly angÍotensån I, owång 'bo inhíbition of converting enzwe by substanees ad"ðed. to prevent d.estruetÍ.on of the generated angiotensin by angÌ.otensinases (Boyd. C.t" g1.. , 196'"1), RenÍn "roneen- tratåonfi or rlaetivity'r estÍmatåons usång these ind.Íreet method-s are useful ín elinåeal praetioe as long as the resul-ts are interpreted 7" RenÍn Coneentratíon

These a,ssays set out to measure the aetual eoneentratior: of renin present Í"n plasma samples" After a preliminary purífieation or partlal separation of renírt, the plasma Ís treated. to d"eetroy end.ogenous substrate anð to inhibit angiotensinases and the renin is then allowed. to react with a stanðard exogenous substrate preparation so that, as far as possible, the renin eoncentration 1s the only varÍable faetor in the reaetion nixture

(Lever and Robertson, 196t+; Cook and Lee, 1)6Ji Brown e! e1. , 1g6t+), RenLn Aetivity

These urethod.s ðo not attempt to quantitate renin as sueh but rather measure the aetivity of the system (renin and its substrate) and. its abålity to produce angiotensin, .A.part fron ånhibiting angiotensÍnases to allow aceumulatåon of angiotensin, and purifieation of angiqtensin Ín one methoð (Boueher €S*g1. , 196t+), no attempt is inade to moðåfy the aetívity of tho system" Tnd.eed", this rryould defeat the purpose of rractivi'tyrr assays, wLri.eh should. eorrelate more elosely wíth aetual angiotensin (and. al.d.osterone) levels than renin 'teoneentrationl' methoðs whieh measure only one component of the system. Renin rraetÍvity" assays have been d.eseribed. by Helmer and. Jud.sorz (196j) , Pickens e3__êt UgAf), Boueher glel Ugøtr), Boueher and Genest (1966) and" Skiru:er (t 967) "

(tit) SIßSTRATE MEASUREMET{T

Available method.s fon substrate measurement rely on the ad.ð.ition of exeess renÍn and. assay e¡f the maximum amcr¡at of angiotensin generated., Fôr aeeurate measurenent, the ad.ded. rer¡in -qhould. be free B. of st¡bstrate anð angio'tensÍnasee arrð the substrate ehoulð not eontain renÍn, angiotensÍn or angÍ-otensinases" fhese eonðítåons are not sati"sfieð. ín any of the avail¿ble nethod"s, although a nethoð. whieh el-aÍms to proðuee substrate free of renl.n anð angiotensinase has been ðescribed (Coot and Lee, 1965> anð. might resolve this problem.

(¿") ESTTMAÎÏ ON OF ANGTOTEI{STNASE AOTTVÏTY .angiotensinase aetåvi'by has been univereally assessed. by examination of the su:rvi:ral of angÌ"oter:sin TT ad.d"eð to plasma" The problems of ínterferenee wÍth angiotensinases by the aqti- eoagulants useð anð the gerrere.tåon of further angiotensån by the

aetion of r"enin need. t0 be eonsÍðereû when using th:ie methoå" Levele of a^r:giotensinase actÍvity have Ï¡een reported" in a

(HteXter 1963) nr¡mber of h¡¡pertensive anð other conditions .9Ë.*-91, " ELevated leve1s were reporteð l"n several hSrpertensåve states, usua1J"y those thought to be assoeíated. with i,ripaÍrment of renal blood. fLawy

in pregnancy, and Ín refraetory oedema.

(") BLOOD AÑGTOTENSTN ESTÏMATÏON

Seornlk antL PaLadinå (1!61 ) deserå.bed a methr:ð whereby angiotensin is extraeteü from arterial blooð eollerote& Lnba )ffi ethanoL by parti-tion separation with an organie solvcnt folLoweÔ by ehronatography oR an ion exehar:ge restn" The extraeteð angiotensin

waå assayed. in the nephreotornizedn anaesthetåzeð" rat by the blood.

pressure respÕnse produieed. b¡f the samples. RereeverÍes of approxÍmately. @o afi aððeå angiotensÍn were eoraststently obtained"" The maÍn åÍffi"eulty wåth thie and other slmilar methods i.s that ,^

9. beeause of the l"ow l"evels of angiet,ensån in p3.asma, remo\ral af rel,ative3.y

3"arge vol"umes ef bl.o,od. is requS-red., wi"th resul-tant st.imulatåon of tire renin-angíotensin system.

A mod.ifieati.en of the method. of Scorr¡i-k and Palad,ini was d.eseribed" by Boueher et*ål ?gík"). lhis method. was quåeker. anð sÍmpler, while retaåning the same degroe of speeificS"ty and. serrsittvíty"

-A mean recovery fnom blood. of \fl" of add-ed. angiotensin was d.emonstrateå by this nethod.. Arterial bloerd" angíotensín levels were found. to be higher than veno,us level-s, whi-ch were too low to mea-cì.rre in all but one of the normal subjeets s'tud.åed.,

lhe method of Morrås and. Robinson (l y6&) offered a high reeovery (mean 8{"), the requtrement of only 50 ml of arteri.al blood., and. a relatively si.mple prooed.ure, al3"owÍng multip1e samples to be 'ilínimum assayed. in one ðay" ffre arnount of angiotensín that could. be accurately d.etermined. in a $0 rr1 blood. sample was g ng, correspond.ing with a eoneentration of 100 pg/m1 of bLocd, Ðeteetable l,evels of angio'bensin were found in the peråpheraL arterial" blo,od. of patients d.iagnoseð as having either h¡pertensj"or:l setloRd"ary to renal isehaemi-a or eoaretatÍon r:f the aorta.

"4, d.ifferent approach to the prob3-em of meas{ìrement of angiotensi,n was that of Reg,rrlí ai"rd lanc (lg64u 1966) who ¡neasured åhe cireulat$ng angÍotenstr¡ l",evel.s of d.ogs by alIowÍng bl:od. to d.rip o'irer various smooth mr¿sele preparations wi"th varÍ.o't¡s sensitåvåties to

ðtfferent pharxraeolr:gåea1 å.gerr'ts, the angiotensån eor.¡eeiltratíon beång assessed" by eontra*{ì.ur* c-¡r relaxatior¡ of the rat colen preparation" xa,

Thås system w&ß sensitÍve to eenr¡er¡tratå.ons cf engàotensån ïï of

0"1-'!.0 ng/mtr when bathed. ån. Krehet sr:l-uti.o.n, wÍth seirrewhåt reåucerl eensÍtívity when bathed. ån bl.oc,å. The ad"vantage of this methoö is that ít perm:its vÍrtually i.nstantaneÕuË measurement of aeute ehanges in the aetivÍty of the renin*angir.¡tensin system, assessing renin aetåv5.ty as well as possÍble changes in the netabolic elearanee of a,ngiotensin. ït is, however, less satÍsfaetory under contl"itions of relatívely sustaíneð stimulation of r"çni.n a*tivity, when only the initial changes may be uieasured. aeeuc"atell, må ít i"s very *umber$Õme.

("'i) STTE OF PRODUCTTON OF RETTIN ( ") lc¡sqlior¡_åe-:Þbe lliflqey Gaor:riagh{:åeh (1 939) suescsted" that ttre håstol,agieaJ- appear* anee of the juxtaglomerular ee1ls was ir:d.ieatåve af a possible end"oerine funetion" He ðemoyrstrated" that renal artery

eonstråetion in the rabbit led- te an inerease ill the number anrl sÍue of the gramrlated" eells in the juxtaglnmerular apparatus

and. that an id.entical picture was seen in the isehaemtc kid"ney of the d"og" This 1ed. him to postulate that the enðoerine aetivity of the juxtaglomerul,ar eells was related to tlre prod.uetion of the hS4pertensive substanee (i,e" renÍn) present

ån the . ùsehaemie kid.ney.

Tobian et e& UgSg) demo'4strated a stråkíng eorrel-atic¡n

between the amount of jwrtaglonerular granulatÍon and. the amount of extraetabl.e renin in the kiðney of the rat und.er eonðj"tions

of renovaseular anð. mÍr.eraloeorti.eoå.d" Índ.uced. hypertensiono 1tr,

Pi,teoek Så _aå (lgyg) she¡weð the s¿me ecrrelatåern between juxta* glomerular gra"nulation and" renín eontent in kidneys of normal and scüftrm:d.efieåent ratsu suggestÍng that the juxtaglonerular eells seorete the pressor substanee renín, and. that the gr"aritrles represent either renån or å, preeursor e¡f renÍn. îhe mieroåísseetåor¡ sttrdj-es of Bång and Koziníerezak (lg6Z) ðeuronstrated that the great majority of the renÍn Ín the kfdney was founå ín the maeiula d"ensa of the d.Ístal tubule or wíthin the wall- of the afferent arteråele, lhis fi.nd.ing was in agreement with the wor"k of Carok anti. PÍckerÍng (lgy9), who used. a suspensíon of iron oxåd.e and. a nagnetic method" to separate the various fragments of & ltrnashed.rr kidsrey and" founð that nost of the renÍn wqs. loeateå in, ûr very near to, the glomerulus.

?kre d.ireet fluoresoent antibod.y teehnåque Ìrr&s suoeessfully apþlied to tkre problem of S.eea1izatiorr of the histol-ogíe site of renín by Edelman and Hartroft (lg6l ), who founrl specifie staining ån the juxtagS,omeruiar r¡e13.s but not in the glomerular elcments or in other structures of the renal eortex"

(b) lrRenÍnti in Extrarenal Îåseu"es Ttlerle et_e& U957) showeð that the submaxil-l.ary glands of some miee eontain hígh amounts of a reni,n:li"ke enz¡rme. Birig anð Faarup (lg6S) d"emonstrated" that there was å cerrelatÍon between the renin oontent and. the propo::tion of granulate¿ d.uets, that both were higher Ín males than ån femaleso and. that both ehanged. Ín the same wåy after eiast,ratÍon anð und"er the influence x2, of varåous hoxmones (Traurbsei:":1d. -9å g1, , 1966). The *ontent eif 'trenånrt in submaxÍ3-lary gland.s appeareå to be ind"epenð.ent of reni.n eontent of the kid.ney anå was not altereå by soðium anð mÍr¡eralo- eortåcoid. admÍnåstrati,on " SeveraL grÐups have shown that the female repro'luotåve crgans of some mammals, includ.i-ng humans, eontain renin, the ooneentratj"on of whi.ch has been found ta be especÍally hå.gh in the rabbit uterus in the last thírð of pregnåney (Stakemann, 1!60;' Gress g Bång anå Faarup 19ð6i Fer:rås anð Mulrow, 1965). -g1. , 1i6Ì*i ,

(v:ii) REßULATTON OF ACTTVITY OF fHE RENIN-ÂNGIOTTT\TSTN SÏSTET/1

(") Sgð.tum_BcJ--sff¡æ

Lueteeher anð Axel.rad (t p![) showed" that a low sod.ium ðiet ån two normal s'trbjeots proåueeð a sharp råse in urJ-nary al-åoster'- one exeret{on and. a fall" j"n urínary sodåtrm" These ehanges were reversed. on the resumption of a nor"mal sod"Íun íntake. Ur:ireary

1 7-ketosteroi-ds oe 1 7-hyðroxyoerti"eosteroíås were not affeeteð a.nil the authors eoneS.uöeð that the effeot on alðosterone was

ånd"epen,åent of p5.tuåtar:y ,¡ortåec¡trophin, The finð.ång of :iri-

ereased aldosterr:ne proåi;totion ft¡i.l"owing sCId.i,un res'bri-e'ü5-on i:n

normaL û!år) was eonfi"rmed by Ïruetseher a.nå eü,nti,s (lgyn),

I{ernanðo eLg& llViZ), ar:,,C. Bas"tber e.&-aÅ ('f y¡p)o A sÍmåi"ar respiense was åemonstrated" ån the sheep by Der.otan 9e-q1. (lgfg)

BLais""-west (962) anã _eå-e¿ " The ro-l"e of the x"en.å.n-'engiotettài.n.systern in the regul.atierrr of

aðrenal fun'rt";å.,oi¡¡ was elarifieö. by the fi"nd.årig that the j"nwes'se t3: relatÍonship between soå.åun bala,:-".oe anð ttre secretiori r:f alðoe: terone was also reflee:teð Ín the eerr¡tent of renin Ín the k5"ðney (Grr:ss , 1958; 1959)0 Aeute sod.ium d.ep3"eti-cn or strong dÍuresis with aecompanying red.uetion in åntravascular voÏume was shown to lead to a pronrpt inerease in plasma renin aetåvity and aldosterone seeretÍon,

These ehanges were d.j-minished after rep)-aeement of bloerd. volume by repeated bl.cod transfusions (Êross g["-g}r 1965g BaÍ1ie -gt:gå, 1966; Brown BJ-et, 1)66i Davås -Êt--el, 1966i Vanðer anü Luciano, 1967a; Mdnarð. g[-9å, 1967), The repl,acement of the fluid lost d.uring mereurial-Índueed d.Íuresis restoreð the en}raneed. plasma renin actÍrríty to normal (Vand.er anå Lueiano, 1)6þ), whereas rapið. ínfusion of a h¡rpotonÍc saline solution into the acrta above the renal arteråes stimulated renin release (farttie et--al,

1966) " Prol"onged" d"5"etary sodiun restri"etion we,s obserueå to eause an elevation 'rf both plasma renÍn aetivÍ"ty and renal renÍn anå (Uavt,* to be acoompanied by an increased. ald.osterone seeretion

$jl, 1961(b); Blair-trest ej-g1,1963; Brown S3..-9.L, tg63i Gross S!,.-å1, 1965; Maråeb anð Mulr'¡w, 1965), whereas sodium l.oad.ing had. 'i;he opposå*e effeeto In resp,f,nse to a high sodåìlin d.ået given together wåth sodåt;tm-netaÍ"nÍ.ng st'eroi.ûs, plasma ren-år: aetÍvity fell to und.eteetabl.e levels aRå renin åisappeareð fr'onr the kådneys (Grc,ss eb--äL, 1965). Scveral, groups of workers d.emonetrated. that aj,åostersne se,"rretÍon was stimulated" by 4ì, exogenoÌls anÉii,Õtensi:n fI infusåon (Laragh gt*gL, 1i6û3 Genest

_gt-_êL, 1i613 Mt¡lrow _e$*&],p 19€,18 BLain-West et__4, 1962), mù it was shown that the å.nr:reaseð ald.os'terone output i.n response to various stÍmr¡lå was markcd"ly red.ueed. after total nephreetemy

(Davås -gt-at¡ 1)61 g Mulnow and Ganon g, 1i61(U) ) " A sÍmilar åmpaS.rment of the response of p)-asma renin ae*ivity to haemor:r- hage after bilateral nephreatomy was ðeseribed by McKenzie ..9!_9L., (1966). Tt was suggested. by these resurlts that the reni"n* angiotensin system, through the aetion of angiotensin Tï, had. a spee5"fi"e stinulatory effeot on the seenetton of ald.osterone by the aclrenal eortex. Severa1 other faotons, namely ACTH, plasma potassittim eoneentratfon, anð plasna sodiun eoneentratåon, were f,ound" to have an effeot on the eeeretion of a1doeterone i.nðe- penð.ent of the renin-angiotensín system (Luetscher a,nd. Ourtis,

1955f Låd"dl"e €&_g1,, lg56t Laragh and. Stocrk, X95"Vt Moran

-gt_g&u 1958t Bartter €t_ê&¡ n9591n Denton -Qt*ê&p 1959t Bl"alr- Tfest gþ_g1., 1)62; KapS-an, 1965)"

ReeentS"y, evi"denee ha.s beer¡ proðueed. to suggest that there may be at least orre furtlier faeton (so far unidentifÍed) whÍ.eh has a signÍficant effeet on the se,oretåon of al-d.osterenc

(ïrittS"ams 19VAg 3l"aå:e.-Triest 19V1) -gf_g&., _Ê!_4., " (b) slsåö-lclcpse A rapi"d.u sígnÍfiean* fa1l. in bl"ood. pressuùre prod.uoed. by haemorrhage or by d.rugs was ðemonstrateå to be fol.l-owed. by an

ånerease Í.n plasma nenå¡r ar-.'bå'víty (Davås -gt-ê&p 1!61g Mulrow 1961i 1)62g Gro.es 19655 MeKeinzi"e 1966) S!*-a1, , S!_g].p " 15,

Tt was riot eestaín whether' {'b was t}re reåuetåon of intr"avaseular voLume or the fa11 in blood. presßure whleh stínula'ted. the seeretion of renån, Broum et*C}. Ug66), shaweð ineonsístent effcets. on p3-asrna renin levels fn man foll.owíng the withdrawal of J¡CI0-500 ml of blood, but elevaterl p3-asma rerrin aotivÍty anð alåosterone (1y66) seeretion rate were ðemonstra*;eð by SkiLknan .e! --q1. after reåuetion of bLooð voll¡me by 1flo"

Loweneð renal- artery perfusion pressure has been showr: to stÍmul-ate renÍn reLease (Sklrr'rrer -9t-3J,, 1963)" Tf the a'nter"y of one k$"rlney 5.s etampeð, the rer¡in eontent í.n the e3.amped. kidney ånereases, whereas it i"s markcåly reðr¡eeå in the unel"amped kídney (RegolÍ eLeL, 1p62; Gross .gå-ÈL¡ X965). fht*se results suggest .that a reeeptor necthanism sensitive to streteh or pressure ån the afferent arteríole nright ae*ount for the aeute release of rcnin assoeåateð with lowered renal artery pressureo lhis situationo however, affeets ur5"nary flow and composi"tion, so that it is also possÍble tu pastulate a nnechanÍsm sensítive to urinary f3-ow and.r/er eomposítion" ExperÍ-mental evÍ¿erree h¿s been obtained suggesting a reæeptor ir¡ the region of the masuLa ðensa sensÍtive to soðÍum eoneentrati.on Ín the tubul-ar flr:id" (Tobian, ß6A:. lhura* et-gL, lg6l+i 196V)' Thi-s reoeptar måy as8Èrme åuçortanee in the release of renj-n seeonðary to soðåun åepletion

and. other faetors ehangång r"rrånary conpÕsÍtÍe¡n st:eh as d"iuneties. 'r6o Vanåer and. Mil-l"er (X 96&) tl.em,,or¡strat'ed" that it wae pÕsstble

tr: overe ome the s'tåmul-aÐt effceit of 1,3w4¿"oå renal s.p'tery perfl":siern

pressure on r"enÍ.n releaec by ånjeetåon of h¡pe:r"toní.o salåne or ðåureties ínto the renal årter"y, thus surggesting that the eomposåtion of the ttibul.e.r fl"uíd. might be of relatívely greater

S"mportarree than ¡:erfusåon pressure unðer these eircumstancles" Funther evåðenoe to support this våew was obtaÍneå by 3ai"li"e S$--el,

3gøA)o who ¡:roüuoed" erråd.enoe i.n el.,ogs that renal eoåÍun metabol"ism plays an important r"ol,e Í.n the a,ente regulatåon of renÍ,n seoreti.etn.

$"nðepend"ent of renal haemoô¡mamÍ.es, However, Ner¡rsome and Bartter

(l geA) showeå that the r"elatisnshi¡r af plasma renin aetÍnity to

serum sodåum eoneentrati"on eoul.d bc reverseð åuring overhyd.raüåon

and, thereforee some fae{:or related. tei boð.y fluÍð ba}ar,¡ee¡ êo8"

åntravaseulan velurûc otr renbl e,rtery perfusion pressure might be

more f"mp'ortant than senrnr soåår"vl eonoentratÍon in åetermining

plasma reni-n aetåuåty unðer these con.åi"tÍonsn a faet eonf irmed

-by the ïsork of Goråern and" Pawsey (1971)"

( * ) Sgr'ipCËþet¿.* NeFTrg-tië--üJåtgs A diurnal rlrythm of plasma renÍr¡ aertivity has been deserÍbeð tn

rran (&orüon qÈ*q}, n966(b)), tne htghest vaf.ues û'r,rurrång i.n the

earS-y mr:rning hours p.nd tkre l-:west Í,c¿ the af'berno,o:n" thanges

from *he reeumbent to tbr.e uprí.ght posåtåon, whether ao'tåve or

passåveu lead. tei ¡;rompt el"evat'iern ot" plasrna rer¡ån aetÍvity

(Corren.e!.'e}u 1p66i ßerüan -e$,-så, X966(a)s BÍ'ewn Så.-?-À, 196€'), d.espÍ.te the faet that b.Lc:;ð pressurs å,oes nct fa]I, Autar¡omåro

reflcxess êogo earotÍå eompressåon or ooJ-å presscr test, åR mar.t '17.

Ï:.ave been shosrn to ståmulate renirt release ar¡d alðos.terone seeretåon err exeretion (Gerdon gþ_Jte "'966(A)i 196'l); the eame effeet was seen after the ad.mi"nåstration c,f a norad"renaline/ aðrenaLine mixture (Gor,lon et a1., 1966(a)t 196T)" Pa*Í.ents wå.th severe autonomi"e insufficieney lver"e four¡å to frequently have a red"uetÍon in ald.osterone seeretíon not responsive to indíreet stimulatåon by salt d.epJ"etåon or di.reet ståmuJ-atíon with eortteotrophån or angiotensÍn ft (Slaton and Bíglierå , 196T). ïn one sueh patÍent, when the orthostatic reaction was preventeð. by the infusion of eateeholamÍnes, plasma renin actÍvity and. urÍnary alðosterone were elevateå (Gordon e,t*-.êI¡ 1967).

Other patierrts wíth autonomic ånsuffieíeney have bcen d.eseribeð. with apparently c ompletel.y normal renin-angi otensín-alåosterone and" sal-t*retaini.ng mechanisms (nttaaal and NieJ_sen, 1g7A) " Plasnna renin ae'tivity was found" to ínerease d.uri.ng exercise, an effeet which could. be prevented" with gangli"on*bloeking d.rugs

(Castenfors g!_É, 1967). I{odge e.t qt (1g66) showed .that bloeking of the renal nerve,s may inhibåt the reïease of renin tn response to haemorrhage in d"egs" Taquini g!_gå 3g6j+) rep*rted d.eereased. renin eontent Ín the d.enernra.ted. kid.ney of the rat compareô wÍth the opposite i'nta,:,,.t ki"ðney" VanrLer ?Eey) d.emonstrated the release of s"enÍn from the kådr:ey in d.ogs in respeirrse to elet: 'tråeal- stimuf,ation of the renal nerves.

lhe foregoång evid.enee sugges*s that an intaet s¡rmpathetÍe innervation is important fcr the release of renÍn" However, e=¡idence has been presenteð (vand"er anð Luetane, t967(b)) trrat trre 18,

sJmpathetie nervous system plays a moå5-f¡ntng rol"e but i"s not essential for the Ínoreaseå renån release proåu+erl by' natrí.ur"etie- indueeð, aeute soðÍ.um d-epletierri" Tt has also beerr ehown

(Greene and. Vandex,, 1)67) 'that normal ehanges in renín seeretion

åtr response to sodÍum d.eprivation and. to assumptåon of the upright posture oeeurreå in a group of patients with renal home.'çtansplants.

A nole for the s¡rmpathetÍe nervous system ín this situatåon cannot be eompletel.y ex*1uüeå, Ï:owever, sirree j"nereaseå sensitivity to eÍreulating eatecholamÍnes or regrowtir of the renal nerves oould. allow such an aetÍon to oeeur. (¿) Hvesxrg the effeet of renaJ- artery eanstrietåon on prod.uetåon of r"cnin by the kåûney has been shor¡rn ts be ur¡rel"ated" üo hSpoxÍa in the acute såtuatåon (Hu.iacbro and. Bratetr-Menend"ez n 19t+2i Df"vny,

1952; Skå.nr:er e_t-_-31., 196A)" A possible role for chroníe h¡rpoxia, however, hae been suggested. by the d.emonstratíon that prolongeð h¡poxÍa eauses íngreaseð ju¿xtagLomerular granul-atåor¿ ín rats

(Otåver and 3reidyr 1965) anå that pï-asma and" renal renín'øere

ånereaeed. ín rats kept ån an environment wÍth red.ur+ed" oxyge* tension (Gould, and Gooümano 'T 97A). (u) 0estroeens

Pl.asnna rerair: leveI,s have been shou¡n to ríse early' Í"n pregnaney, returntng to ::crr¡ra1 after d-elivery (Br"own. _gt*_ql, 1963). Tlie

*nerease ån plasma reni"n eoneer:tratior¡ d.uring pregnaney ås not as marked. as the i.n-orease in p.lasma seni"n aetivity; the greater

åncrease ín the latter para"rneter has been sharvn te: be Õ'u.e 1arge3-y "19,

to the inereased levels of renin substrate (Helmer and. Juð.sor,, 1967)o A slight varÍation in plasrna renån üuri-ng the noruaL menstruaL eyele has been reporteð (nrornn: gþÆ, 1961+) " A.signifieant Ínerease ín plasma renín aetivity values and. alåosterone exeretion was seen fol"lowi.ng admÍnåstration of

ethinyl oestraðiol" to no¡:rnal sub jeots, but no âpparent ehenge was åe'teetable after the admånùs'bration of medr"ox¡¡progesterone aeetate" å. substantial pereentage ,of nerrmal subjeets given

oral" eontraaeptÍ"res shovyeð inereased. pl.asma renin aetivity anð

aLðosteroRe exoretion rates d.uring tkre first anel- th:ird. weeks of

adminÍstration (Crane and" Harris, 1)6p). These studåes eonfÍmr the effeet of oestrÕgens on the renÍn*angiotensín system, aetång prfncípally through inereased. prod"uetíon of renin substrate. (r) Polese¿uu ïn marr, hågh d.oses of pctassium gåven d.urÍng the aðmlnis*

tratÍon of a low-sod.ium d"i.e't, d.im:inished. the high plasma renin aetåvÍty (Ve¡rrat g!._g.1, 1966), SimuT-taneously, the aldosterone sebretion rate rose, signifying the d.ireet effeet of a high plasma potassitam coneeR'tration on the aðrenal ee¡r"tex, wkr-ich Ís especåally pronouneed" d.uring sodium Êepletåon (tannon -et_g]., 1966), If , together with the feeåírig of a sod.å'um-restrl-etecl.

ðiet, potassÍfm rryas remoxreå by glvÍng an íon*exohange Í"esårr, pï"asma renÍ.n aetåirity jtnereaeeû more marked.ly than a1åosterone eeeretion" The aåd"ition cf potassíu¡n under these eonåitÍone stirnulated. al-d"osterone seoreti,on, whereas it lowereð reni¡r aetivity ån the plasma (Veyrat Si__el, 1966)" Iïì patients wåth 20. renal artery stenosi"s or malÍgnan't h¡¡pertension, in whom the plasma renin leveL. was high, potassåtrm admini.stratíon also Ieð to a markeð (Çanr:on 1966) ír¡ercase in the aldosterone se6iretÍ.on rate st-elr " fhese observations ðemonstrå.te that, under oertain experimental cond.itions, eÍtLrer an ad.ðltíve or an antagonÍståe effeet between pS.asma renin aetivity anð the plasna potassíum eÕneentrå,tion on the seeretie,n of aldosterone ean be aehieveð' The effeet of tho plasma potassÍr,in eoneentrati"on on the ald.osterone- producing eells of the aårenal cortex is, therefore, ind.epenð.ent of renin release or the plasma renin level.

(e) Feeåb aek ControL of Renin Seeretion There is no evLdence of a díreet feeðbaek meehanÌsm betl'ceen plasma alåosterone lerrels or å.ts seeretion rate and plasma ren-ín aetivÍ.ty" 0n the other hanð, h:igh d.oses of alðosterone or other sodium-retaining eortieold-s, as well as ssðium ad.ministration, red.uce renin concentration in the kid.neys and pLasma renin activÍ-ty, provÍd"ed. they are gåven for a suffieientLy long períoå¡ th:is suppressíoR of rer:ån proåuetion ås thought to operate through

the inereased plasma vol".ume cotlsequërlt upon these manoeÈìvres" Angiotensin has been üemonstretcð tci exert a negative fced"-

baek effeet ern nenÍr¡ release from the kÍ"ðney whi"ch Ís ind.epenåent of ehanges Ín nenal arteråal. blooð pressure or a1Õos'frerore seeretion (ßenest -ej-g}-, 1965; V¿rlåer a¡rð" Geelhoed", 1 965). The speeifíe rneeiranåsm by wliåckr 'bhis effee't Ís exerbed anå the relatj"ve åmportanee of this nie&ns of e.entro.L of renÍrr seoreti"oTL arc at present unknowr¡, 21"

( r.) ïnteeratÍon of c ein{:r"o1 Meoh¿nisms

Consi.ð.erÍng a3"1 the avaíle,b1e evid.enee, it would. seem that there are nultÍple faetors affeetíng the proctuetton ef renr,n by the kådney, md that these include at l-east the renal artery perfusíon pressure, tubular sod.åum coneentration, bl"ood. volume, pl"asna potassåun eoncentratfon, plasma angiotensin coneentration, and" aetÍvity of the s¡mrpatheti"c nervous systen" fhe relattvs Í,mportanee of eaeh of these faetors probabry varies aecordÍng to the physiological" state of the subjeet at that particular tÍ,me, wf"th particular refer.enee to state of hyd.rati.on, sodium baJ-amee, bl"ood. pressure, ånil so on. 22"

(z) RÄDTOTNMüMÍOASSAY gAC}INTQUE

(i) GaNptu\L A reaL unrlerstanding of the physíological roles of hormroncs was hampereð for meny yea.rs by an inability to measure aeeurately their lcvele ín bicJ"ogåeal flr¡j-ðs' Bioassays or the in*ireet methoel of examinatíon of theår effeots on tanget orgaJls were ueeð &s a mcåslure of tlic:i-p aetitoíty" Most of the avaå1ablc åssâys were relatåvely enurï-e bÍoassays, laekÍng sensitívity anei specifieity anð often being unab1e to deteet no¡mal eoneentratíens of the hormones eoneertaed" The availabílíty of protei.n horTnoncs in a purÍfied. fopm ancl the nceognS.tion of their speoíes speoifÍoity have leil ín reeent years to the proiluetion of speeifi-e antísera whieh eoulð be

used. to measure the hormones by immunorogieaf methocls. However, the conoentrations of protei"n horrnones in bíologÍeal fluÍd.s are so low tirat the only two ímnnlnologioal

methods wh:ioh are reaðily eapable of d.eteetíng them are the

red" .blooð. eeIl haemaggJ"utination ínhibiticn methoil anð the

naitÍoírnmun'oa.sÊey proeeðune o

, The red bl"ooel eeIl haemaggltltlnatíen i-nh¡-ibítåon

nethoð makes use of aggl-utination of sensitåzed. ezrythroeytes as thc mani-festatíon of an antÍge¡l-antibod.y reaction. Ït was

shown by Boyelen (lgy ) tirat ta^ns,):ie aeåå-{:neateel cx'Srbhroe¡rtes to 23"

whåeh speeåfto preteins are ade,orbed" are a.gg3"'utånated" by the

spec+åfåe anti-protein an'bis:era a:rd. ttrat an'ü{ge:l $.n sma,Ll" arnou:nbe

eran be d"etceteå by tlie i¡rhÍ.bå"tå<¡¡r of' the iraemaggJ-ut:lnatieir reå,otåon"

This effeot is d"ue to antlgcn e,onbÍtlång with antibody ilun$.ag pre-

ineubation so that few free antíbod"y moleeules remaån avaålab1e

for reaetåotr with the subsequentl"y aüded. sensåtåzed. red. bLr:e¡d eellso

The rcd. bIo,od. oeLL haernaggl"r:t:ination tnleibåtåori method. has been

applåed" to thc mcå,surcme¡:t of L¿i¡¡nar'¡ gnowth homre::e (Reað anå stoncn

19585 Reael asrd Sryai:n 196CI) a.r¡d adrenoeonti.eotnophie ho¡rnone

(nfeGamy gt* s¿. , 1962) ån plasma and, of Lruman ehoríonåc gonaåo-

tr,ophj"rn (Wtae a¡:il Gemøe]-J"u Xp6t; Xg6Z) ar¡d tn¿man ltateÍ.nÍzårig

k¿ormone (mae e.nel ån uråne e*cnzel-J-u 1Ç62g ï/iåc gL_g! ", 1961). ït ð.oes not, however, prov$"ðe the seneítåvåty and" preoi.siorr of

the raðåoímmuRoassay teotrnlque whieh has n,ow almoet eompletely repl"aeed" åt.

fhe rad.åoímmunsassay meth,ret is based" on the observations,

first repcrted. with ínsulin-15xt (u***on S-Lgl.," , ngg6).u.t;hatn åzr

the abeenoc e,f preeeipåtatios) cf antÈ.gcn-anbiboely elompr,exesu

antåbod.ies å.n 1ow eoneentnati.';n ane d.eteetable by theåx" a'båi-åty to x3trl*t*butleð bin¿1. hormone, anet furtherm're ttrat suoh bånåång is

ocmpetitåvcly ånhibi-ted by unlabelll-ed. harm,one. This eompe.t{ti¡u"e 'bc binð.ing may nepreselrtiaå as fo "l-¡r:w.s: 2l+.

Labell"eel. Pcptid,e Un-Labell"ed Peptiete

À

An'tlb,ody I

I ü ,/***o\ Label"l-eeL Peptåele' Unl"abel"leel PepticLe

+

Free Pept$"ele

tabell"eel ünlabelleð

Tf a system Ís set up sueh that, ån tl:e abeenoc of unlabell"eð honnoneu less than 1CIØ of the l"abel"Led homrone present ùe bounð to antåboðy, i.e" anttbod.y bínel{ng-sítes are saturated"u anå Íneroasíng amotrnts of unlabeLlecl hormoRe are ad"d.eå to this system, there will be progresslve tl"Ísplaeenent of label"leð homtone from the antiboily biniling-sites" Tf a mcthaeL Ís avaílabl"e for separation of antåboðy*bound" from fs'ee homone, this ðíspS"aoement can be measureel anit the neeults e:epressed. graph:iea1Ly in the fonn of a stancLard eurtre" If unknown samp3-es are ir¡oubateel ureder the eane oonclitions with the sa¡ne emou!1ts ef antisern"¿m and labell"eel hormone but wåth the unknown sample in plaee of the stanclarð, the cl.egree of displaocnent of label1.ed hor"rnone fnom the antibod.y ean . 25. be measuincd a,r¡ql u usirrg thås figure u ùlee arnou:it ef he¡nnone ån tire

sampl-e ean be detez'måned by eorapa:eie,olr wi'üh the stsnd"ant o.urve"

Tirc radíoirnnnunoassay teohæåque wâs or:lgínally d.eseríbed.

fon ånsulån (Ber"son and. Tale¡wr 1g18g T'al"ow e.nd. Benson, lpgg) anå has surbeeqtler':.t1y been applåcrl to a wide variety of proteín honnone;s n eog, glueag,rn (Unger -Q!_eI," , lg|gç Gnrrui"sky.gåjÅ" , 1gG1), grsurth lromrone (Utiger gt_*gl" , 1962; Glåcrk -gt- qå" , 1965¡ Hurntcr and Gr"eenwooð , 19&-r) u paratl4yr",oåd h,o¡"mone (Ï}ensoïi gg_gå" , 'tf)6jg shenwer¡d -q.!-*gå" e xg66), th¡æo:id" etinrura,"bi.ng ïroerro're (uttgen €S*åå",

1963a CIdeJ-r -eË*g&" , 1g63; tltlger, 1g65)u edrencerortteotncphio L¡ormorne (Fel"benn 1g65a Yatrow g$.*q&.. , lg€n+)n anð many ot,Lier"s.

The pråneipa3" ad"va.n*agee <¡f a"åd"åoi.atrnLr"n,oas.ss,y over" bå.oaseay pnoeedunes &re hrågh seresl'båvity anú sper:if.'åeùty" Against thås

ùt mu,st be realåzeð tla.et ti:e inn¡äùno.1og.i.e s"eaetivåty may not eeirselate e3"osery with the bioJ-ogåo aetivítyo as .the speeåfie e¡ni¡To aeåel eonfigtaratíone fon eaeh of thôso propcntíes may reeåde at åifferent si-tes on the horrno:le moleeu3e. Tf tile mol-eeul-e remaina

{gltaet it wor¿lci be expeeted 'that ttre tw.c urethode weuld. gi.ve å¿entieal g"esultsu but separati,on of thc tw,o aetåvi,t{es oo¡¡.1d. oecur"e eog, elüe to brcakde¡m of the Lre¡rmo¡re mol-eeule so tl¡at i*åclogåe ae.èivtty ås l-os.ü but immunologåo aotivS"ty åc netaåned, cre due to the ¡:resenors of ej.üren laåologie or åmmur¡ologåo inl:tbi.tc¡re" und.cn these e,enåitåo¿rs the a"esr¡l"ts obtained. by nad.åo{runìånoå,sså,Jr and" by b{oassa¡r might r¡ot be ån 26" ol"ose agreemento For this reasonu the generally acceptcd. et:nventi.on is to r¡se the profix '0årnmun,orea

4eg¡¡íremegþ s_ f or Rqlíglmmggqæ.S.+g lhe requirements for a sueoessf¿:l ra¡lioÍmmunoâssay proeedure ane as follows:

(u) Pune peptide fon use as antigen. (b) Speaifíe antíbody of high titre and affínåty.

(e,) A method" of labeIl"ång the peptåde to a suffieiently hÍgh speeifåe aotívity withoutt pnoðueång sÍgnífioa^nt'rðamage" te the moleeule" (¿) A nethocl of separation of antÍ"boeLy-bounå from free peptide.

(.) Puritv of Antieerx All antisera &re heterogeneotls anð the hsnmone antibod-y pnoôueed expeni.mentaj-ly us'utal"3-y reprosents only a small fnaeti"on of the total antS"body pncsent" Atr-though heteroger;eous antibodies ar,e inclueeð when a sÍngl-e proteín a"nti"gen ås useð, th:is d"ecs not Ínterfcre wíth the immarnoassay. Howevetr, eontamination of the hormone preparation usef, f<¡n immuniøatjLen with a pnartein of po'bent antigen- iei-ty may lead to proåueti"on ef an anti-sertrnn eontainíng antíbodíes 27,

to the sontamåna'nt ín eonoer¡ta"atå.er:rs mueh hågtrer than the oÕT1o€l]-

trati.on of hormone antiboåies" Tf the hcrmone prcparat3"on useð

as a standard" in the radi"oinmun,cassay is free of Ímpuriti"es o anti-

bod.íes to eontamÍnants i¡l the ínmur¡i"zíng antigen do..¡.rot prescnt a

problem, A smal"l resídual- eontamånant ín the hoc"mone standarci may interfere wíth the assaye but 1s less troublesome in the raeiio- immunoassay than i.n the neå bi-ieod eelI haemaggJ-utination*i"nhibåtj"on

nlethod".

(u) Produetiron of fu¡ti.bodÍes

The prod"uotion of anttboðies to the langer preteån hormones trae not pz"esentcd. a major probl"cm and many are suffieiently antigenåe to enable the proeluetåon a¡f antibod"ies with¡:ut the use of ad.juvants"

The small-er peptid"e hormones, howevere ã,re less antigenie and ít has ustrally been found neeessax"y to eouple angS"ote:,rsin to a earråer (Ðeodhar, preteÍn 196Ai Haber -gþ-gå, , 1965) cr to adsorb it onto ehareoal (Boyd -g!-gå, , 1967) ån onder to raåse antibod.ies of suffieient tåtre anð affÍnity for a raåioimmtlnoassåy ta¡ be developed.

The sensitivåty of thc raååoimnunoåssay teehníque has been

åemonstrateå to be ultimatel.y depende¡:t on the energy of the e::tågenr= antíboåy reaotion (Berson ar¡ð YaLrow, 196U)" tru$,en ind.ivådual antåeena eontain heterngeneous antí.boå{es reae tå.ng ïsåth varåabJ-e energåe,s u anå the po'üentíal sensåtivity of the assay w:!.11 d"epenrû an the enengy of neaetùon of, those antíbodåes present ån irågh enor*gh eonrcentnat{oia t,e l¡c d"cteotable at thc d.iluticn of anti.sen¡m employed." Tkris i"nformatåon 28"

eân be d.er*vecl fs'om the sJ-ope c¡f the stanåaæel eÌåmre or the s3-ope ef the cutrve obt¿uined when label]-cå peptåd.e i.s ånen¡l¡ated with ðoubl-tng d.:i,l.t¡ti.ons of, antÍsenum. ïl¡e tttre of the antj.serum is not important

Ín thc seLeeti.on ef the Eer.u¡n to be used" ån the ra.d.ícimnunoaesay end. tlae onl3r be¡aefit of a high ti.tre antisemm¡ íe that it allows a large

numben of samples to be assayed" with a giver: amount"

fhe sensåt{vity of the asså,}r is ðeter¡n:!.ned. by t}re fraetíona1

ohan¿ïe Ín bound.: fnee nat:{"o or pereenta.ge 'i¡erus}d wh:ieh Õoeung with the aeLùttåon of a small e¡mount of standas'd. hor¡none and thås ås la:ngest

when the åzri"tial pereentage bot¡nd. ís (Berson a¿ed yalow 196ì+) bV, , " rhus, for hågh sensit.i"vity åt ås pr"eferabte to use a dilutÍon of antiserum tlaat wi.ll bånd" approximatery 5V" øf the smarLcst aðeqgate

qtaantÍ.ty of la,bel-}ed" hoauor¡e ín the abse,nee of un1abelS-ed. honmone,

(") Lal¡e1nånE of, Her:rmone

Raêi.oeotålre homno¡"res f"or ¡¡s@ fn tlae naåi"oå¡nmu¡roassay teolrniqa:e

have usualtry been prepared. by J.od.Í.natÍon ef t¡rnosåne ncsi,d.ues in the tr31T. 131r pol¡rpeptådc with ru125T. Tr-re ad'antage of t* that åt Í-q

nel.ati-vc3.Jr eesy to ob'tain the high speoåf'åo aotivåty usually ïÌeeessår"y

fon sensåtivity (to minirni.ze the arnount of labetrred hormone used.)" fhc d.isad,vantage is thato ewå:ag to the short iratrf-life of the åsotopeo åt ås neo@sserJr to prepaz"e fx"eslr3-y lab,el-l-eel hosnene at fncqucnt íntes"- tr251-x*ïr*r.1ed" verso ria th.e case of' hormosne, & single pnepanatJ-on may be useð for weeksu the only l$.midatåon beå.ng the etabilfty ef the etone& tnaeetr, i.Iourevenu *he n¡axÍmum speeJ-fie aeti"vity that ea"re be aehricveð is i-nversctry retrated to the þrarf-nj"fe of the teotope anå to ,a aehieve eq'uÍ\rel"ent sp.reei.f5"e aotivi"ty rcqr,lire¡s labell"ing wÍth moæe 125x '13'trI atoms ,f tþr*¡r pu* merleoutre of poJ-¡rpepttde" ïfåth mo]-eer.rLes oontaånång J"in:îted a¡nou:*ts af t¡æosåne, thts may not be easåly aetrùeved. anð, even when possibJ..e, a hågh ratå.o of ioðine atoms pen motreeul,c eot¿l.d" theonetieally alter the affårråty of the anti"gen fon 12'5T antåboðy. Thås pe,tcn*i.al üf.-ssd.våntage cf as a l¿¿be1 is partially of,fset by the greater effioieney of eot*ntång of thts åsotope eornpared 171* * ïtl-ïn J o

Tlicl åodånation å.s al,rnost unåver"*al-1-y earvåed" ot¡t by the eh},onarsine T method e¡f Greev.lwr,.od S$*e¿ þVØ) whåoh alleiws high speei,fåo aotivåties to be a'ttaåned. vuþú1e tisårrg relatívely smal-l ameitul.'ts of, åsotepe"

( ¿ ) S ep ary!:Lo¡:r_ o-{-Þ o'q}èl .*a.pd... Sre e*g*çqfgoÐe. The or.5.gånal me*hod used" by Yalow and Sernon (t959) f,or separatÍ.e¡r¡ elf bor:,nd ar¡d. free peptiåe was ohx"omatcel.eotrophoresís, and" thås remaíns å ver"y eatisfaetery rnethod wlien the peptåfle eor:e;crned"

ås aðsort¡ed" to pape1" The d"i"sadvantages of tÌ:ås methoð are that onl;y small volunes of the reaetien mixture ear:i be appl"åed" te the på.pcre wåth resulta¡rt loss of ser:såtivåty" that i'b is rather tcåious and that it ås not reaðily appli*abl.e trr large nun.f,åer"s of sam¡rI.es" .A wåd"e vaníety of othen methad"s Ìrave ber:¡: ustid. for" separatåo¡:. of beuvlå anå fnee honmoneu å:äe3ì-u.d"ång the foì.Li.'wing: CIeeted eira,re,oal (lït*rtrer& gL3å.u

'n965), Lak': (Rosse3.ån .g[*g&. , X9€'6) u SepÌ:raðex-coupled. ar:tiboååesr (ïiråd.rr, a^r:.et Fers"atb., 1p66)n sc,3-:id*phase mc¿tÏ:'od" (tat'b g!--e}" , fg66)o arrd d.er'¡rbjle- ant$-bod"y rnet?¡e¡el (Steem an-d Tal¡r,age, X95B)" In t,hcr clroíee cf methcå 30 Llseð, åt ís usual- to seleclt the tcelmåqtie fciunå t'o provååe satisfaotox"y sensiti.våty arrð pneoÌsi.on fqrr i;he partieular h<¡rmcr"re whi"l.e retuiriing sÍnçl,i"eåty anü ease sf operatierr:"

(r,r/ Á,NGTOIENSTN R.ÀDTOTMMUNO.ASSAY

Deod.har (1960) was abl-e te raåse antibod.Íes to angiotensin Ïf by eoupl"ing the aeltapeptÍd"c to bovåne y-g3-ebteiÍn to ferrm an antigerr"

Thís antlgen rvas måxeô with Freu¿indrs ad"juvant and. årejeeteå intra- perit,oneatJ-y Í"n rabbits, ltre y*gl.obuli¡i was ísolated when the anåmal.s were su'bsequently'bled." The presenee of a^ntibodieË Tuas denonetrated" by a preeå¡:Ítin neaetÌon be'tween the rabbåt y-93-etrulf-n anå the angio'tensån-bovåne gåJü¡r¡e. gS"ebulån *omplex, by pelrtiatr, Í.nhibítiern of this preeipåtÍ"n r"eaetåon by angíatcnsÍn IÏ, and" by a preoÍpitån reaetiorr between rabbit ge,mrnsì. gl.roTrulín from the Ímmuni.zeå rabbit a¡¡d an angiotensin TÏ-proteån com¡:lex eontaining a protein other than bovÍne ga¡nma globiilin" ïhe antÍboåy was fer¡.¡nå to have greater aetívity ån neutral.izing the free aoi"d for"ri of arrgiatensin TÏ than the amÍðe fe¡rm anð reaeted. to the same exten't wíth s;¡cthctie vu.15- anrl natur"a.l ís,oIeti5-angicterisån f srld" tkl-is was feLt to bé in agneeruent wi.th the sturlùes of Landsteir¡er tlgZZ) who d.emonstrated tirat the amino aeírl with the free eax"boxyl gr,oup at ttre earboxyl end" of' the peptid"e lar"ge3,y' deter"miriee the speoificity of tlie antibod"y" The antt*

Ï:orly was used. by'De,oelhar 'üo investÍgate t.he effcet of an antåogensin inluibit.er on the ble¡,od. pressïìrÉ) of h¡pentensåv'e aRi.mals" Goc'dfrien¿ e!-éL (tgee.) produe,eå an'tåbcåic,s t<¡ bradykinån and angåctensln ån rabbits by ttie u;s(: of eorLj"lgates eentaåni-rrg albtimiri 31" and. the hapten covalently bounðo The eonjugate was.suspended. in

Fneund.ts eompJ-ete ad.juvant anü injeeteel ínto the toe4aels and. leg nuseres of rabbits, foJ"J"owed" later by íntraperitoneal injeetion of

the eonJugate Ín adjuvant" AntÍbod.íes were el.emorrstrateô. by eomplenent-fÍxati"on and inhíbi'üi"on tcehniques. The speeíficity of

the antÍgen-antÍbocly reaetíon was Índ.icated" by lack of eornplement flxatÍon with heterologous hapten-alburnin conJugates and laek of Ínhibítíon by heterol-ogous haptens" The authors eonsiðered. that the methocl of eonjugati"ng hapten wíth protein using the earbodiÍmiðe reaetÍon had. ad"vantages over other eonjugating proeeclures. Thus the

reaetion oeeumed" unüer very nrild. cond.itions and. the methoð was

eapabS"e of conjugating a wid.e rangë of compound.s, anð of eonjugatíng

haptens tlÍreetly to proteins wåthout ínterposing að.d,ÍtionaL groups between the hapten and the óarrier. The antibodi"es produeed by this

methoeL were used. by the authors to stud.y the ínmurrochemistry and biol"ogÍcal" roles of small pol¡¡peptides.

Gooðfriend. e-t al (lggq reported Ímmunoehemieal studies of angiotensin" lhe antigenie ileter^roi"nants of arrgåo*tensÍn were invest- ígated by inhÍbÍtion of eomplenent fixation by anaS,ogues of the poly-

peptiele, Anong the strrret'r-rs"a]- features of angÍotensin tested., those which had greatest importenoe for serologíe aetivity Tyerel

eonform&.tion as ðeteruÍned. by an optical isemer Ín the ni"d.d.le of the moleeure; the presenee of the two amÍn

Haber e!_gå (lgín) produr:ed antigerri.e branch-ehain eopolyrners

of angiotensin and. po1.y*L-lysine, ustng earbodiimide conðensati"on between the ca,rboxyl-terrnÍnal end of angi.otensin and" the e*amånç: groups of' poly*L-lysine, æ,d eerripli.ttg the anj.no*termj-na1 enð of

angiotensån to poly*ï,*lysine via m*xylene ðíisoeyanate" TLre antíger:s were suspenðed" in Freund.rs eermplete ad.juvar¡t and injeeted" into rabbits intramuscul,arly, intraperitoneally and- into toe*-paðs; ad.d.itåonaL intravenous injee;tion of antigen was given Ín some eases. ,rAntibodj-es I were deteeted by alteration of the elution positian of label1eð angÍ.o- tensin from a G2$ Sephaclex eolr;mn when míxed" wÍth imm¡rne serumo ft wa.s shovun that d.i.splaeerrer:t of l-abelled. angíotensin from the antibody eoulô be aehi"eved- by the ad.d-itíon of inerements of unlabelled" angic- tensín prior to gel fil-trati"on. ft was suggested" that poly*L*1ysi"rne was preferabl"c to other ea¿rå.ers berlâüsÉi it was rrer'c intr"i.nsícra1ly antå- genic and the e-amino grÕìlpË provi.ded an opportui:ity feir r:otipl.i.:rg peptid"es speeifieally by eíther thetr earboxyl- or. theír arn-t:no- ter.minal enels.

lhe mcthCIil was th.en exte:n-råed te an as$åy system f,or angåetsnsín

(fianer _Ê!__eI.. , 1965), Gel fj.ltratiçn on a Ê29 Sephad"r+x e ol-r.¿nm was used te separate ba:r;nå from free a,ngåetensi"ri after ír:eubatíon wåth antr;åbod.y. fhe methoð was nhown to be usefui for measuring quantitati"vel"y tbe 2Z

reå.rtion between antibod"y and. proteirr a:rtigen and. al.s,o between

antå.serum and. the small pol¡rpeptide a^nttgen"

Val"loton et_g¿ ?geZ) reported a radioÍmniunoåssay pr,oeedure for angS"otensån IT and. were able to measure plasma le¡rels for the first

time" Antibod"ies were raÍsed. in rabbits by injeetion of copoJ-¡rmers of

ang5-otensin with poly*L-lysineo va15-an6iotensín rl e¡nid.e

( "H¡rpertensåntr-Ciba) was used" as a stand.ard. and. also as isoteipieally 125I. labelled traeer after ioöination wÍth Separatíon of be¡urrd. from free angiotensin after ineubatÍe¡n with antibody was achieved by gel filtration as before, BÍnd"ing aetÍvíty of the antibod.y was found. to be the sarne for bovine angiotensÍn rr and hunan angiotensin rr but

there was dÍminished" affS.nity for angiotensln T and. eompounds obtained. by enz¡rme hydrolysÍs of t'H¡ryertensínt'. Angiotensin was extraeted. from plasma by use of an ion-exehange resi-n prior to neastpement in

the radioimmunoassay proeed"ure" Further plasma samples were ineul¡ated.

at 37ae far 3 hours prior to extraetåon of the angiotensÍn as a meâsurement of pl.asma renin aetÍvity. Normal values ferr plasma angier- tensín II were d'eríved and. resurl'ts suggested, that normal values were to be found in'essential h¡perter:såon, raåscð values ín nalågnant h¡¡per* tension and. hepati"e eirrhosis, arrd. low vaL.ues in pri-mary alùosteronism, Pla,sma renin aetivíty values tended to paral1el the plasna angioterisÍsx fI levels" The leve.ls reported. in thås påpcr were subs.bantÌ"a1-1y higher than those o'b'taineÕ by later" authors and. were subsequentl,y ad.justed. to lovrer levels when it was realized" that ad.seirption of angíotensin stand"ard"s to glasswarë iñras giving falsely el-evated valuesn 3l+. eatt and" Coghlan (1967) proåueerl antÍbod.ies to angiotensin IT in rabbÍts by immr:nízation with. vaLS-angÍotensín II amiåe ("H¡rpertensin"-

Oiba) coupled- to porcine gânma globulÍn by carbod.íim:Í"ðe cond"ensatÍon. The a¡rtibodíes reacted equally well wåt?r0(-aspartyl angiotensin TI, and. exhi-bíted. only slíght eross*reaction with angiotensin I" The antibod.y lryas useð for a rad"ioimmunoassay proeeåure, ion exeha.nge ehromatography being used" for separation of bound from free angio- tensin after incubation wíth the". antibocly, lhe authors tliought that the antÍgen5"c portion of the angiotensi¡r merleeule whieh reaeted" with th.is partieular antibod.y was elose to the 0-terrninal end. of the oota- peptÍd.e, Sinee angioterrsÍnases a,et by removing arnlno acid.s from the N-terminal enð of the meiloeule, bíologiea1ly inaetive metabolítes of angiotensin fT míght retain immunologieal reaetivity" The preserree of such fragments in cireulating blood. eould lead. to d.Ísparity be'tween the results of bioassay and radíoimmunoassay of angíotensÍn IT unless ad.equate Ísolation procedures are performed., Boyd et gt (lgøl) raised" antibodies to angioter¡sin II by injeetion into rabbits of val5-angiotensin ïï auride (,'H¡rpertensin,t-

0iba) ad.sorbed. onto chareoal and mixeð with Freund.rs ecmplete ad.juvanto some ínjeotions were gÍven into popliteal lymph nodes and. some into the spl.een. lhe antÍbod"y was used to set up a radíoÍmmunoassay teclrnique, using d-extran-eoated. ehaneoal- to separate be,und. and. free peptiðe (Herbert qt a1" , 1965)" Ven,rus blood samples were eolleeteå into cooled. plastie s¡æinges containing ËDTA ancl díurereaprol to inhibit angi-otensirra.ses. The angiotensin was extraeted" from tire plasma by 35"

ad'sorption ontc Fui "i"erns earth and. subseqliently eluteð wj-th ammonåa ån

methanol. The assay was sensåtj.ve enough to deteet 30pS of angiotensin ïT and. u¡as nc't influeneed. significantl"y by angioten-qÍn ro

CIat't gfg[ (lg67b) reported plasrna levels of angieitensin TT Ín normals and. in severa,l pathologieal cond"j-tions usi.ng ttre radÍo* i.mmuncassa.y as prevÍ,or.rs1y d.eseribed, but usirrg dextr.ar¡-eoated" ehareoal

te separate bound. frcm frce peptid.e in p.l,ar:¡e e¡f' the iern-,exe?pnge resino arteri.al blood. wa.s ceLleoted inta c;r:ld- ethanol crontai.rirrg 125r

angiotensin TI aß an inter.r¿l. ànd.i.eator" Angåotensirr wa.s ad"conbed ilsfi*sephaclÊx'r cnto the eatj,on exehange resån and"u after washÍ,nge r4ra,s eltated wi.th o.0þM triethylanii,rie and. d.r'icd. in a rotary e.såpûrator,

lìeeove.ry nf labelled. angioten,sin Il. was 65-"1ú/r" The radioimmnnoassay xJlr pr,aeed.ure was earuied out usi.ng *rrgiotensin ïr traeer and.

fà Íscleu'-angioterrsin fl standards" the lewer 1Ímit of sensåtivity was 5opg"

I{ollemans (gea) rai.sed ant:ibod"ies in rabbåts by injeetåon

of eopol¡rmers of valS-angioterrsj.n fr amj-de (,,1î¡rpe:nt;ens'i.nrr) and pcry*L*

I'ysåne, eourpl-ed. by earbodi.i.mi.rle *crråensatioR" 'fhe radÍ.oåmrnuneassay üeelmique reporteå had. a sensitivity of approximatel,y z.gng, l'he antisera tested strowed. d"iffercnt affinity fer vaj-5-angiotensin ïï

amåd.e anð i-soleu5*ang-iotensirr ïïu and. it was suggest,ed" that the usc

of "H¡4pertensir¿rr fcr etandard.s mÍght 1ead. to erres" in the aõså.yo

Good"fråenå e!*å.1. (t geg) desoribed a rad.ioimmunoås"say preced"ur."e

erapabJ"e of d.etec'ting f*1Opg of angíotensÍn TT" AntÍbod.ies wëre ra.i.sed i¡r rabbi"ts by injeetion of angÍotenså,n coupled. ta rabbit ser."im albumín 36. by the earbod.ij"r¡ri"ðe proeess" No i.mmtxiol.ogic d.i.fferenees between ÃÃ vaLr-- a::d. ísoleu/-angiotensån IT were rl"eteoted". Ðífferent antisera had. variable affirråty for angicitensin anå variable speeifieÍty as ðeibersníned" wiùh analogues" It was felt by the authors that the teehníc1ue used. to separate antiboðy-bounå and free 1abel.1ed" hormone largely d"eterrnined. the reprod"ueibility and. convenienee of the assåyo

A number of teehniques vrere eval-uated. and. the authors fÍnally chose the antibod"y*eoated. tr:ibe mcthoö, pri"marily nn the ba,si-s of inez'easeð sensåtivåty,

Gereke g-3_el (lpgg) d.esoribed. a ra.di,oimmunÕassay teehntque oapable of measuring angiotensÍn II Levels direetl-y ån 0"1 ml of plasma. .Arrtj-sera were proðueed in rabbÍ.ts by inJeetion ef val5-angiotensin IT amid^e ("H¡¡pertensfr¡t') eonjugateü with rabbÍt serun albunin by the oarbcd"Íimi.d"e reaeti,on plus Freurr.d.rs aðjuv'ant" Vene,¡ls bler¡d. was eolleeted" Ínto EÐfA to inhlbit angÍotensinascs, Separation of bound. anð free peptid.e following incubation with antibo

3oyð anð Peart (1969) ðeseribeð the deve3.opment of a raåioÍmmuno* a6Éey proeedure for angíoter¡sin I anð the appJ-ieatj"on of this technåque to measurement of plasma rentn aetívitiy. The aurthors tleveloped. th:i.s assay beeause of the problems assoeiated. wÍth the applieati.on of the angiotensin II rad.Íoimmunoå.ssay to measurement of p1asrua renÍn aetÍvÍty, na"nrely inhíbåtion of conv'erting enzJrme aotívÍty by the EDTA and BAL ad"d"ed" to inhiblt angiotensinases a:rd the ðåffieul'by in reste¡r"ing this aotåvlty witLrout al.so reaetivating angiotensinases. Antibodies to angiotensin Ï were raísed in nabbits by repeated. injeetion of angå.otensin T ad.sorbed" onto chareoal and emuf.sified" wåth Freu.nårs eomplete ad.juvant, The lov¡er limåt of

ôeteetion of the asñay was BOpg and. there ï¡as no ero,ss*reaetion with

à val/*angictensin TI amÍd.e. Bloed. sampl.es were eolleoted. into EDTÀ and" BAL. Ttre plasrea samples were ex'Lraeteå with Fullerrs earth at uero time anå after Íneubation at 3/Õe for h houns. Recoveries for extraetÍon were apprerximat,eby B$n, The results obtaÍ.ned. for plasma renín aetivity by reuåioimmunoessû.y ï/oro eomparable wíth tho¡se e¡btained" by bioassay, altheugh the radÍoímnun{iåËsay valties tend.ed. to be some* what lower.

Page 9þ-gL (1969) repor"ted a revi..seð rað.i-oimmr:rjoå.$ålay prercedure ferr angiotensån ïï. Tì"r* pr"evir:us1.y reported level-s frr:m

Lli:i.s; group (VaII¡bon e-1""-nJ" , i')t,l) irr,Lu l:t:r;¡: í'¡iì;;r: L;y Ir:i.¡':Ir L¡clc¿lt-lsc o,'

ad.serption of' a.ngi.ntensin standends c¡¡: ei-J,.åcr:nizecl gïasswai:e during

the drying step" The effect cf' va:'ieius ar¡t.ic;eagurlants was sl,udåeå and the reeovery of aådeå angiotensån from blr¡oð sampïes was best in 38"

the presenee of EDTA (recovery 7¡+.-8æ/,)" The sensitivíty of' the method

was 1 Opg per sampleo Levels of angiotensin TI were cleteeted. in a1L patåents except anephric subjeets" lhere was ffi eress-reaetion with

angiotensin I. The rad.ioimmunoassay technique was applied to measure.-

me¡it of plasma renín aetivi'ty; generaLion of angiotensin rr was greatest in eitrated. plasma but vÍrtually none lras generated. ín the

presenee of TÐTA. In the plasrna renin aetivåty met.hoð, plasma samples

were ineubated. in parallel wíth aliquots to whleh stanclard.ized" hurnan renÍn had been ad"d"ed.. It was assumeð. that the faetors affeeting renin aetivity, eonversion of angiotensin f to angiotensin TT, and ðestruetion of angiotensin IT were íd"entieal in the aliquots. Hnðogeïìous renin

eoneentration was estimated. from a eomparison of the maximum veloeity of angiotensÍn TT generation between the unknown and. the sample with

stand.ard. renin, assuming that substrate was not lårniting. 0hanges

in plasma renin activity and. plasma angiotensin rr were as expeeted. with ehange Ín posture and. sod.Ír:m i"ntake, the changes ín plasma renín

aetivity beÍng proportionately larger than the correspond.Íng ohanges

in eireulating angieitensån. A very low but d-eteetable enrlogeneius renin aetivity was demonstrated" in twr: out of thtee anephrie sr:bjeets, including one male, The souree of this reni-n aetivåty was obserure. CIatt C!_gI (gAg) reported plasma angÍo,tensi"n Tf 1evels ir: nonnal subjeets and" i^n pati"ents witii h¡rpertension of' var¡rårrg aeti.ologies. Art;erial bl.oed. angi.otensin revels were approximately zffi fu.gner than venoì-rs levels. Arberíal angiotensin rr levels i-n

þatients wiLli essential. h¡4rertension showed" a wid.e scat¿er, wÌttr no 39. change in mild eases anä a tend.eney towards hÍgher l-eveLs i.n severe eases" PatÍents wåth ma}ignant h¡rpertension showed. raised. leveLs before treatment but normal levels after treat¡nent. Most of the high angÍotensin levels assoel"ated. with h¡pertensíon were encountered in those patients Í.n whom the h¡rpertensíon was due to und.erlying renal d.ísease, either renovascular or bålateral d.iffuse renal disease" Low values of angÍotensin rr were found Ín one patient with prtmary al-äosterontsm and smal-l- quantÍties were d.eteoted in the pJ-asna of two anephric female subjects. Êocke et a1 (1169) reported. further erçerienee with their rad.Íoinr¡unoassay technÍque" It was shoïun that plasna angiotensín II concentratlon was d.ireetly related. to plasma renin actl"vity anð

ínversely related. to the state of sodiun balanee. There was some oross-reaction between the antibody and angiotensin T which had 1/lOtU't of the i.nnunoreaetivity of the oetapeptitLe, and. the antÍserurn aLso cross-reactecl with hexapeptitle and heptapeptÍde fragments; experiments, however, showed. little Ínterferenee wíth the assay. Sunclsfjor¿ (tgZO) reported. a further radíoimmunoassay for angiotensin ïr. AntÍbod.Íes were raised. usÍng ?'H¡pertensinrt-ciba coupled to poJ-y-L*1-ysine as the antigen" AngåotensÍn was extracted. from plasna by ion exchange ehromatography prior to assay. The normal- range for healthy aclults was found. to be in agreement with reports by other groups emplo¡ring extraetíon proeeåures prior to assay (Boyå s!-4" , 1967; catt et a3- ", 196Vi Pase g[_e]., 1969). The 40. advantage of th-ts partÍcular method was that naximum.bind.i.ng of antigen to the antíbocLy was achieveel. wlthån onê houn, thus ninímizÍ"ng the nlsk of non-specific clamage to angiotensi.n which night ocoull durlng more proJ.ongeð ínoubation. It1 ,

CHAPTER. 2

A¡ÍETOTEIISIN TADTOTMMIINOASSAY MEMTOD

1. ANfIBCIDY PRODüCTTON

2o TODÏNATÏON

2 SEPARATION OF BOUND FROM FRSE PWTÏDE

4" ASSAY PROCEDT]RE

5. SPECTFTCTTY OF THE ASSAT

6. SEIVSTTTVTTY OF THE ASSAY

7" RUPRODUCTBTLITY OF THE ASSAT

( a. ) ïlithÍn Assay Varåation ( b ) Setween Assay Varåation

8. ASSAT OF ANGIOTEIVSTN TT TN PLASMA SAMPLES

o MEASUREMENT OF RENTN ACTTVITY" RENTN CONCEI\TTR.ITTON" AND REI{TN STTBSTRATE BY STOASSAY 42

cIfAPrm. 2

.AT{GIOTENSTN RADT OÏMMUNOASSAY METHOD

(r ) ANTÏBODY PRODT'CTTON Antåbodies were procLueed l"n rabbÈ'ts by injeetíon of va15* angiotensin fI a¡råcie ( "H¡rpertensinti-Ciba) ad.sonbeil onto eharcoal ancl enulsifietl. wåth Freuncl¡s eompS"ete adjuvant, Slx rabbi"ts were useð ancl injeetions were given íntranuscul"arly or subeutaneously, initially at weekly intervals for four weeks and. thereafter at two- weekly Íntervals. The anÍma1s were bled_ from ear veÍns eaeh nonth and. the sera lryere tested for the presence of antiboðies by the following methods:

(") Bgpes_El"es:EsophoresËë

ïn ar¡ eleetrophoretåe system using Tfhatmann JMM paper ancl veronal buffer pH 8"6, the majoråty of the 1251*1"¡*LL*¿

angÍotensin fI nigrated in the alpha-globulin region, with a further small fracti"on, presumably non-specifíealLy bound., ín

the albumin reg5"on" T[hen the labelIed. angiotensi"n was måxed.

wåth eontrol rabbl"t semm, i"ts el"eetnophoretåe mobåI"åty ðið

not al"ter but when it was måxecl with serun from suecessful]-y

inmunized. rabbits, the majors"ty of the label"led peptide was

found in the gamrna:gJ-obul,in regåon, suggesting that it was bound to antibody (trå,g. l). t+3

FTGURX 1

DISPLACEMENT BY ANTIBODY

ORIGIN ¡

; ¡.GL0B ALB.

500 Non -immune Serum

0 125¡ 4¡6¡OTENStN (cpm) 750

500 lmmune Serum

0

Alteration of electrophoretic mobi-lÍty of 12 5r-R rgi otensin ïï when mixed" ¡vith immune serum. Ì+4'.

(r) å of the 10n otensin fI the Rat oassav Svstem

when immune serum at a di"l"utåon of 1:100 was m:Í"xed. with

standard. solutions of angiotensån rr at room temperature for two månutes, there wås a slgnÍfioant reduetÍon Ín the pressor rësponse ån the rat båoassay system (ri.g"e). The reductíon

i"n pressor effeet was not seen when stand.ard. angÍotensÍn TT was måxed. with controL serum, sugges.tin6 a speciff.c neutraLization by the antiserum rather than a non-specifie effeet

due to angÍotensånase aetå.vÍty. This Latter effeet became

evid.ent with both immune and. control- sera when higher ooneen- trations were usetl,

(u) Gel I'iltratå oin 'T 25ï-*"gåotensin lhe el.utíon posÍ,tion of ïr from a &25

sephad.ex eol"umn was d.etermåned, Mix$.ng of the Label-l"eð pepti.d.e wåth normal rabbit serråm did. not ehange the el.¡¡tåon position but, when nixed with seruin from sueoessfully tmnunizecL rabbits, the radioa'otåvity appeared ån the voåd vo]-ume, suggestång bind"ång to antåbody" .ô,d.d.åtåonal- evådenee that the ehange ån el"ution posåtion wås d.ue to antibod.y binding was obtained. by ehangÍng the pH of the voi-d vo1.¿rme fraotåon to pH 2.0, whieh w111" break antigen-antibody linkages, and re- applyång to the ea]-umn, ln whi.eh ease the rabell"ed. angi.otenså:n lyas reeovereð frorn its origÍnal positåon (trig.5). )+5 J t- + o U

f- + o

f- + o (J f- + o

+ 1 (\o r\)

f- + o(\

of- + C\ U

1 + o(\

FÏGURE 2

NeutralÍzation of pressor action of angiotensin fÏ Ín rat bioassay system by irnmune serum. c Control serum A = Antiserum )+6,

FIGURN 3

200,000 (a)

150,000

100,000

50,000

0 5 10 15 20 25

60,000 (b)

40,000

20.000

0 5 t0 15 20 25

FRACTION NO

Alteration of elution position of 125t-lngiotensin ïï from a Ç21 Sephad.ex column when mixed. with antiserum. 47.

(¿) Construetion of a Stand"arcl Curve Incubatisn of labe3"l"ed angÍotensi"n IT wS.th inereasing clål"utíons of antåser*¡m resulteci ín progressively less binåing of

the pepticle, When a suitable &il"ution of antiserum was ehosen, 1z5T-^ngíotensin sueh that approxinately 5úÁ of the aclðeil II was bounð to the antiboð.y, tbe adðitíon of inereasl"ng aurounts of

unLabe3J"eð angi"otensin IT to the systom proflueecl progressive

clåsplaeenent of the 1abe13-ed pepti"de fron the antiboðy, enabll"ng a etanðarcl curve to be eonstrue tea (fig,4).

"â.ntÍboeLíes to angiotensån If were ðemonstrateð. ín some animals as soon as såx weeke after the inítial inoeulatÍon wíth antigen, anå were ðeteetabl"e in most animaLs after three nonths" Shere '¡¡as wi¿le varia*ion in the titre of antibod.ies proäueeil, however, so that in only two rabbl"ts were antåbodies of suffieíent titre and affÍrrity for applåeatíon to the radioimnunoassay proced.ure producecl" Alternative routes of injeetion, eog. into 1.ycrph noð.es or l¡rrnphatics, the perítoneal eavJ"ty and the sp3"een, were used. at various times in an atterrpt to further raise the leve1 of antåbodies, but without any eonvincing evíd.ence that thÍ,s mad.e any si"gnÍfieant differenee.

ïnd.ivídual animal variatlon seemeð to be more Ímportant ån d.etermi.n5"ng the ease of produoti"on of antibodi"es than the injectíon teehnÍque useå, but this was not systematíea1J"y examined." 48. 50

40

30

'1, BOUND

20

10

0 0'2 0'1, 0'6 0'8 1'0 ADDED ANGIOTENSIN (ng)

FrGr$m L Stand,ard. curve for radioimmunoassay of angiotensin If. ]+9 "

(z) ÏODTVATTON

Shis was perforured" by a mod.ifåeatåon of the chlorami"ne f nethod. of Green¡qooð et aI (gel) o In tþ,ls method, the oxidizång agent 125y. obLoranine I is used. to att,seh t* the t¡mosine in the angiotensin rI tr 25T noLecu1e" Hågh aetåvity fu* proteÍn iodination ïÍas obtained from

the Ractiochentcal" Centre, Anersham (fUS-¡) and was used. as seon as it arrLvedl"

A representative iocLinatÍon j"s earrüecl out as fol"lows: 125t To 2mci of is ad.clecl 2op1 of o.!M phosphate buffer pH 7"5 anil then

5pem of vaI'-angiotensÍn IT a$åde ("Ii¡rpertensÍnrt-Cåba) or isoLeu5- angiotensin II (Schwartz Bl.oreseareh) in 20¡13- of O.O5M phosphate buffer pH 7"5, foll"owed" by 100¡rgm of chLorami.ne f in 20¡1L of 0,0!M phosphate buffer, fhe reactÍon l"s all"o¡recl to proeeed. for 10 seconds and. then stoppecl by the aildition of 0.1 ml of sod.ium metabåsuLphS-te solution (2.4*g* per ml in O.O$M phosphate buffer)" 0.2n1 of potassiun iodÍde soLutlon (tomgn per ml) åe ad.decL to dilute the unreactecr 1251 the nrixture transfemed to a"î 5 x 1cm coLumn of ê1o Sephacl.ex ^nd' equilibratecl wåth 0"07t{ barbítone buffer pH 8"6" An al-l"quot of the reaetion mixture Ís usually taken at thås stage and. applieð to a paper strip for subsequent eLeetrophoresis and ealeuLation of speeÍfie aetívÍty of tbe labell"ed pepticle" The reaetf"on m:ixture is eluted fron the ê10 eol"umn wåth O"o/M barl¡åtone buffer pH 8,6 and. 1! d.rop fraetions eol"l"eeted.; 20¡11 a]"$"quots of these fraetisns are eounted.

ín a well-type eounter (neto Seåntåll"atåon Counter, f¡pe N55OA"). 54"

The rad.ioaetivåty ås eluted fs'on the eol..umn Ín.twe p,eaks, thq first representing labe]"3"ed angj"etens:i.:n and the seeonð free l"sotope (Figr5),

Part of the pealc freetÍon frem the GtrCI eoL'um¡r ås appl-åeä to a seeonð

column (zo x "l cm G2g seph.ad.ex) equilíbrated and elutecl w:ith 0"07M

barbåtone plÌ 8"6" 2ml" fc"aotÍoc¡s are eol,l"oe.ted ancl 20¡rj. al-íquots eour¡ted " The peak fraetåon fron the Ë2| eo3-umn is d,iluteü. ín buffer eontaining 1fi ]n:uman seru¡a atbuml"n anå stored. frozen for subsequent use in the rad.ioinnunoassay proeedure,

Speeifåe aetÍvåty of the l.abell,,oð angiotensln is ileteru.rineå by

eleetrophoresåe of a ema13" a]"iquot of the ioð.inatåon mixtune on ïtlhatmanr: Jffid paper ån versnar buffex" pH 8.6, wlren two peaks of radioaetivÍty are obtained, eorrespend.ing to Labell.ed. peptide an¿ free 125ï' Fre¡m the &rêas þe*eath the tw¡r peaks, an estimate of the

fraetåon of the ísotope attaehed to the angiotensin and" thus the ãveråge speeÍfåe aeti"vÍ"'ty ean be ea3-eulated", À similar estimatfon ean be obtained. from the areåB beneath the two peaks from the ê10 oo3"wnn, and" thÍs wae for-¿nd. to agree with that obtaineå by eJ-eetru- phonesås" A speeåfie aetivíty of approxåmatery 300¡rtå per pgm f,s oonsistently attaÍ.neð..

lhe l"abel"led. angåoter:sin ås rcutåne3"y eheoked. for ¿amage after

íod.ination by ehromatereLee'brophonesÍ,e on whatma.r:r¿ 5MM påpcrÞi tk¡e radioaetÍvíty ls found. to be sonfined to a sång1e pcak at the origin and the peptíd"e ås the:: eonei-d.ered d.amagc-:ftreeo 0ver the eourse of several weeks0 storage, a sniaI1. e¡aount of rad.íatåon daruage oeeurs and thås ean be ðemorrstra'ted. on chroomatueleetrophoresis ås a seoÕn¿ smal"L. 51.

FTGURE 5

400p0 0 G10 Sephadex Cotu mn

30 0,000 c I l0sec/ 20ut 200.000

100p00

0 5 t0 15 20 25

FRACTION NO.

Todination of Angiotensin ÏÏ 52, peak of rað.íoaettvÍ.ty mtgnating with the proteÍn fractf.on (ftg"6)" The teehnique of purifíeatåorn of the label"3-ed. peptide by (gaber investigatecl" extraetion wfth antåbod-y "g3-g&, 1962) was This teehnique uses antiserum to angiotensin II to extraet from the iod.ination nixture the l-abel"l"ed. hornone wlth the greatest affinity for the antiboclyo foLl"owecl by aci.ðtfication to bri"ng about dissoel"atÍon of the antigen*antibocty complwes anð subsequent separatÍor¡ of these two fractions by ge3" fÍ"I"tratíon. fhe method was found to yteld. Labellecl peptåde with slightïy tnereasecl affinity for the antlbod.y but the ånerease ån sensítÍv5"ty aoh:ieveð was only narginal- anå the purifieation prooed,ure was not useð r,outinely.

fniti.ally, both va15-a^ngåotens$.n ïï amÍcLe and Ísoleu5-angioten* sin II rrqere i-od.inatecL for use in the radi"oímmunoassay proeedureo but åt was subsequontly founcl that the Latter form was more resis tant to non-speeÍ"fie clarnage than rtH¡ryertensi"ntl and i"t was, therefore, usecl routÍnel"y ín the a.ssay system" ß) SEFARAÍTON OF BOUND FROM FREE PEPÎIDE This forns an S"mportant ftnal step in the radioimmunoassay proeedure after incubation e¡f stand.ard.s or plasma sanples with antiserum and l"abelIeð" peptide. The method used by Yalow and"

Berson (lg|g) was chromatoel-erotrophoresås and. thÍs remalns a preeise anð effieient methocl of separatÍon of bound" from free peptiðe" The ehroma*oeleetrophoretio prooeðure, however, has the åisad.vantage that it is not read.Í"J-y appl"ieab3"e to large numbere of sampl"es and, therefore, a wåðe variety of al-ternatÍve method.s have been useclo 53

FIGURE 6

aooo

COUNTS/ lO0 ¡cc. .ooo B

o I @ ORIGI il '..l ¡ooo t

COUNTS/ IOO scc. .ooo A

o

125t-¿neiotensín Chromatoelectrophoresis of II to conflrm freedom from d.a.mage. 51+.

It wae clsoåcled to use a nsd.i.fi"eati"on of the d.extran*eqated. oharooa[ methoð ð.eseribed by Herbert e$-e& Ug6Ð for the rad.åoÍmmunoassay of ånsul"in" Tn thls method., sri"orofined. charcoal" l"s eoateil wLth d.extran and. a stand.aril quantåty of this suspension Ls ad"dled. to the reaetion urilxtures after laeubation 1n the assay proeedure. The free angio- tensfn is avÍ.clLy adsorbeð onto the eharooalu but aeees* uþ tfre larger antåbocly-anglotensin complexes ås blockecl by the coating of ð.extran, CIentrifugation aLl"owe easy separati"on sf bound from free peptíd"e.

Thåe nethocl is quiek, effeots al-most ineta¡:taneous sepa.ration of bounð and. free hormone, ancl is rea¿lÍl"y app3"5"cable to large numbers of sam¡lles, ït d.oes, however, require netículous technÍque, for d.issoctation of the antiboð.y*angiotensin complex read.ily oecurs if the l"neubatfon nlxture ts left in eontact with the clextran-eoateå chareoal for more than a few månutes prior to eentrifugatåon" lhe extent of this d.Íssoeiet{on can be eeen from FÍgure 7 whl"eh shows the effeet of leaving the ineubatton mixture in esntaet with the ð.extran*eoated. charcoal for inereasÍng period.s of tåme prior to eentrifugatio¡r" At room tenperature, there is rapåd. d.iseociatåon of the antigen-antibody complex so that the bÍnd.ing is not suffÍcient for the rad.ioåmmunoassay proeeil"ure wåth:in three nånutes" The d{ssocÍation

ås less rapið at ¿+oC but the pereentage bind.ing is stlj-L sfgni.fíeant]-y reclueecl by any d.elay"

lhe cl.extran-eoated ehareoal" wa.s preps,red" by rntxång in equa3" proportions a 1Oê per 10O nl suspension of neutraS- pharmaeeutieaL grad.e ehareoal (Uerek) ån barbÍtaL buffer pH 7,1+ and a,¡a 0,!G per 100 nl soLutåon of ðextran, moleoular weåght 60r000-90r000(Koeh-Lite). ÉÃ

FIGURE 7

r

40

o 4"c % IOUND ^ a 20

Room Temperoture ^ ^ o o5lot5

T rME ( t't lN. ) BEFORE CENTRIFUGATION

Effect of prolonged contact _with dextran-coated. charcoal prior to centrifugation on þ binding. 56. lhe d.extran-coatecl chg¡ooa1 was stored. at l¡.o0 and. d.id not seem to d.eteríorate slgnlfíeantl"y on storage for several weeks. Tfhen used. in tþe rad"Íoimnunoessay procedure , 1 oQ ml of the d.extra4-coatecl chareoal suspension was ad.iled to each reaotion tube fol"lowing the incubation step and the mLxtures vrerê subjeoted. to oentrifugation at J1000 rpm for 3! ninutes at l+o0. It was found that, ín the absenoe of antÍ- sqrumr ttris a^mount of clextran-coated charcoaL eould. consistently ad.sorb gfà of the LabelJ-ed. angiotensÍn when freshLy iodÍnated, th:is pereentage fal3"ing to about 9{o over the course of several weeks antl presumably ånd.lcating some clegree of d.anage to the labelLed peptid.e on storage.

(+) .A,ssAY PRooEpuRE

The assay was eamLed. out in 6 nl poL¡propylene tubes (camelee- Aclelald.e) ¡utrich dlcL not sígnifieantly bincL angíotensin when carrier protein rÍas present in the buffer. The buffer used. was 0,09tr{ barbitone pH 8.6 contatnLng 1/o lnwaarL serum albumin. rhe tubes were set up ín duplicate eontalning: d.Íluted. antiserum, 0.1m1¡ 125r-*ngio- tensín rrr 0,1nJ- (approxf.natel.y 10r0oo oounts per 100 second.s); stand.ard. or plasma sample, 0"1m1; and. buffer to a total vor-ume of 1 "0m1" fhe mLxtures were Íneubated. at ho0 for l+B hours and. then separated. wíth d.extran-coatetl chareoal as ðescribed. previously.

After eentrifugatíon, the supernatants were el.eeantecl into 6 nl poly* propylene tubes and. both precipitates and. supernatants were counteð.

ån a welL-t¡rye counter (ncto Sclntillation Counter, T¡rpe IV55O¿,-)" Shorter periods of incubation yieLd.ed satisfaetory results but there tend.ed. to- be sone l"oss of sensitivity Ín the lower range ancl., 57" therefore, the S.onger period" of ineubati-on was employed routlnely for low actÍvity sanpleso

(¡) SPECTT'TCTIY OT T}M ASSAT The specífie$.ty of the assay for angíotensln IT was cheeked by courparlng the displ.aeement of label"3"ed angiotensin II fron antåbod"y Ëq proclueed. by val)*angiotenstn II an{åe ("Hspertensinn), isoJ"eu/- arrgiofensin II, and Ísoleu5-angiotens5-n I (F1g.8)" It can be seen that the d"ispLacement prod.uoed by the two forms of angÍotensin TÏ was vÍrtua1Ly íðentieal at all ðose leve3-s, inðicatång ícLentl"oal affinity for the antibod.y, whereas there was only slÍ"ght dísplacement of Labelleð peptide with the aðdition of rel"atíve3-y large amounts of ísoLeu5*sngiotensÍn I, ind"icatÍ-ng only slight cf,oss-rêâotion of the antÍbody with the d.eoapeptide, of the orðer of 1*{o" It hae been reporteti by Caf"n g[ e! Ug69) that bioLogÍ"ea11.y Ínactive fragrnents derived. frorn the break-åown of anglotensån ÏÏ nay retain inmunoreactÍvity and. thus give falsely elevateð levels of ângiotensin" lh:is problem was ínvestigatecL by ÍneubatÍon of a known quantity of angiotensin II wåth al,pha-ch¡nnotr¡psÍ.n at 37aC until no pressor activlty *å,"'de*onstrable in the rat bioassay system, followeð by measurement in the radioimmunoassay" llhen this was done it was found that displacement of labellcd peptide coulð still be ðemonstrated., to the extent of 5-1€/, af that seen p::,ior to ehymotr¡rpsÍ"n d.Í.gestion" This persÍstent lmmunoreaeltivåty of bÍ"ologÍ"eaIly i"naetÍve fragments represents a potenti,al souree of error in the radåoimmuno- assay, although 1t dÍd. not appear to be a sÍgnÍfieant prôblem Í.n praetiee, 58 FTGURE B SPECIFICITY OF ASSAY

100

90 tt. l- 80 '--- _.- - -l

70

60 .-- vatS AnqioII amide isoteu II olo lNlTlAL '-* !-Angio r-t ¡soteuS Angio I COUNTS 50

40 \ T 30 --. { ---_. 20

10

01020304050 ADDED ANGIOTENSIN (ng) Specifícity of assay. Comparison of üisplacement of 1251-1"u"11ed" angiotensin II by isoleu5-angiotensin Ir, val5-angiotensin II am:ide, and" isoleu5-angiotensin f. 59.

(6) SH\TSITI\¡ITY 0F THE ASSAY the Lower i-in:it of sensíttvity of the assåy was 10 påcogranrs of angiotensin II in buffern fn view of the extraction procedure employecl prior to assay, wl"th resultant concêntration of the angio- tensÍn ïï from pl-asma, thls enabLed. plasma concentrations of l-ess than 10 pg/nL to be measured wíth aoeuraoy,

(z) REFRODUCTBTT,ITY OF lHE ASSAY (') I[ithin Assav Variation

fhe withín assay varåation was cletermÍned. by the assay of replicate sanples witlún the one run" Representative

results are shown in Table 1, It can be seen that for samples in the normal range the eoeffícient of vartation is approximately 5 to 1(o whereas in the case of higher activity samples the varíation is greater" This d.Ífference occurs because the

higher values fall on the flatter part of the stand.ard curve where a relativeLy small aLteration in the pereentage bíndÍng

of the labelLed. peptíd"e will l"ead to a much greater proportional change in the value of angiotensín II obtained. fron the graph" For thÍs reason, when maximun accuracy was requíred., high

aotivity sanples were assayed. in d.ilution or with more concentrated. antiserun so that the values obtained. fel-L on the region of the stand.ard. curve with the steepest sLope, thus minimåzing the variabílíty in the assay. 60.

TA3T,E 1

WITHIN ASSAY VARTATTON 6s/^t)

SAÛIPTES

( a, ) ( b ) ( c ) ( d ) ( e )

28 2l+ 35 39 123

27 23 37 l+5 100

27 20 29 38 137

29 21 32 3t+ 115

31 zl+ 34 l+6 99

25 21 33 l+1 145

3o 27 36 43 120

28 ,2, 3a 39 119

29 22 35 I+7 108

l+B

27 20 3+ 39 127

l+2

; 29"1 22.5 33.5 !1'ls 119,3 n 10 10 10 12 10

S.,0. 1 .61+ 2"06 2.42 l+"O2 14"A6

v 5,BtÍ, g 7 11 "16% "2ú 9,6ïÁ "7fà x Mean v= Coeffåcient of varÍati.on - 100'S x n = Number of observations s. Dn Stand.ard- devÍatíon 6t (u) Between Assav Yariation

lhe between assay variation was ascertained. by the assay of replfcate samp1es i.n d.ifferent assay runs. Representative results

are shown Ln Table 2. It can be seen that the between assay varíation is consicLerably greater than the within assay fÍgure. For this reason, sarnples fron physioLogical stud.ies or other

situatÍons where values were to be compared were alL assayecl within the one assay rr¡.n where possible in ozd.er to achieve maxÍmum accuracyo

(s) ASSAT OF AI{GIOTENSIN II ÏN PI,ASMA SAMPLES

For the åssay of plasna concentrations of angiotensin ff, an extraction proceclure was employetL to isolate the peptid.e from plasna prior to íts introiLuotion into the railioimmunoassay system¡ the Fullerfs earth method of Boytl et aL (1g6il was routínely used.

lwenty ml venous blood. samples were collected. into cooLed. pLastlc s¡m'Ínges contaínfng 0.6 nl of 0.2M dimercapro!. (Koch-títe) and 1 .0 m1 of O.JM ÐTA whích were useal to inhibtt angiotensinases. lhe samples were trensfepe¿l to cooled" pol¡rpropyt-ene tubes and. oentrifuged- at Loc to separate the plasma. To 1o ml- of plasma tn 15 nrl por-¡propylene tubes (camelec) was added. 100 ng of acícl-washed" Fulr-erts earth, followedL by nixing for 10 mÍnutes at Loc" The supernatants were típpecl off and the Fullerrs earth washeil in turn with distilled water and nethanol" The angf.otensin was subsequently elutecL from the Fullerts earth with 2: ml of À-Ø, o.B8 s.e. a,mmonia in methanor (v/v) by mix{ng at roon tenperature for 2o minutes, It was found. that a second. elutton 62"

rAsrE 2

BBflTEEN ASSAY VAÎTATTON 6e/rù)

SAMPTES

( f ) ( g) ( h)

45 37 5o

4z 29 l+7

33 2l+ 59

37 36 39

U+ 2A LO

31 28 li+

3B 27 49

t+3 32 53

¿þCI 21 39

39 26 l+8

50

l+1

X 28,0 l+6 39.2 "6 n 10 10 12

S.D. l+"38 5,¿+4 5.94

v 11 .1Va 19,t+y" 12"7ffi x Mean V 3 Coeffieåent of variatÍon æ9-q X ÏI Nunber. of observations s"D. Standard ðeviation 63" with ammonía in methanoL was necessary at this stage to ensure recovery of the rnajoríty of the angiotensín II, The eluate was dried l-n a¡ aår stream at room temperature after the addition of 0.1 ml of 1ft lnunan serun albumin to prevent d.rytng losses. lhe dried. eluate was subsequently dJ.ssoLvecL in human serum albumfn buffer prior to assay in the rad.ioímmunoassay system.

Control- bl"a¡¡ks were set up containing: (a) water,

(b) prasma from anephrie nale subjects, and. (e) re-extractecl plasna. These blanks were found. to produce df.spJ-acement of labelled peptide of the ord.er of 2 to 5 per cent, corresponding wfth originaL concentrations of up to J pg/ml, None of the blanks used i.s conpLetel"y satÍsfactoryo she water blank invariably gave very 1ow values, suggesting that the emor clue to the nethod. itself Ls m:inj.mal"

The anephric pS.asma has the disadvantage of being obtained. fron a subject other than those whose samples are to be assayed." The use of prevÍously extracted. plasma as a blank Ís not satÍsfactorf as the peptícles or other substanees which may interfere wÍth the assay couLd be removed' corrpletely on the first oeeasl"on and their presenee would., therefore, not be reveal.ed by the blanke

lhe extraetion prooed.ure was checkeð" in two ways:

(t) Recovery of Labelled angiotensLn TI added to anephric plasma Ln l"ncreasing a,nounts and. then extracted."

(b) Recovery of unlabel]-ed. angiotensin rr added. to anephrie pJ"asma i"n increasf.ng amounts, extracted. and. then assayed. fn the rad"ioi.mmunoassay system. 64. the resul"ts of two such experÍments are shown Ín Figure p. It can be seen that there has been quantitative reeovery of both the labeIled and. unlabelled. angiotensin ff at all d.ose levels. The overall reeovery for the extraetíon procedure was approximateLy 8f", whích is of the same orclêr as that reported. by Boyô et al (1g67)

9 " MEASUREMNM OF RMITN ACTIVTTT. REIVIN CONCTNTRATTON. AND RET{IN SITBSTRATE 3Y qI0ASSAY

Renin activl"ty, renin concentration, and. renin substrate were measured by the method. of skinner (lg6l), except that the bÍoassay step was performed as descrÍbed. by Gordon et el Ugeq. Blooð samples were colleeteð into bottles eontaíning 0.2 ml of 1{. ammonium

EDTA to lnh"ibit the action of angiotensinases, the plasma was separateð and. stored frozen until subsequent proeessing. fn the skinner proeedure, seleetíve denaturation of endogenous substrate anð angiotensinases in the case of renÍn concentration estination or of 'the angtotensinases only in the case of renin aetivity estimation was achÍeved. by d.ialysis of plasma samples agaÍnst mrA- " containÍng buffer at pH 3.3 or l¡"1 respectivel"y, fol-lowecl by heatfng to 32oC. The samples then und.erriçent d.ialysÍs against EDTA* eontainÍng buffer at pH 7"5 æà tras-ylol and. neomycin were add.ed. to each sa.rnple. rn the renÍn aetivity methoð, the prepared. plasma samples were then ineubated" at 37oc for 2l¡. hours. rri the renin coneentration method., stand.ard substrate, preparecl aceording to the method of skÍnner (lg6Z), TÍas ad.ð.ed. to each sample and incubation was carrfed out for a peråocl of 6 hours, Renin s.ubstrate was d.eteminecl by the adclition of prepared renal renin (skínner, 196T), suffieient 65

FIG.IIRE 9

4O,OOO a

a

All 2O,OOO RECOVERED

o o 2O,OOO 4O,OOO ADDED ANGIOTENSIN ll (../100 sec')

200

Ail roo RECOVERED

o o roo 200 ADDED ANGIOTENSIN ll (Pg')

Recovery of labelled and unlabelled" angiotensin ad"ðed to anephríc plasma. 66"

to drÍve the reaetion between the renÍn and. endogenous substrate to completÍon, followed. by Íneubation af, j7ot for {.0 minutes, fhe angio-

tensi-n generateå d.uring each of these proeed.ures was meas,ured, in the rat bloassa,y system"

The rats used. for bÍoassay were pure Trewis straín animals, weígh-

Íng between 1 þ0 and 200 Gm each" fhis straín of anåmals had. been founrL to be more sensitive to the pressor aetion of angÍotensí.n than the other strains tested." Bilateral. nephreetomy ïyas performed. on each animal s everal hours prior to beíng used. Ín the bioassay,

AnaesthesÍa was achj.eved by intramuscular injectÍon at 5úÁ urethane, ealculated. on a scale aeeorrling tr: body weight, the d.ose varying between 0.35 and. 0.40 ml for the range of weíght as above" A traeh- eostomy was routinely performed.. lwo fíne polyethylene eannulae

(rmoo¡ were íntroðueed. into one jugular vein and. heparÍn 0.h ml (e5o units per mr) was adm:inistered through these" A larger polyethy- lene cannura (rn !o8) was inserted. i"nto a earotid. artery and. blooð. pressure was moni"tored through a straÍn gauge attached. to a Hitaehi. recorder nod.el QPD 55 with an input of 1 nv, The animal was gÍven pentolinium (0"2*0"1+ ml of an a.flo sohution) and. phenoxybenzanÍne (0'2*0"4 n1 of an o"1fi solutåon) intraperitoneally in order to prevent the effeet of the s¡rmpathetic Rerv"ous system on blood. pressure.

* rntramedj.c, clay Ad"ams Division of Beeton Diektnson & co"

N.J. OVASì+. 67,

The eoneentration of angiotensLn in the samples was d.eterml"ned" by comparison of the heíght of the bl-ood pressure increase produced by the samples in rats prepared. as above wíth that produceð by

inJeetion of a standard solution of val5-angiotensin II anicLe

(ttH¡rpertensin"-Ciba). Both the stanciard. solution and. the sample to be assayeô were ad.minÍstered into the jugular vein via microlitre

s¡æinges fitted. w'ith repeating dfspensers*. All samples were assayed" at two dose levels and" were matched. as cl-osely as possÍbIe wíth an injeetion of stand.ard. angÍotensín solutÍon at each 1evel. llíthÍn assay variatíon in sensitivity of the bioassay preparation was checked by comparison of the blood. pressure responses to stand.arcl angÍotensín before and" a^fter each assay, results beíng d.iscard.ed. ff a varlation of greater than 2 mm in d.efleetion was found. Final results ntere expressecl ín the form of ng/lO} nl/J hours in the case of renin activity or renín coneentratÍon estimatíons, anil ng/nI in the case of renin substrate concentrations, ALl- assays rcere repeated in at least two rats and" samples from physíological or comparative studies were all assayeð. Ín the one rat on each oceasion wherever poÊsib1e, in order to minimize t},:'e vari"ability of results commonly encountered. with the bÍ"oassay system,

* Hamålton Co,, WhÍttíer, CalÍfornia. 68"

CHÄPTM. 3

NORMAT T.ANGE OF PTASMA ¡,NGIOTENSIN TT

AND RESPONSE TO PHYSIOTOGTCAT STIMUTT

1 NORMAI, RANGE OF PLA.SMA ANGTOTET\TSTN TT

2 O RESPONSE OF PIASMA ANGTOIEI\TSIN TT TO PHYSTOTJOGTCAT STTMÌNT

3. COTRETATION BEI\IIEEIV PLÀSMA ANGTOTEI\TSTN TI CONCEIVTRATTON AND PLASMA RM\TTN ACTIVTTY 69.

CHAPTER. 7

NORMAI RANGE .OF PTASMA ANGTOTENSTN TI

1 NORMAL RANGE OF PLASMA ANE rosrNsTlv rT Ïn ord.er to ascertain the normal range of plasnra angiotensín II

concentratíon, plasma samples were colleeted" from forty-four no¡.ma1

subjects wLth no evid.ence of h¡pertension, agecl between 17 anð.45

years" All subjects were on an unrestricted. sodiun d.iet and. the

samples were coLlected. between 8:00 am and" 11:00 a,n ín the upright positíon.

There was consid.erable varíation in the values obtained, rangÍng fron lapg/nt to 63pe/nL, with a mean value of 25.2 j 1l+.5 (S.D.) (ftg"tO). lhís value is comparable wÍth that reported by several

other groups (Boyd g!_31, 1g67; Catt g!3!, 1967r page e!_gl, 1969; Sundsfjorð,, 1g7O) (naUre 5). Plasma angi.otensin II concentrations in normal subjects reported. by groups assa¡ning plasna samples d.írectly (Gooðfriend et a1, i968; Gocke et al, 1969)(raute J) have been h:J.gher than those reported from the above groups, where an extraetíon procedure was employed. príor to assay" lhe reason for tkr-is d.iserepancy ås not knonrn for eertain but it may be that the inerease l"n sensitÍvity which enables the dírect assay of plasrna samples has been attaÍned. at the price of deereaseå specificity. Angiotensin levels measured. by bioassay have also tend.ed" to be higher than those obtaÍned by radíoimmunoassay (Genest et*,êl, 1964i Morris and Robinsan, 1)6la; Massani et -el, 1966) (ralre 5). fhe tl.ec¡'eased speeífícÍty of the bioassay procedures 70.

FÏGURE 10

300 a

a

200 PTASMA a a a Ail 1rs./ mt.l a

a a roo

3t a a a a a a I a a taa a a o t. a a a a o NORMAIS ANEPI,IRIC c. c. F. NEPHROTIC þIYPERTENSION CIRRFIOSIS coNN's

Plasna angiotenõin Tï concentrations in normal subjects and in pathologlcal conditLons. 71 .

rAStE 3

PIASMA ANGTOTTII\TSTN TT CONCEI'TRATTON IN NORMAL SUBJECTS

AUTHOR naNçE (pel*r) MEAN (pel*r)

3oyd. et al t 1967 8-56 Catt et aI, 1967 5't+7 21 ! 1t4" Gooctfriend. pt aL, 1p6B <5 - 126 45 Gocke M, 1969 18 - 110 5l+ Pase g!3!, 1969 2'l + 11 (SupÍne) 25 + 14 (uprisht)

Sunclsfjorl., 1970 12-57 27 t 1t+ Present Stud.y 4-63 25 ! 14"5

Mu1row, 1t6l¡ o-60 23 + 3

Genest et al., 1)61ç 70 + 20 Massani e+ aL, 1966 95 2tz 72,

employed. probebl.,y aecerèåæ.t,s ferr these hi"gher values, althou.gh, in on9

sueh study, the valu.es eråtaineå agreed elosely with the rad.toimmuno*

essåy values (Mulrow, 196]+),

2" RESPONSE OF PLASMA ANGIOTENSTN TT îO P}TYSTOTOGTOAT, STIMUTI

Ïn oråer to d.eternrine the effeets of upright posture and. d.ietary

soååum restrietiÕn on plasna angi"otensin IT eoneentrations, two normal sub jects were equí1Í.brated on a d.iet ealeulateû to eontaån 10 mrq of soål"um per 2[ ]:,ours. "ana]-ysi"s of a d.uplieate 24. hour sampïe of the diet eonfirmed. the Low L,evel of se,d.Íum intake (g.i *¡q per" åay).

0n the d.ay before oorumeneement CIf the sod"ium restríetion and ð.urång seven d,ays ef low sod"Íum diet, blooå sampJ"es were taken for meåsure.- ment of pl"asma angio'tensín fI levels: (r) at 8:0CI am fol]-owÍng at l"east I hours of overnight strie'b reeumbeney, and. (U) at 11:00 am after J hours sittíng, atand.ing or walkj"ng. Angj-otensån IT was extracted. fre¡n the plasna samples and. assayed. in the rad.ioÍnmunoassay system by the usual method., Aliquots of the pLasma sampl-es were useå for measurement of plasma rer-in a,g bivÍ.ty by the methoð of skÍ.nner (tg6z) " lhe resurlts of *hese studi.es are shor¡rn Ín FÍgures 11 and. 12. ït ean.be seen that the urÍ"nary soåi.um dropped. quåekly to very low levels, eonfírrriÍng the effåeraey of the sod.ium restriertior¡, TLre plasma a^ngiotensin Tr d.ata showed that the values at 1'l :00 am after J hours of uprÍght posture n¡ere eonsistently higher than those at B:00 am after overnight reeumbeney" The magnítud"e of tkls ínerease varieå eonsÍd.erably, averaging approxirnately !@" std 5 r-b 0q Hì ooP.O cl cl' 300 oSo (¡ Fb D¡ Sodium Restriction ts. Dietar Sodium Restriction lOr 200 F. 200 URINARY Na H pcl' OT (mEq l2t hrsl o< t00 100 OtD ÞÞoo cl H' hd 0 a HC H I trl 0 I - - - Êr il f) 0 - - cr ts' r-J Þd oo H 5 cr! 80 Subject B .cl 80 Subject A t-- ol-r. cl- t F. o 60 60 , b o t a .t H PLASMA AII lr0 F (pg/mt) 40 a' s ''----)' (t -.o- " ta p 20 o' 20 O----a.

0 0 6 7I 123t,5678 1 2 3 4 5 DAY OF STUDY DAY OF STUDY { \N 7)+.

FIGURE 12

ao A too

PLASMA A II P. R. A. 1rn./nl.l 20 'oo (ns./ loo ml./ 3 hrs.)

o o

o-o P.R,A. .-T PIASMA A II

too tooo

800 to I

ôO óoo PLASMA A II P. R. A. 1on. / nt) (ns./t00ml./3hrs.) 40 ¡loo

20 z>1 200

o o I 2 I 7

DAY OF STUDY

Comparison of plasma angiotensin ÏÏ concentratÍon and plasma renin activity during d.ietary soôÍum restrÍctíon" 75. Both the ree'¿:mbent and. the upright plasrna angùotensin TI levels

showed, a. progressive inerease d.uríng the peråod of d.Íetary sod"ium

r"estråetfon.(rig"lt), The inerease u¡as more marked. in the case of

subjeet (3), where there ru&s a tend"eney for the values to fall some- what during the last two ðays of the study perioå, although stij-I remainíng elevated. above eontrol levers" These changes are of the same order as those reported by page et-_êl (lg6g); the inerease in angÍotensin TI ecncentratÍ.on was more marked in one subjeet than the average i"nerease reporterl by those authors " The peri-od of d.Íetary soåium restrietion rvå,s, howe.ü'er, longer in the present study"

These ehanges in plasma angåotensÍn TI eoneentration in response to posture and. d.ietary sodi"um restriotion &re sirnÍ1ar to those previ.ously reported" for pJ-asma renin aetiuity anð alðosterone exeretion (Brown _Ë-t_,glc 1g63; Cohen -gt_gl., tg6t+i Gordon e_t_êl, 1g66(b)). The eonsistent Ínerease ån the Levels at 11:0o am compareð. wi"th the levels at B:00 arn ås presumabry d.ue to assumptisn of the upright posture rather than to the time of d.ay, sj.nee levels of plasma renin aetlvity are fal1Íng at this time Ín re*r.rnbent subjeets (Ëordon gå31, 1e66(b)) " ggruwr0N 3, _8ry84{ _qusuA ANGI0.r.ENSAN_ rr_ 4p _ rl4s.¡@ AqgTvIrI +ET{JI{

Plasma rcnin aotívåty d.eterrninati.on,s were perforneå on aS"iquot samples of plasma from the physi,o3,ergi.caL stud.y, the angÍotensån generated" in våtro being measured. by the rat bi.oassay sys.tem

(skinner, 1967). comparison of these results with the plasma angio* tensÍn rr varues showed. that eÍmårar ehanges were seen in be¡th 76" parameters of aetivity of the renÌn*angiotensin system, The renån activity responses were proportionaLly greater in one subjeet (n), an effeet consistent with that reported by Page e!_el- (116l). Figure 12 shows the comparÍson of these values in the two subjeets, in the reeumbent positÍ-on Ín one instance (A) and in the upríght posi.tion in the other" fhe reason for the proportionall"y greater rise in plasma renin aetivity than plasma angiotensín TI may be related. to the longer half- life of renin in the cÍreulati"on, to variable tissue extraeti-on of angiotensin, to variations in aetivity of converting enz¡rme and. angiotensinases in vivo, or may be relateð to vari,ations in cond.itions ðetermining the amount of angiotensin generateð on incubation of pÏ-asma in vÍtro" CaÍn gl_gt (ly6g) reeently published. eviðenee that-'the material measureå Í.n venous blood. in a rad.ioi"mmuno*

åssay for angÍotensÍn ÏÏ eonsísted. of a mixture of angiotensin TT and.

Íts breakðown produets wtrich could" vary in compositíon from sample to sample, In light of these possÍble variables, ít is not surprisíng that the plasma renin aetÍvity and plasma angÍotensin Tf estímations d"o not correlate more c1ose1y" 77"

ü$PÎFR -,&

PLASMA ANÊIOSSTSIN II f,E:lJlStS JN PATI{0IJ0çICAL C0NÐTfI0I\TS

1, CONoÞSSÏVB OAÎDÏ.EC FATISRE

1) 1o æCTRRT1OSTS ET' THB ITUER, 3, FIBPffRCITTC SYFIPRO¡IE

&, ERrl{asl AlÐqggnRp¡çT$e 5. HTFM.SDîffiTO$ 6" æAI{ÏEPffi.IC SUBJEOÎS 78,

crTAgrqR. ,h

PL.A,SMA ÁNGTOTEIVSTN TT LEVET,S TN PATHOLOGTCAI CONDITTONS

fhe angÍotensi"n If rad.ioÍinmunoassay was app)"åed" to the measure-

ment of pl"asma angiotensin TT coneentrations in patients lvith a variety of cond.itions in wh:ich d.leturbanees of the renin*angíotens5"n system were thought li"kely to o*eur, namelyo congestive earðÍae

faÍLure, nephrotÍe s¡mðrone, hepatic eírrhosÍs, prinary alðosteronf-sm,

essential and. renovaseular h¡¡pertension, and" in anephrÍ"e subjeets.

The results of these stud.i.es are shown graphieally f"n Figure 10.

1" CONGESTTVE CARDTAC FATLT]RE

Plasma ren-in aettvity has usually been repori;ed to be elevated. in patients with eongestÍve carûi-ac failure (Brown SÍ_gl, 1g6t+, Ve¡mat Så*4, 1961+; FaseåoLo .e.t*_êl., 196l+)o Tnereaseð renin secretion has also been demonstrated Ín d"ogs with experimentalry

proüueed. tuigh-output cardiae failure (Davås _g!__gl, 196Ì+)" eongestÌ"ve eardiao faålure could. leað. to i,nereaeed. prod.uetion of renin ay tne kidney through a drop ån renal artery perfusåon pressure ].eadÍng to stimulation of .baroreeeptors at tÏ:e afferent arteri.ole level or by an alteration i"n sod.ium loaå or coneentratiop at the maeula d.ensa

slte. Evidenee has alse been presented" (Sctrneid"er _gt_q].u 1969) tù suggest tbat d.eereased. raetabol"lsm of rentn may eentribr:te substantial,ly to the inereaseü plasma renin of experÍmental hågh*output eard.i.ac failure, 79" lhe trrue sitt¡ation in uneompS.åeated. eongestive eardÍae faílure in m¿n is d.i"ffi.cult to aseertain beeause of the aLmost ånvariable

treatment of these patients with a low sodåum diet and díureties whích

will tend. to prod.uee an increase ån renÍn l-evels of íts own aoeord.. Treatnent of congestive cardåae fal.lure has been reported. ts produee eåther an inerease in plasma renån Levele (Brown e!_e}, 196ù or a fall (veyrat et--êl., xg6e), rt seems l{.kely that the elevated renin

levels in eonges*i.ve heart faå,lure are at reast partly rj".ue to the

therapeutís manoeuvres used. i"n treatment, a proposítioa supported.

by the study of Vandongen and G.ordon (tg7O)"

Elevated. blood. angåotensån levels in patients with eongesti-ve eardåae failure have been repcrted. by groups errþloying bioassay nrethod.s (Genest gt-e1., t96l+; Massani _e!-g]-, 1966). Tn the study

of Genest et-al- Ugeü, the b3,ood angiertensån coneentration was reported. to usual"ly deerease following total or part5.al relief of the oeclema. Plasma angåotensin TT eon*entratj"on was subsequently reported. to be marked.ly elevateð i.ri patier.rts with seïrere congestS.ve eardiae failure wh.en measured. by the rad.ioåmmuRe¡assay method.

(catt el_-êl, 1969) " rn the present stud.y, plasma angÍ-otensÍn ïï eoneentratíons in a group of 15 patients with congesti.ve eard.iac faj"lure ranged. from

16 pg/mL ta 3O3 pe/m\, wåth a mean value af 1ZZ pg,/m1. (ftg.tO),

The majorå.ty of these patÍents wer,.e al.neaåy on treatment wåth l,ow- sod.Íum dået, iligÍ'talís and åi¡.iretå,es when the p]-asma samples were taken, and. it was d.åffåeult to d.er:í.d.e with certaÍnty whetlaer the 80" increased. angiotensån rr leveLs were largely d.ue to the sti.mulatory effests of the therapeutie manoeuvres or to the und.erly:ing disease

prooess. some support for the fo::mer proposition ï¡as afford.ed. by the find.íng that the plasma angíotensÍn Ir concentrations in the threo patients from whom plasma samples were taken prior to treatment were

26 pg/nlu 4o pg/mL, and 86 pg/n] , i.e" they were within the normal range or only slightly elevated, and. the higher values found fn patients who were alread.y on trestment may have been at least partly d.ue to the treatment itself"

2o IfnpATIC C¿RRHOÞIå

Elevated. levels of pJ-asma renín have been reported. in plasma from patients with cimhosi.s of the liver and ascites (Brovnr t ql, lg6l+i Faseiolo et-4¡ 1964)' Reni"n levels ín patíents with cimhosis but no ascites have usually been reported. to be normal or only slightly elevatecl (Brown et al, 1g6h) but signÍfíeantly el"evated. values in this sltuation have also been reported (Imai and Sokabe, 1968)" Blood angiotensi.n eoneentrations have been reported to be elevated in patients with cirrhosås and aseåtes (Massani -g!__Ê1, 1966) and this elevatíon has been confÍrmeð by more reeent radíoimmunoassay measure- ments of plasna angiotensån TI (Catt _9!_4, 1g69g Goeke e!_gl, l96gi sundsfjord, 1970). Factors wh:leh may contribute to the elevation of plasna reni"n and. angiotensin includ.e low plasma volume, impaired metabolåsm of renin by the d.amaged. låver, and alterations in sodium ÍÐ"l*nc" or soð.ium handrång by the kådney (schroeder es_ÊL 1g7o). 81 " Plasma angiotensån fI eoneentrations were measured. ín seven patients with eirrhosis of the lLver (fig,tO). Values ranged fron

I+O pe/nl to 194 pg/n:-o wi"th a mean of 106 pg/rcI, confirning the elevated. levels reportecL by others. In this small seríes, there seemeil to be no d.ifference between the levels in the patients with ascites and. those wÍthout.

3, NXFHROTIC SyNDRCIME PLasna renin levels have been reportecl to be elevatecl ín patients with the nephrotic s¡rndrome and generalized oeclema (Ve¡rrat et_al, l96t+; Imai and. Sokabe,- 1968). Elevated blood angiotensin levels have also been reportetl in this situation (Massani -gS-jl, 1966i Sundsfjord, 1970).

Six patients with nephroti-e s¡rndrome and. generalized. oed.ema were found" to have plasna angiotensin II eoncentratíons ranging from

26 pg/nl to 166 pg/ml-u wÍ"th a mean of 9Q pe/mi-" The leveI of plasma angiotensin fï was not sígnificantly eorrelated. vsith the d.egree of proteinuria or the severity of the oed,ema.

4o PRrr{ABYltlgÞgE'oNrSM Pbimary aldosteronåsm is assoeÍatetL with autonomous h¡¡per- seeretÍon of al-d.osterone from the adrenal eortex from a cortåcal aðenoma or bilateral cortj"cal h¡¡perplasia. The inereased ald"osterone levels result in h¡4pertensåon and. tncreased plasma volume, with resultant feedback suppr.ession of renin seeretion, so that plasma renin levels are very low in this eond.ition (Conn et_gl, 196t+) " Plasma angiotensin TI eoncentration has been shown to be low also in this condi.tion (Catt xg6g) _9!__å1,¡ " 82,

Plasna angåotensin ïI coneentrations were assayed. in one patíent with primary ald.osteronåsm" the patient was a 1g year ol"d female who presented. with severe h¡rpertensíon, nocturia, h¡pokalaemÍa (mad"e lçorse by a high sodiun intake), and suppressed pl-asna renín activity, not responclÍng to the stinul"l of sod"åum restrietion and upright postureo She was ultlmately fo'und. at operation to have bilateral aðrenaL cortj"eal h¡perplasia, Plasma angÍotensin II eoneentratíons of ta pg/nl in the recumbent posítÌ"on and 5 pg/m\ when upright were found" after a peråod. of five d.ays on a d.iet eontaínÍng only 10 m"Eq" of sod.ium per 2l+ hours. Plasma renin aetívity estÍmation on aliquots of these bl,ooð. samples ¡nieldod values of ¿+5 ng/tOO nl/24 hours and 47 ng/lO} hours respectively, Both these parameters "ù/Zl+ of aetivÍty of the renin-angiotensin system were thus inappropriateLy low fol"lowing díetary sodíum restrietion and, it appears that the angÍ.otensin ïï rað.ioi"mmunoå,Bsay would" be a suåtable substÍtute for the reni"n activity estÍ.mation in this clinieal sÍtuation"

5" ,r!ßq'gET{ËrON Plasma renin levels har"'e been reported. to be mostly wÍthín the norrnal raTlge in patÍents with uneomplicateå "essentåalrt h¡rpertension but often elevated. in. patåents wÍth severe or malignant h¡pertension (Hehrer, 1961+i Ve¡rrat e!__elr lg6t+E FascioLo -g!_gl, 1g6t+i 'Brdmr At-ê], 196U)o In the ease of patíents wåth h¡ryertension secondary to renal artery stenosås, h:igh pS"asma renin values have been reported.

(Helmer, lg6t+i Ve¡rrat e!_-al, 19€'l+A Brown -gt_,q1', 1g6t+), but some values were noted. to fall" wå.thån 'the normaJ" range" Htgh levels of plasma angÍotensin have been reported. in patients wÍth renal artery 83"

stenosis (Genest çS_a&., '196+g Massanå qt*g¿u 1966), but in one stuðy (Mulrow, 1964), normal levels were found- iin a group øf 16 patf"ents

wíth h¡rpertensåon anå reRovaseul-aE" d"5-sease, eåx sf whom were eureö or åmproved. out of '14. subuuitte,l to surgery. Results obtaÍneð using the raðl"oåmrnunoassay proered.ure for assey of p]-asma angiotensin rr

have revealed. elevated. plasm& conoentrations in severe and. malignant

h¡pertension and. in renovaseular l:¡ryertensåon (catt e!-êr, 196g; Geeke g-t-g¿, 1969), EJ-evated plasma eoneentrat{ons of angiotensin If have also been repcrted" i"n patien'bs with uneonrplicate¿ reessential"

h¡ryertension (catü xg69g 19To) .9!_e1, " Ïn the present stud,yu p1-asnra angictensin II eoneentrations were

assayed. in a group of patients with h¡rpertensåon and. the results are

shown fn Figure tr 0. Tt ean be seen that plasma angiotensín Tr

soneentrations vary frorn values wåtlún the normal range to quíte high

levels" lhe hÍgh pereentage sf cases wåth erevated. angiotensín TT values may be partly expl"ai"n*d. by the faet that the majority of the ea.ses had. been referred. for ÍnvestigatÍon of p*ssi"bl-e renovaseular

h¡pertension. In the grÕup Õf ,seven patients Ír¡ whom renova$euLar

h¡rpertensÍon was unoqt"riv*ea1Ly established", the p3"aema arigiotensj"n IT eoneentration was ur,l.versally elevateå abo,ve ,the normal range" Tt was not possib.l-e *o oategorize æxaeb1y the aetio]-ogy of ilhe h¡rpertensåon in al-1 of the remai.nd.cr of the eåses, buü sone of tihem had- evíðenoe of renal paren',:h.¡mal dåsease as shown by red"uetior¡ in renal síue or evidenee erf d.ysfunetåon sn renal funetåon tests" trYhen. the patÍents witi: rer¡ovase,;Lar h¡rpertensio:r: and. those wtth suspecteð renal pareneh¡rmal ðj.eeasê werê exeludeå, there remaån¿*d 84"

å. gror:p af 13 patíents wåth ttessentÍal"fr hypertension" In this group of pati"enüs, approxínately half the pS"asma angiotensin Tf levels were witirin the normal range and. the rernaind.er were elsvated. above normal, so that å mean value af t+Z pe/nl was found. This fÍnding is in agreenent with the hÍgh mean values found^ in t'essentialrt h¡pertensJ-on by Catt e!_-s,1 (tgZO)o fn the case of malignant hypertension, elevated. lovels of plasma angiotensin II were always found, a finding Ín keepíng wíth that reported. by Catt qL-eL (1 969) and Goeke et (g6g) eL " The rol"e of the renån*angiotensån systern in clÍ.nleal h¡¡per* tensive ðÍsease ås not eIear" fhe renin d.ata suggest that the system d.oes not play a major role in mild. to mod"erate rtessentialf' h¡rpertension but that ít may contrÍbute signåficantly to the severÍty of the d.isease ån accelerated" or malågnant h¡rpertensíon, when features of seeondary h¡perald.osteronism may be superimposed on the und.erlying h¡rpertensíve proeess. Plasma angiotensln rr data, however, have shown elevateð mean levels in patients with i'essêntíalrf h¡rpertension and. a hÍghly signåfi"eant correlation between the plasma angS"otensin ÏÏ eoncentratåon and 'bhe diastol.ie blsod pressure (Catt et*,al, 1970). Although the elevated" angiotensin rr 1evels need not exert a h¡rpertensåve effee't (e.f" val-ues in subjeets on oral, eontra* ceptives (catt ei*êl,u 197CI)) and may represent merely a seeondary effeet of the h¡çertensíve processe a possi.bl.e eontri.butory role of the renin:angiotensån system tå trre blc,od. pressure el-evation of Itessentialrr h¡pertensåon cannot be eompLetely exelud.ed- at prescnt. 95.

In the case of h¡pertension seeond.ary to renal artery stenosis in man or in experimental renovaseular h¡pertensi"on in ani"mals, the renin d.ata suggest that the renin-angiotensin systen is of príme lmportance in the d.evelopment of the blood. pressure increase. Support for this role of the system has been provid.eê by errperiments demonstrating a cLrop ín blood. pressure in anÍmals with er¡rerirnental h¡npertensíon on infusÍon of antirenin or antianglotensin antibodiee

(Deocthar et a1., lg6t+i HÍ1L et al 197A; Tforcel et al, 197A) , " Howeveru d.oubt has been east on the valid"ity of th:!s concept by the recent cl"emonstration of the prod.uction of experÍmental reno- vascular h¡pertension i"n anåmals ímmunizeð agaÍnst angiotensin fI, together with the laek of effeet on the course of establishecl h¡rpertension of active immunizatÍon against angiotensin ïï (MacDonaS-d et a1, 197Oi Johnston et_êl., 1970)" The fai.lure of antirenin antibod"lee to affeet the blood. pressure ín anÍmals with h¡4pertension ðue to renal artery constrictÍon and contralateral nephreetomy (Wetser S!-gL, 1969) ad"ds to this doubt regarding the role of the renin-angiotensin system ín this situatÍ"on, lhe suggestion fron the latter stud.ies has been that the renin-angÍotensin system is not the major faetor in the prod.uetion or maíntenanoe of errperi"mental renal hSpertensi.on. The problen raised. by this reeent evidence índ"icating a less important role than prevS"ously aecepted for the renin-angiotensån systen has not been eorapletely resolved. at the present tåme" 86, 6, ANrF,t{Rrc str.g"ryîg

Btlateral nephreetomy has been shown 'bo cause a raarkeü reðuetion in plasna renin to very law or undeteetable levels (Lever et_gl, 1963). Very low 1evels of angiotensin TI in plasma from hunan strbjeets with bilateral nephreetony have been reported. by Catt et al (lg69). Plaema angÍotensin II eoncentratÍons in five anephrie patients being maÍntained. on the ehronùc dialysls progra¡n at rhe Queen

ElÍzabeth Hospåtal were fouRd to be O, 0, J, O an& ¿+ pA/mI" Four of the five patients were femal-e,s and. d.e'teetable levels of pl-asma angiotensin rr were found. in two of this group. The sígnificance of this finciing is uncertain. Although a source of a *renj-n*l_ikeft enzJme fron the female genåtal 'tract Í.s a possibility, this has not been conelusively d.emonstrated. to affect plasma renin levels, 97.

CI{ÀPTER 5

fO MEÀSTTRNAIfiÍT OF PLASMA RENTN ACTIVTTY

1, enNERA+

2o RENAT VEISoUS PÏ,ASÌIA REI{ïN ACTïïrFY 4êfr9s 88"

EHAPTER q

APPLÏCATTON OF ANÊÏOTEïTSII\T TÏ RÁ}TOTMMUNOT.SSAY

TO MEASUREMENTT OF PT,ASMA REIVTN ACTTVTTY

1 o ÊENp8$L

One of the orÍ"gånal aíms Ín setting up the rad.åoinm'¿¡noassay for angiotensÍn Tï was for the purpose of replaeing the bioassay s*ep in the measurement of pl..asma reni"n aetivåty and" plasma renån eoneentration. llntÍ1 reeentl.y, the only method. ,of, assessång the aetivíty of the renån-angi"otensS"n systen has been by bioassay of the angåotensín generateå by plasna when i-ncubated. tm. vi*r,o after åntrïbÍtion of angiotensinases (Bouoher _gå*elrlg6hg Brovm et*êLp l96t*; Skånner, n96V). The båeiassay teehiaåques åepend on the pressor response produeeå by'Íneubateð samples eompåreet with stanðard. angiotensin prepara'tåons in anårnaL.s wi"th pharmaeological.l"y bloeked. s¡rmpathetie nervous sys'tems" []ie båoassay teehnÍques &re tedåous, laek precÍsion and are nob readily app3"Ì"eable to Large numbers of sampS-es " Comparåson of the resulbs obtalneå by bioasnay and. raåÍo- immunoassay for the angÍcter:sin generated. d.uring the Skínner nethoð for pl"asma renån aetÍvity, have shervun tha* the values obtained. by the radÍoÍmmlrneassay methcid" have been eonsister¡tly 3-ower, usuall-y of the ord.er of 15" of those obtaåned. by bioassay, ïllhen samples fcrmång pårt c,f a seri"ee a:f phys:1,a1.¡:gtr:aL, stur,lå,es have beera exam:incr1 by the two teohnåques, however, the sa.rne general-'&renås havc been seer:, suggesting that *heæe may be some e,,orrelatåon between the bwo rnethoð.s" 89"

In orcler to eheck whether the substanee being measured was actually angiotensÍn II, severaS" Íneubated" plasma renin aetlvtty

samples were assayed. at ðoubling d.il-uti.ons in the rad.ioimrnur¿oassay proceðure; ít was found that the points obtaÍned coincid.ed. c1oseL.y wíth the star¡clarcL curve for angiotensin II obtained uncler the sarne cond.:itions, suggesting that the two substanees were åd.entical

(nts.r 3) .

A llkely e:rplanation for the diserepeney between the two methods is that the entl prod.uet of the plasma renin aetivÍty proeeåure is a mixture of angiotensin ï and" angiotensin ïï, only the latter being measureð in the rad.j"oim¡nunoessay but both gÍvÍng a respense Ín the rat bioassay. It has been shown by Boyd. et el (gel) that the presenee of ed.etåe acid. anê 2n3-damereaproJ, in ineubated. plasn,a samples l-ead.s to a higher value for angiotenstn by bÍoassay than by rad.ioimmunoasså.y and. it was suggested. that this d.Ífference vras d.ue to fornation of angiotensin r and. preventi.on of conversion to angÍotensin fI by the 2rJ-dimereaprol and. ed.etie aoid" whioh inhibited. convertíng enuJ¡rne as well as angÍotensinases. Subsequent work from this group (Boyd -g!_el, 1968) showed that it was difficurt to obtain a complete return of aetivity of eonvertl.ng enzJrme by the add.itÍon of varíous metal åons without also restoring angiotensi.nase activity, Sånilar observatåÕns were nad.e by Page g[g[ (t g69) wno denonstrated. inhibition of converting enzyme aetivÍ"ty by ÐfÀ.

If the fraotíon of generatect angiotensin in plasma samples present as angiotensi"n rT were constant, it woulci be possi"ble to use the rad.i"oimmunoassay proeedure to obtain a measure of renin aetívi"ty. 90. IDENTITY OF INCUBATION PRODUCT WITH ANGIOTENSIN II

50

1+0

30 o/o BOUND \

20 lsoteu 5- Angiotensin II

Dituted PRA. SampÌË

10

0 5101520253035

ADDED ANG|OTENS|N (ng )

FTGIIRE 1

ïdentity of Íncubation product with Angiotensin fT 91" ExperÍence has shown, howeveru that the fraetion present Ín this form

is variable and. the eorrelatåon wå'th bi"caËsay results is not el"ose enough to it¡stify the use of the rad.iotm¡nt¡noassay for th:is purpose"

Attempts to reverse the inhibitåon of eonverihing enu¡nae by the ad.d.Ítion of various metal ions has confirmeô the experience of others

of reaetlvatíon of angiotensinases, The alternative approå.ch of

procLueing conplete inhibÍtåon of eorriêrtíng enzylne aetivity anð. measurenent of the generatecl angiotensin in a rad.ioimmunoassay

system specifJ"c for angiotensi.n I would. seem to be the more satisfaetory ansurer to the probleno (Boyd et al-, 1969t Haber et a1, 1g69),

2o REIlAL VU{OUS PT,ASMA ru}TTN ACTTVTTY RATTOS

Corparåson of pï.asma renin aetivity values i.n both renal veins has been founcl useful ín the d.iagnosis of renovaseulan h¡rpertensíon (Uicnetatcis gLpl, 19672 Fåtø, l96TE tfi.aer et*¿L, 1967)" Bilateral renal veín catheteråzati"on anð sínultaneous sanpling from both sÍdes Í"n patíents wïth restrieted ðietary sodåum and controll"ed posture before samplÍ"ng has been used as a sereening test for possíble renovaseular h¡rpertenõi"on" P1asna renin aotivtty has been measured by the methocl of skånner (1967) exeept that the bÍoassay step has been performecL as cleseribed. by Gonelon et_gl Ugee). A signifieant d.ifference 1n the values obtained. from eaeh sid.e has been found. to be a useful test for renovascular h¡rpertensíon (Pawsey g!_el, 1g7A). sinee the rat bÍoassay proeed-ure fer measurement of generated. angio-

tensin is relattvely imprecise and, eumbersomee the possible application of .the angiotensin rað.ioi¡nmunoå.ssay to the measurement of genera'hed. 92.

angiotensin in these renal venous sampS-es was investÍgatecL. Since

the proeedure i"nvoLves catheteråsatíon of both renal, veins and. the

wíthdrawíng of bl-ood. sampS-es såmultaneously from the two sid.es, it seemed 1Íkely that the eond.itÍons d"eterrn:inÍng the proportion of angio- tensin ï and" angiotensin II generated during an iiLentical period. of

ineubation Ín vitro woulð be the sa^me for the two sanples, and. the

problem of vary:ing proportions of angiotensi.n I and. TT mentioned.

previousl.y would. be raÍnimized.. fhe ratio of pT"asma renin aetivíty in the right renal veín to that in the left renal vêin should. then be very sÍmtlar for measurement of the generated. angiotensin eíther by rad.ioÍrluilunoassay (specÍfic for the fraetion existíng as angio-

tensin IT) or by bioassay (measures both angiotensin I and. angio- tensin rr)" rn order to test this h¡pothesÍs, a comparative study was eamied" out d.otermini.ng the ratios of renal venous renin aetivÍty

in 27 pairs of samples by both bioassay and. radÍoimmunoassay. The levels of angÍotensin generated. ín vttro by the Skinner

technique and" measured. both by bioassay and by rad.ioimmlino&ssay a"re

showa i.n fable l+" comparåson of the absolute level-s ðetermíned. by the two method.s shows a coneiôerable variatÍon ån that part of the

total generated. angiotensín meesured. by bioassay n¡hich is measurable as angiotensin ïï Ín the rad.ioirmrunoassay, usually of the ord.er of 1q" There is hewevere vepy goed. agreement between the ratios [frigh síð.e : low sið.e) ð.etermined usång the two methods; the correlati"on is shown in Fig. 'î[. From the regression line, ff a value of 1"5 is taken as sågnifi"oant for bioassay (nffi.efrelakis _e!_e!, 1967), the eÕrresponding value is 1 "6 for the rad.ioimmunoassay. 9'5 " TASLE ]¡

ComparÍson of values for renal venous plasma renin aetivity (pn¡) and. ratío of high síd.e to low sid.e obtaínecl by rad.åoimmunoassay of generated angiotensin ïï with those obtained" by båoassay in the rat pressor system

BÍ"oassay Rad":loimmunoass ay @e/too mVjnrs) 6s/tao uLL/311r,s) .8*,.&"[. ,Lr.!rJ. Ratio 3rE*I' !*^U" Ratio 1. 405 900 2,22* 13 27 2.08* 2n 685 563 1"27 48 33 1 J+5 3. 167 172 1 .O3 17 17 1.00 h, 1081+ 1395 1.3 l+2 6o 1.43 Ã 1 658 1765 1.06 217 200 Q.92 6, 1258 2155 7 ,71* 1U6 250 1 "71* 893 1 7. 775 "15 96 96 1"00 B" 1O6t+ 1128 1.06 133 188 1 "r+1 o 978 1119 1"11+ 127 171 1"?+ 10, 1 725 557 "30 229 175 1 .31 11 . 255 93 2,72* 67 23 2.91* 12. 800 1 1 921 "15 3B 33 "15 13. 3065 2737 1 ,12 283 258 1.10 14. 22gA 1335 1 "72* 134 7t 1.94* 15" 701 B 26/+o 2"66* 563 17t 3.29* 16. 730 679 1 490 1 0¿r 1 "08 "21+ 17" 1 784 715 "10 71 58 1.22 18. 592 t+58 1"29 109 71 1 .51+ 19. 848 780 1.O9 63 67 4"95 20. 589 635 1.08 233 233 1 .00 21 . 708 779 1"'î0 292 263 0" g0 22¡ 188 x63 1 I 84" 150 1 "15 "23 23. 88 1,O2 67 1 90 75 "X2 at 2l+. 111 93 1 "19 108 0.85 25, 2319 13t+1 1 26A 116 2 "73* "21+* 26. 9l+ 11"t 1 ,17 o? {00 1,O9 27" 85x I 2503 3"32* 700 '159 L.¿fo* rF SignifÍoant ratio 9l+.

FTGURE 1l+

RENAL VENOUS RENIN RATIO ( GENERATED ANGIOTENSIN )

5

4 r =0.72 n= 27 RADIO 3 o p<0'00.|

IMMUNOASSAY o a 2

1

01 23 45 BIOASSAY

Renal vein renin PRA ratios; bioassay vs raðioimmunoassay. 95" Using these eråter5"a, 7 out of 27 ratÍos are slgnJ"fåeant by both methods of assay" Thie exeellent comclatlon between resuLts fon renal venous renin actf"våty ratf"os using båoassay and" rad"i"o-

Lnnunoassey suggests that the latter proeeclure couLiL inel.oed. be appJ.ieel to thís test to replaee the more cumbergome bioassay" 96.

CHAPTM. 6

RM{IN-Á,NGIO.ïM{STN SYSTMI IN PRSEN¿,NCT

1 TNTRODUCTION

(r) cnwnnar

(¿t) ToxAEMTA oF PRmNANcY (r) Established. loxaenrta (¡) Renin Activity Prior to Developnent of Toxaenia

2, PTASMA ANETOTH\]STN ÏT CONTH\TRATIONS TN NORMAT PRTENANCY

3. PTASMA ANGTOTENSÏN ÏI CONCUIITNATTONS IN PRE-ECI,AIUPTTC fOXAM{IA q7

CHAPTER. 6

REI\ITN--ANGTCIfTNSTN S'YSTEM TN PRESNANCY

1. TNTRODUCTTON

(i) General-

The exeretåon sf al"d.osterone Í,s inereased tlrroughout pregnaney

and returns rapid.ly to normal following d.elivery (Vennång and. D¡rrenfurth,

1956; Jones eå_g¿.' 1g5g; Iüatanabe ej*et, 4"965), The i.nereased

seeretion oceurs as early as the fÍ.f.äeenth week of pregneriey and. the aotual f.evel reacheå d"epend.s on d.5"etary sod.Íurn ångestion in the usual way (Tttatanabe gh_g} , 196Z)" The d.emonstration e'f a eorineeti.on bettseen the renån-angÍ,otensin system and aldosterone eeeretion (Laragh gt_er, 1)60; Genest gL-*t, 1)61; Hi"ggåns e!*-el, 1962) ïues soÕn followed by studåes of the renån*angi.otensÍn

meehanåsm during pregT)ancyo Brown et*aå (lgøS) showed that the plasma

renin eoncentratåon was raised. throughout pregnaney and returne¿ to normal- within two months of parturition" lhey showeð also that, d.uring the f.ate stages of gestation, the plasma renin 1,eveL.s Ín many of the normal pregnant wÕmen were withín the normal rånge, and. sugge,sted. that thÍ.s mÍght be d.ue to a d"eereased" demand. for sod.i-um at thls stage of pregnaney, The increaseð aetivity of the renån*angiotensín system durårrg pr.esnanûy was confírmed. by

Genest e! .aÅ ?g6r), who showeð that there was a progressive and slgnificant ri.se in plasma renin aetivity from the fc,urth .to the twenty*fi,rst week of pregnaney, followed. by stabålåzati.on at thås high level anð then a tend.eney to ðer:rease slJ"ghtly after the thì.rty*first. week of pregnaney. wåner (lpøS) ðemonstrateó. a two to eigkrt*fold ínerease Í"n pS.asma reni"n aetívå'Ly åri pregnaneyo with values being gB,

elevated. from the si"xth week cnwards" The high renin valules grad.ually fel"l- to controS" levcl..s wåthin a week af.ter deltvery. An expla^natt,on of thås el.e'watåon of plasna renín aetivity

ån pregnanoy was províd.ed. by the d.emons'üration (Helmer and. Ju,å.son, 1967) that pregnaney was aceompanåed by a marked inerease in renín substrate. The íncreases Ín p3-asma renin aotivity were, therefore, of much greater magnítud-e than those Ín plasma renin eoneentration, si"nee the amount of angÍ.o'bensín generateô per unÍt of renin is d.ireetl.y related. to the substrate coneentratÍon" Confårmation of the probable rol-e of oestrogens Ín the inereased" substrate levels

d.uring pregnancy was obtaåned. by treating non-pregnant women with oestrogens or oral contraeeptåve preparati.ons containíng oestrogens, Th:is caused. renin substrate levels to rise ínto the range founå Ín

pregnancy" The Íncrease in renin substrate d.urÍng pregnaney ïras

confirmed. by PÍckens g!--ge ?gøb), wiro showed. a three* to four*fold Íncrease above normal control values d.urÍ.ng the last trimester of pregnåney.

Geelhoed and" Vanrler (1968) showeü that p)..asma renin aetåvå'ty was eonsistently elevated. d.uring the third. trímester of pregnancy and that the renin levels d.urång l"abour we::e sÌ-gnåfieantly hígher than the

thÍrd. trimester values for the same wÕmene No sågnífåean.t ehange

oeeurreð ðuring parturitårn or for the first week piost partum,

Índ.ieatång that nei.ther the plaeenta n'er the foetu,s ürafi the souree of the elevated. plasma reni.n" The plasma renån aetivity had. returned. to normal by sÍx weeks post pantu$, a mueh longer peråod. than that reported by others (Sfiner, 1965) " 99. Another postulateð e:çlanation of the inereased. actÍ.víty of the reni"n-angiotensin system and. the ínereased- seeretion of ald.os- terene duríng pregnåney is that ít is rel"ated to the high levels of progesterone" rt has been shown (lanð.au g!*gl, 196b) that progesterone has an action antagonístic to that of ald.osterone, probabi"y by a meohani"sm of competítive inhibition at the d"Ístal renal tubule, æå that ad.uiini"stration of progesterone ín physíological amounts to norrnal subjeets is aeeompanåeð" by a natriuresi"s and. fol"l"cwed. by

Íncreaseð mineralocorticoícl aetåvÍ.ty, A elese sorrelati"on between urinary pregnanediol and. ald.osterone seeretåon when measurerd. símultaneously ån pregnant women has beer¡ reported. (Jones €t_-q}, 1959), r¡shereas there Í,s no correla'tion between the seereti"on of these horrnones and. oestrcgens" 0n the basi.s of this evíd.enoe, it has been suggested. that the increasecl aotJ"vS.ty of the renin-angiotensj"n system

Ís neoessary to ac?rieve the retentíon of sodium required. for normal homeostasis in the face of the antagonÍsm by progesterone. ( Íi ) Toxaern&e_ og_qrggïìe$qÏ

Pregnaney toxaeuula Ís a eond.i"ti.on eharar*terized. by fJ"uÍ"cl retention, generalized. oecl.ema, elevated. bloocL press{rre, proteånuria,

åruitabilåtyu head"acheu and somettmes by the eventual" d.evel-opment of eonvulsÍons anil oomao The prominence of both oed.ema and. hSrper* tension Ín the s¡rmdrome of pregnaney toxaemia sugges*ed. a possible rol-e for the reni"n-angiotensin system in this eonðÍtion"

(") Eetablished Toxaem:i"a

Bromr €t_êå (lg6f) fotind. that i.n nene of seven.teen

patÍents with pre-ee3-anrpsåa and h¡pertensi-on ð.i"d. the plasna 1 00"

reni"n eoneentrati.on exeeed. the range found. Ín normal pregnaneye and. plasma renÍn aetivåty was nct hågher in three patients with

toxaeni"a stud.ied by Iifåner (1g6il comparecl with normal pr.'egnaney"

Helmer and Judson Ug6r) four:rd. tl:at renin substrate was not

signlfÍ"cantJ-y d.ifferent ån pregnant wonen with raÍsed" bloocl pressure when compared. wÍth normal pregnaney, but that the

lowest renån eoneentration and. renÍn aetivity Ievels were found. ån the prêsenee of toxae¡nia, However, in toxaemia of pregnancy, another feature, proteínuria, is d.ue to a glomerular lesj"on which is ådentifiabLe (Diecknan pollaek on li"ght -91--a1B lgsrg and. Nettles, 1p6o) and on eLeetron (Rttcfreir-, 1)6t4.i Mautner

1959) mieroseopr, æð whj"ch results tn f"mpaired sod:lum exeretion

(Mc0artney et al, 1g6tþ)" Thus, the more severe the renal 1esíon the more sod.ium retention oceurs and. the greater the d.egree of suppressåon of renin (Brown -gt-gl, 1g6D), rnterpretation of renÍn levels in establishecl toxaemía is complicated. by this possibly over-rj"d.ång faetor"

(u) Reni.n ActÍvitv Pri"or t o Developnsn't of Toxaem:ia

Gordon e!-gt (lgeg), Ín a prospective stud"y, found that mean plasma renin activity in the rn:td.d.le trimeeter was hÍgher j.n ten

üromen who subsequently d.eveS"opeð toxaemj"a than ån l¡B women wlth subsequent uneventful pregnancy" Fo3"l.ow:tng sodium restrietùon and. adrninÍstration of a d.iuretÍc, the pl-asma reni_n aetåvity vaL,ues rose Ín both groups, bnt the d.ifferenÕe "!vas aeoentuated." rt was ¡\," '), 4I '.. ^+:-ilF 'fi \i: pregnaney suggested that the elevateð reni¡r aet$"vå'üy of ís on#¡. \. t.' ' ,i of several aetio1ogi.cal fat:tors Ín the d.eve)"opment of toxaemÍa

cf pregnaney and that th,ose women wÍth more striki,ng elevation

in early pregnancy are mÕpe prone to develop 'toxaemÍa later i"n

pregnaneyo to PL¡,SMA ANGTOTM{STN TT CONCM{TRA,TTONS TN NOT.MAT PREGNANCY

The present stuðy was und.ertaken in orðer to further elarify the role of the renín-angi,otensin sys'tem in pregnancy" Tt was felt that, as all the known effeets of the renÍn*angiotensln system appear to be med"íated. through angiotensån TT, meâsurement of the latter substanee would. give the best indi.eati"on of aetåvity of the system"

îhe stud.y was d.Í.vÍd.ed. Ínto three parts as fol"lows: (u) Meas,urement of plasma angåotensin TT eoneentratj.ons Ín a group of women representÍ-ng alJ" stages of no:ral pregnâney ån orðer

to ileteet any trende wh:i"eh måght be evÍðent, These patients

were aLl, attending the Anten,âtal Clini"e , The Queen Elåzabeth Hospital and. all had" uncompl.åea.ted. pregnancy; in partíoulor,

they had. ns evååenoe e'rf hrypertension, album:inuråao or fluÍ.d retentå.on. (U) A prospeetÌve stud"y of plasma angåotensin TI levels ån a small

group of patients wi.th normal pregna?]ey in or"d.er to eonfírm that

any trend.s sugges'ted by the survey outS"ined ín part (a) abor"e

were valid.ated" by repeateå sampling in the sarne patients"

(") A prelimi"nary study of pLasma angÍotensin TI levels in a small- g:poup e,f patå"en**s with est,ablished. pre-.ee3-amptic toxaemia" 102.

Plasm¿ sarlples from sr.rbjeets Ln all three grÕups were eoLleoted. fron a,nbulant patÈents, between 81 00 a¡r and" 'l x : 00 am" Tkre samples were eolleeted. Ínto ce.¡o1eð s¡rringes containÍng rÐTA and. BAT,, trans- ferreð to coo3.ed" porypropylene tubes and. the plasma separated. as stÕn as praetÍeable" AngÍotensj.n TT ïra_s s:cfp¿otcd" filom the pl-asma by the I'ul1er!s earth methc¡d and. the coneenttation present assayed.

ín the rad.l"oimmunoåssay proeeðure in the usua3. way.

The resul-ts from part, (a) <¡f *he study are shown in grapkic form in Figure 1 $" såx'üy*six patåents with normal pnegnaney uneonrprieateå by toxaemi"a anð repre*sentång a]-l stages of pregnan(3y from six weeks to tenn were inerud.eð. ín tlie study" rt ean be seen from the fi,gure *hat TVo of the values for all ttrree tråmesters of pregnaney are above the range fon nonnaJ-, non-pregnant .$romeno

There is an apparent trend. forthe p1asma angiotensån TT values to be somewhat kigher d"uring the seeond. trimester tkian d.uring the fårst and" thÍrd., and, thi,s i"s sholrn in the everage values for eaeh trimester (rig.l6). when subjeeted te si.ngle eiassification analysis of varåanee, howev'er, ttrt.s d.ifferenee i.n mean values of angiotensj.n rr in the three trÍmesters proved. to be non*sig:nifi.eant (pÞ0"05).

The results of repeated. essay of plasma angí.otensin TT through* out normar pregnaney in the three subjects whe compl-et.eð paæt (b) of the stud.y are shown ån Table g. vaL,ues are el..evat*d in two and hågh normal in the other, anð these hågh leveLs are matntai"neð thr,cughout pregnaney. There Ís some variabål.j"ty but this d.ces no.t fol"low a eonstant pattern. o aa aa aa c 103, aa aa

a a

a

ôl c) a a aa a a z a It- aa aa t l- ot Ø t¿¡ a a o a t a - ¡J- o a tn € v aa u¡ t¿J a

aa = a a

a aa a o a a a

o o o o o o c{ F

FrguRE 15

Plasnra angiotensin II vaLues d-uríng norrnaL pregnancy ¡d F Þ co 20o/

Þ a s ûq ¡å a o o cl- a o Ë a It }J- a a O' P L a ASMA a H É a a a H q)H a aaa a |.J. d a Þ Þd roo a aa .1" a E A ll Ë a o a 3t r-J o\ ips. nt.) aa / aa a a p a a t o H a a a a rld I o a tsJ a o o a a æ o o 5 O þ a 5 I a qo a a a to o

o f 1 05,

"îsÐåg_-5_

Pt" ti th:roushout nreananev {n *hpeo sub:i ee w{ th nn¡+mnl . Ðresna.nev

Subjeet Weeks of Gestati"on Plasma Angiotensin TT

I 48 pe/ml" 12 l+z tt A 16 6zn 24 7A rt 32 4t rr

I 95 ps/nr 16 Bl* It 3 2i+ 121 rt 32 100 fr ¿r"0 72 r1

10 6S il 14 79 It c 18 9U tc 26 7t ¡t 36 80 It 1A6, The resr.rlts of tkrå.s stud.y are consÍstent with the fåndings of ínoreased. activity of the rerz5.,n*angåotensin system d.uríng normal

pregnancy in previous stuðies measuring pS.asma renin aetivity, plasma renin coneentration, and renin substrate. The finding of inereased. plasma angÍotensÍn rT val"ues throughout pregnancy is consistent ïd.th

the renÍn activity d.ata, alth

reporteð by some authors (Genest -e!*_Al, 1g6DE Vriiner, 1g6il, The increase in plasma angåertensin rr oeeurs early in the course of pregnarley and. was d-emonstrable as early as ti:e seventh week tn the present stud.y. The inereased. angiotensi-n rT levels d.uri"ng pregnaney cou1d. possíbly be d.ue to the ínereased. stimr¡lation of al'd_osterone prod.uction requíred. for sod.ium homeostasÍs in the preser¡ce of the antagonism of the perå,pheral. effeet of ard.osterone by the hígh levels of progesterone (tandau e!_aå., 1g6r), or could be due to an inappropriate inerease sescrnd.ary to the raÍsed. 1evels of renÍn substrate anrt faÍluro ta suppress renån seerotÍ,or:.

5" ANGTOTg\lSTN CÙNCHVTRATTCINS TN PRE-EC T,AMPTTC TOXANTúIA

rn part (e) of the stud"y outlined" previouslyn plasma sarnples were obtaíned. frorn si"x women wåth establíshed. toxaemÍa of pregnaney as eviðenoeå by a blood" pressure readÍ.ng of grea'ter than iJaþa mn

Hg together wåth pro'teinuria anð"/or fluÍd. retenti.on. The pS.asma angiotensÍn II values in thi,s grÕup otf pa.båents were not significantly d.ifferent from those ef normal pregnaney (f:"g.t 6). 107. The fånd.Íng that plasma angiotensj.n II i"s not signifï.cantly

higher in pregnant women wåth establåshed. toxaemfa comparecl. wåth nom¡al pregnâ.ncy ís consístent wåth slmi"lar reports regard.tng rentn values (Arown g[-g!, t965; T[åner, lg65; Hetmer and Judsono 1j6f)" lïhile thÍs result would seen to ånd;icate that the renin*angiotensån system Ís not coneerreeê with the d.evelopment of toxaenÍa of pregnaney, ít is possLble that these values are in faet inappropriately high in the presenee of increased sodium retentåon (Davey -gt_gl, 1951). Before a rol"e for the renÍn*angiotensin system in the s¡md.rome of

toxaemia can be compl"etely ðåscounted., further stud.ies embod"yÍng careful sod.íum barance d-ata wil"]" be required.. This is, of course,

d.iffieult or impossfble to aehieve since toxaemÍa is usually recogniaed. only onee it is fu1ly d.eveLoped." Prospective studies sÍn:ilar to that of Gord.on et al ?geg) but somrneneing just prior to the onset of toxaemÍa would. be neeessary in ord.er to provÍd.e an answer to thi-s problem. 108.

CI{APTER 7

S}MEP STUDIES

1O æTNTRODTICfION 2o ç4Ìtr(lI,aîIoN 0F REI\IAL TTMPHATICS

3" -, AND ANGIoîENSIN II C0NCEImRATÏON r

4 RENïN AND ANG|Ï0T-ENSIN rr I¡ErrEts rN SHEEP FoLtOrENG soDTUM -DEPTETTON

5. mFECr 0F MER.CURLC CHLORIDE INFUSToN 0N PLASMA AND tyMpH RENTN AND ANGTOTENSTN TT

6 -EFFECT OF CHLORTDE ON ON REIVAI

7, IF'FECT 0F MIRCIEIC CIILCIRIDE INFUSIoN 0N TIRTNARY RENIN tuCONCH\ITRATION B. DISCUSSToN 1 09.

CHAPTTR. 7

STMEP STUDIES

The stud.ies d.eserÍ,bed. ín th5"s ehapter were carrÍeð sut in

eollaboration vr¡:i"th Dro GoHo MeTntosh, from the C.S.T.R.0. Division

of NutrttÍonal Bf.oehemistry, Kintore Ave., Ad.elaid.e, who performeð

the surglcal proeedures d.eseribed..

1 O ÏNTRODUCTTON

The use of fine plastie eannulae for the cellecti"on of l¡rmph i"n

conseious rats was i"ntroduced by Bollman g[_gl (t?ug). These

technf.ques afford.eå rnarry ad.vantages over the prevÍously used. glass

eannulae and, were adapted" to the sheep by Lascel.les anð Morris (lg6l),

who extend.ed. the seope of these preparations to enable lynph to be eol-leeted. from a wÍde variety of organs. Lever and. Peart ?gaz) showed the presenoe of a pressor materÍa1 in renal lyrnph from anaesthetiøed dogs. The material was not

detectable in l¡rmph d.erived. from other organs and was showr: by

båoehernical and" pharmaeologiea3- rnethod.s to be ind.ístinguishable from

ð.og renÍn. constrietÍcn of one renal artery 1ed. to an inareased.

coneentration of thís materÍal in the l¡rrnph from the åsehaemíe kåd"ney.

HÍggÍns qt-g& Ug6lr) ðemonstrat,eð tn thoraeie d"uct 1¡rmph of

åogs vnith second.ary h¡perald.osteronåsm d.ue to thoraej"e inferÍor ver.ra oavå constriotåon the presenee cf irrereased. pressor and. al,d.osterone* stimulating aetívitÍes eompareð with normal dogs, A marked. d-ecrease or d.isappearenee of the renån-låke aetivity from thoracie d.uet 1¡nnph 110, luas prod.uced by nephreetony, Índ"icatíng a renal origin of the material. the results were thought to Índ.icate inereased rel.ease of renin from the kidney into renal lynph of dogs wíth second.ary h¡ryeraldosteronisn. lhe relatÍ.ve Ímportance of thÍs route of renin secretion into the blood" stream has been d.i.ffícurt to assess because of the difficulties of quantitating l"Jrmph flow and of rneasuring small arterío-venous d-ífferences in the enz¡rme concentration across the

kid'ney by currently avaiLable, r'elatively insensitÍ-ve assay method.s.

Ïnvestígation of the intra-renal handling of renj-n could. be expected.

to help clarify the situatÍon. Rappeli and" peart (rggs) investigated.

the renal" excretton of renin ínfused Íntravenously Ín rats. The

ad.ministratíon of both mercuric chlorLd.e and maLeÍc acid., agents wÍth a.:r ectl"on at the renal- tubular level, l-ed to a sixfol_d. increase ín the amount of renin appearíng Ín the urine. fhe authors felt

that the most l"ikely erplanation for this result nr&s a d.eereased. renal tubular reabsorption of renín fol-lowing glomerular flltratÍon. Lumbers and Skinner (t 969) descrÍbecl urinary renÍn concentrations in hurnan subjects whlch nrere epproximately ï, of plasma levels.

Consíðerebl"e varíatíon in renal output and" clearance of renin was found. but levels remai.ned. simirar over several months in any one subject. Natrí.uretic adminlstration l"ed to ftvefold. elevations of plasma renin, twofold. Íncrease l-n renín exeretl"on, and causeð renin eLearancs to fall to 47Á of control vaLuee. The authors suggested. that rerrin wå,s filtered. at the gJ"omerulus e¡,nd sel-ectively reabsorbed. 111 . 0n the basås of the above evid"enee i"t seens reasonably eertain

that renin ís fÍltered at the glomerulus and. subsequently parti-a11y

reabsorbed by the renal tubuleso However, the orígin of the reni.n

present in renal lymph fs uncertaÍn, The possible sources of rymph renin arec (r) direet access to l-¡rmph from plasma; (b) access to

lymph foLlowing tubular reabsorption of filterecL renin; and (c) secretåon of renin Lnto the renal interstitial tissue fron the

juxtaglomerular regíon and thence into lymph. The present stud.íes were und,ertaken Ín an attempt to clarify the intrarenal hand"llng of renin, to id.entify the source of the enz¡rme in l¡mph, and to

d.eterrnine whether the cond.iti.ons were such (*.g" renin substrate and.

convertlng enzJrme avaÍlabte) that angiotensin Tr coulð" be prod.uced. localJ-y ln the kÍ"dney. ThÍs woul"d have ímportant impl-icatÍons in lntrarenal regulation of blood. fLow and sodi"un reabsorption (lhurau , 196¿+).

2N CANNUT,A TON OF RUVAË LYMPHATTCS

An area of skin over the flank of the sheep was clípped. of wool- and sterilized." An ÍncåsÍon ïvas made below the lateral proeesses of the lunrbar vertebrae para13.e1 to the spåne and. the kíd.ney vras approached. and- mobílÍzed. retroperitoneally, The efferent l¡rmphatics were id.entified and. followed" toward.s the renal hÍlus, where one or two read.íly aecessible l¡rmphatics were prepareð for cannulation whíle the others nrere ti"ed. off. The number of renal l¡mrphaties usually varied. from two to six. One or two l¡rmphaties were caïlnulated with polyethylene tubing (usually 0.55 mm ï.0", 112 " 0.90 mqr 0.Ð. ) at a point about 2 to j cm from the hilus of the kidney.

The ea^nnulae were tied" fnto plaee, anchored. wtth stay sutures, and. brought out through the psoas major muscle above the line of incísion.

3, AND RTNAT r.m[ïN c ACTIVTTY ¡"ND

RenaL lymph was col"leetect from normal sheep after cannulation

of renal hilar lyraphatåes as descrtbed. above. tymph and blood

samples were coLl"ected" simultaneously Ínto cool-ed. pol¡rpropylene

tubes contai.ning rDrA and" 3AL" samples nere assayed. for plasma

renin activS"ty, plasma renin eoneentration, and renÍn substrate by

the raethod. of skínner (t 967), and for angiotensin Tr by rad"ioimmuno-

assay. lhe strueture of sheep angiotensÍn rr has been shown to díffer from human angiotensín rT only at the posítion of the fífth amÍno acÍd, where valine is present instead. of the ísoleucine of the huuran forrn (cai.n g!é, 1g7o). The antÍ.bod.y used for the radio-

Ímmunoassay was shown to have id.entåcaL affinity for these two forms of angiotensín If.

Results of the stud-y are shovm in Table 6. fhey may be surnma^rized. as fol-l"owsc

(t ) Lnnph renin eoneentration was consistentl-y higher than

plasma renin eoncentratíon (6*16 timee as htgh, mean

12 t5.me$.

(z) Renùn aetivj"ty was signÍ.fåcantly hígher i.n renal l¡nnph than in plasma but the d.ífferenee vÍas less markeå than in the

ease of renin eonoentration values (J&*B times as hågh, mean g tåmes) " 113.

rA3T,E 6

Renåra Renin Renin .4, ïï Aet5"vÍty Coneentratåon Substrate Coneentration Ge/tom¿,/5nrs) @s/toosllÆhr"s) (ne/mL) (pel*r)

Jugular Pl"asna t+64 2s7+.5 1 0l+ 1 Renal Lynph 1 66Ì+ /+2 r¿¡00 98

JuguS"ar Plasma 112 t+TO Renal" t¡rurph 5V5 7 1625 25 2 Jugul"ar Pl"asna 157 66a Renal" Lymph 778 7,670 21

Jugular Pl"asma &a 186 Renal Ly¿rph 2 r51O 135 3

Jugul.ar Plasna 3¡+5 1 ,125 55 9 4 Renal" Tr¡rorph 2 1607 6,gua ,+O 21 9

Jugular P1asma 43o B7o 1 73 11 5 Renal Lprph 1 t 9ll"0 9 t 3t+o 1 6l+ 58 11l,+. ß) Renin subetrate was slÍghtly lower Ín renal lymph than Ín

plasma (mean 10) ng/n\ vs. mean 1'JO ng/ni*).

(+) Anglotensín fT coneentrati.on was nuch higher in relal lymph

than ln pJ-asma on the two occasions on which they were compared", Pl-asma Ievels were comparabre wÍth those reported. by Cain et_al (lgZO) (tr+ g tt (S.D. ) pa/*a), These results confirn the prevåous report of hÍgher renin eoncentration in renal 1¡¡nph than in plasma (Mctntosh and Momis, 1971). The hÍgher renin activíty values in renal ly¡nph than in

plasna ínðieate the presence of ad.equate renin substrate in the lymph eompartment, which was confi.rrned. by measurement of substrate"

The observation that the d.ifferenee in l¡rnph:plasma ratfo was not as rnarked. as in the ease of renån coneentration could. be e:çlaíned. by the lower eoncentration of substrate in J"¡rmph compared" with plasna, a situatíon which was not unexpected- Ín view of the lower proteÍn coneentration in lymph than in plasma (Mcrntosh and Morrís, t96t).

The values obtaíned. for renÍn substrate in jugular venous plasma, how- ever, were lower than those prevÍ"ously reported. i-n normar sheep. This find.ing Í"s probably explaåned by the demonstration that the renal renin useð ån the assay of the renín substrate was not entirely free of angiotensånaee actåvity" The final value was thus the result of generation of angiotensin together with breakd.own of a fraction of this generated" an8Íotensin. The substrate fj"gures probably ind.icate the general trend" eorreetly, but are not aeeurate in terms of absolute values. 115 " rhe d.eraonstratÍ.on of angåotensin Tr in renal- 1¡mph by speeÍftc

rad"ioimmunoassay suggests the presenee of convertång enz¡rme in l¡¡mph or ínterstÍtial tissuei orr alternatåveLy, the angiotensÍn rr may ga:in access to the lymph after it has al-read.y been formed.

1 251-trb*l1ed. This possibÍ"Ltty was Ínvestigated by f"nfusion of angÍo- tensín rr into the renal artery of a sheep for one hour and colleeting urine and. renal l)mrph. lhe renal lymph, urine, and" plasma samples were eounted. in a well-t¡rpe counter and. the followÍ.ng results were obtaLned.c

Cg¡qL¡/190 sec,/ml

Renal artery plasma (at terni.nation of 37,62O perfusÍon) Renal vein plasma (at termínation of perfusíon ) 37,o3o

Renal l¡mph Iil+'55o

Urine 522,57O

Ït ean be seen that there was a smaI1 arterio-venous d.ifference across the kíd.ney, that the coneentrati"on of counts was Lr:igher in renal lymph than in prasma, and. that a great d.eal of rad.ioaetivity was present in the uråne. rn order to aseertain whether the rad.ioactivity represented. intaet angiotensin TT, sampS-es of urine and renal lymph were incubateil with an exeess of angÍ"otensin II anti* bod.y and" then subjecteð" to gel fl"ltration on a G25 sephadex column. The results are shown in Fígure "l/. Tn this system, intaet angiotensín tr is bound to antibod.y and. appears in the voåô volume, whereas fragments of angiotensín rr with less j-rnmuno-reactÍvity are not bound. to the tt 6.

lo,ooo LYMPH

ó,ooo

C. / 100 sec. / ml.

o 2,OOO o

o

too,ooo URINE

óo,ooo

C. / tOO sec. / ml.

2O,OOO

o o 5to 15 20 2t

F RAC T ION NUMBER

FTGURE lJ

Gel filtration of uríne and. l¡nnph containing labe11ed. peptíd.e after incubation wíth angiotensin fI antibod.y 117 " same extent" ït can be seen that, i.n the case of renal lJrorph, a small

fraction of the raclioaetivity appeared Ín the voici v,olume (approxåmately 1fft) whereas almost none of the radÍoaetåvity present in urine demonstratecl persístent imnunoreactivíty. It was concludecl fron this e:rportroent that the urÍnary radÍoacttvity represented completely tLenatured. fragments" The renaL lymph contaåned" a snall fraction of the radioactivÍ"ty wÍth intaet åmmunoreaetívity, but at least part of this could be aecounted. for by the ability of fragments to retain theÍr reactivíty with antåbocly, ft was conelud.ed that the contribution

to l¡moph angíotensin rr concentratÍon maðe by peptide gaÍning

d.irect aoeess in this manner woul-d. be slight, l+. REîfrN AND ANGrqTElrSrN rr LEITELS rN SHEEP FOLL0ti[rNG S0DilJM DTFLETTON

sod.åum d.epletion was ind.uced. ån three sheep by productíon of a parotíd. fÍstula" Plasma and renal lymph samples were collected" and. assayed. for renin coneentration, renin activity, and. angiotensin Tr concentration. The effect of sod.ium d.epletion on these parameters is shown in Table 7. rt can be eeen that there rvas an increase in both plasma and. lymph renin aetivíty, renin eoneentratåon, and. angi.o- tensin II concentratj.on fo}3"owång sod.åum d.ep1etÍon but r:o eonsístent ehanges ån renin substrate. îhe changes are as would. be e:çeeteð. follomlng the stånuration of renLn prottuctåon by the kÍdrrey consêquent upon sodium d.epletåon. 118"

r,Aqrp*-7

Renin Renån Reni"n A IT Aetivity Coneentration Substrate Coneen'traticn nel oomlr/5hrs ne/taaù/lws ns/nr pa/nr

4q_øe (Parotåd. åraÍ"nage commenl:eð. B/6ftA) Jugular plasma '112 ¡+'7o n7^ '7 '"' Renal l.ymph 575 u6z5 25 -,n'" ¡6 JugttS-ar plasma x57 66t Renal lymph 778 7,6'7a 21 ,n ¡" Jugular plasma +t¿ tr ,214 ' '/' Renal l¡rmph 723 9,2gA 169

Jugular plasma 6aì+ "l ,&CI0 12/6 .1 Renal- l.ymph !9 2x5 'tr6,6öCI

A8:026 (Farot3"d. d"rainage commeneeû z/z/ZA) Renaf. lyrnph 36a 3,256 82 J/2 Ju6u1-ar plasma 3o5 1 ,27O x "50 Rerel vein plasma 2BX 1 ,'170 153 Renal Lymph ,þrhB0 19,1'-70 5/2 27 Jugular plasma 2 r27o 3,6bo 52

AB-j"41 (Parotåd d.raånage eommenseeL lO/l/lO)

Renal lymph 2 9 6a7 6,9tço ,+0 219 S/1 JuguT-ar pïasma 5U5 tr ,125 55 O RenålveÍn plæma 365 11254 27

Renal" 3.ymph 3 t 760 9U 9/1 Jugular p3-asma 5BV Rcm-al vexin pLasma 522 1t4/t R"enal J"ymph 4Ð096 21/1 Jugular p3-asma 1 ,326 u6 16 22/X Rena3. lynph 3,o7a 20 B6 Jugular pl.asma 1 ¿'r/oz /¿ | r'fIçCl 6reCIo 22 5o &enel I¡nnph x eTou 15,76A 21 'tr ,t|"tr 119. tr T trRïc clil ÏON ON ¡,ND TYMPH

ïn an attempt to clarå.fy further the intrarenal hanð.ling of renin anil to assess the eontribution of reni"n reabsorbed. from the renal tubures to the renal lymph renin, mereurfe chrorid.e was admÍnÍstered. to three sheep in ord.er to assess the effeet of a tubular blockfng agent on plasmau lFph and urínary renín, and. pLasma and. 1"¡nnph angiotensin II.

lhe results from these three sheep are shown in Figures 18, 19 anð. 20.

A8-521 the d.ose of mereuríc chlorid.e given in this case was

too large (t em) and the ani¡nal quickly developed toxic effects and d"ied. wi.thin 2l- hours. The results

obtained. prior to death are shov¡n in Figure 1g,

ït can be seen that during the short period Ín whÍch observations Tvere mad.e, there was an inerease ån renÍn activÍty, renín concentratÍon, anð angiotensin II

coneentration ín both plasma and. lJmph, the changes

being of relatÍvery greater magnitud.e ån renal 1ymph.

AB-509 This animal survÍved much longer foll.owÍ"ng ad.minås-

tration of a lower d.ose of mereurie chloriau (Tr mg of mereuric ion), FollowÍng the infusion, all parameters showed- a prÕgressíve rÍse, again with relatívely

greater changes in renal lymph than in plasrna, exeept

for the varues on the fifth d.ay after infusíon when there ïvaÊ a sharp decrease i4 all three parameters 120.

400 o-o PLASMA l-¡ LYMPH

Ail 200 (pg. / nt.l .f.

o

3O,OOO 2,460

20,ooo P. R.C. hs./loo ml./3 hrs.) to,ooo

otr o

óooo

4000 P. R. A. hs. I too ml./ 3 hrs.) 2000

o -l o I 2 + INFUSION

T tME ( DAYS )

FTGURE 1B

Plasma and lymph Aff, PRC and. PllA followíng ad-mínistration of mercuric chloride. 121 , óoo o-tr PLASMA t-¡ TYMPH Ail 400 1on. / nt.l 200 a¿t-

o

t5,ooo 24,85o

lo,ooo P. R. C. hs./too ml./3 hrs.) '\ ./' 5,OOO \¡

g-.,,,,.d-g-tJ-'- o

óooo

P. R. A. 4000 hs./loo mt./3 hrs.) 2000

o -l o I 3 5 7 + INFUSION TtME (DAYS)

FTGURE 19

Plasma and. 1¡rmph Aïï, PRC anð PRA following adminÍstration of mercuri-c chlorid"e, 122, óoo tóoo t530

Ail 400 1rn. / nt.l 200 7" o tr-o PTASMA t-l LYMPH t5,ooo

to,ooo P. R.C. (ns./ loo ml./ g hrs.) 5,OOO

o ,o5o

trt' óooo t4,3t5

4000 P. R. A. ("g./ loo ml./ 3 hrs.) 2000

o -t o I 3 5 + INFUSION TIME (DAYS) FTßURE 20

Plasma and 1¡nnph AII, PRC anð PRÂ follow"ing ad"ministration of mercuric chlorÍde. 123.

meesured. ån 1¡rmph wh:i1e the plasma values continued. to ise or remained- stationary"

A8*51! The response seen in thi"s ani"mal given a smaller ð.ose stí1l

(Ø *g of mercuric ion) was d"Ífferent from that seen ín the other two. lhe plasma values of renin actÍvity, renín

concentration, and. angÍ"otensån If concentration rose foLlow-

ing ínfusion and. remained. high" In renal lymph, on the

other hand"o all three parameters showed an initial decrease

lastíng one to three d.ays followed. by a mod.est later rise I to levels which signÍficantly exceed.ed. the pre-infusion levels only ån the case of renín concentrati.on, Al"1 three animals gíven mercurie chlorí,d.e suffered. severe scouring wåth narked loss of flui-cL and" eleotrol¡rbes from the gastro-i.ntestínal tract and. d"eveloped. acute anuria wíthån 24 hours. This fluid. and eleetrolyte loss would. be expeeted. itself to aet as a stimulus to renÍn secretion" 6 " m'rncr QI Mq.c-IErc cruoRrDE TNFUSToN 0N Rmr.A,L tynmn Ftotï RATE ANp REATAT Hrsg0tgq:I

Following the infusion of mereuråc ehl"orÍd.e, there ïras an initåal inerease i.n urínary output ån each anÍma1 stud-ied.. Thi-s increase, however, was of short d-uration and. was followed. by complete renal shutd.own within 2l¡ hours ån each instance. The renal lymph flow rates remaíned at the control levels d.urin6 th:is stage of d.iuresis but rose to signifÍeantly elevated. values after renal shutd.own haå ensued., The changes in lymph flow rates are shown ín Table B. 121+.

rÆT,E 8

Day of Stuclv L.mplg Flow R.ate (mL r\ å.8-509 L8-519

-1 1,3 1 "7 0 MerourLc Chloråcl.e Infusion

1 2"1

2 25"5 2.O

l+ 33"o

5 33"O 35"O

7 19 "2 125.

Renal håstology was eheckeð in each animal after death. ït was found- that the mercurie ehlorl"de infusion had. prod.uceð severe d-isruptíon of the renal archi.tecture ån each ease, ïråth particularly severe effects on the renal tubul"es, which showeð complete coagulative necrosis. The interstltial tissue space was marked.ly Íncreased., an

effeet consistent with the inereased 1¡æph flow rates observed.

7. mFSCT 0F MmCüRrC CHL0RTD N CONCAITTRATïoN ïn two of the sheep given mereuric chl-orÍd.e Ínfusion, hourly uríne collections rvere mad"e over the first såx hours following comnencenent

of the infusíon, Íhe uríne samples were eoneentrated. by vacuum d.iaLysÍ"s and. the concentrated. samples ïuere submÍtted, to the Skinner (lg6l) method. for determínation of renin concentration, the angio- tensin generated. from sheep substrate being measured. in the rat bíoassay system. In one instanee (¡.9*çOg) the prepared sarnples were found to consístently give d.epressor responËes in the bioassay so that no values for renin concentration could. be obtaÍned.. In the other animal (¡,4*¡t !) values for urinary renin concentration were as follsws: U 1 386 ng/ 1Oo $tL/ ] hours tJZ 798" rr rr IJ3 X63" n n Il 4 1191 ¡r tt rr u5 3326" ' i'

tJ 6 51I+ u It rr fhe results show that there rvas an åncrease ín the urinary renin eoncenttration, reachÍ"ng a peak value in the fÍfth hour after commenee* ment of the ¡oereurÍe chlorid.e i.nfusion. x26.

B. DTSCUSSION

These stud.ies have confårmed" the previoirs observati.on (McIntosh and. Morrj"s, 1jll) ttrat the coneentratåon of renÍn in renal lymph is higher than in plasma" They have also d.emonstra'ted. that the renín actåvity and. angiotensin fI levels in renal J"¡rmph are hígher than the correspond.ing plasnra figures. The renin substrate results were inconclusÍve, but there ïvas a suggestion that the levele were slåghtly lower Ín renal lynph than Í.n plasma.

rhe response of reni"n eoneentration, renin aetivity, and. angio- tensin rr eoneentration ín both plasma and. renar lyrnph to sod.ium d.epletion was eonsistent with increased. seeretion of renin from the kåd.ney in response to this stå.mulus, all parameters showÍng a similar d.egree of elevatÍon. the adnÍnistratíon of the renal tubular toxin mereuric ehloride led. to j"nereases in plasma reni.n and angiotensin IT in all cases but changes i.n renal lymph whi"ch were vari"able and. passibly dose- d.epend.ent"

l,he interpretati"on af these resul-ts presents a riurnber of d.iffÍeulties" The para1lel ånereases in renin aetivity, renin concentratíon, and. angiotensin rT e,¡neen'tration in plasma and. L¡rmph would. be consùstent with an orågin of s"enån from any of the postuS-ated. sources and. this would apply al-so to the situatj"on of d"Íetary sod.ium d.epletian where a1,1 parame'ters showeü similar ehanges. The labelled. angiotensin rr ån.fusion experiment suggested. that clireet aeeess of íntact angÍotensir¡ rr ta renal tymph from p]-asma ïras rrrilåkely. 127,

A eontråbuti<¡n to renal ly¡rph renín from tubular reabsorption could.

not be eompl.etely exclud.od but thi.s ís un1åke1y to be of any magnitude for the following reåsonsr (a) a:"oetcing of tubul*ar reabsorption of renín with nereuríc chl.orÍó.e 1ed. to an increase in urinary renín but

the lynph renin concentration rose over this períod., ana (b) inereased. levels of renin were seen in the renal lyrnph after conplete cessation of renal flow had oeeurred..

ït seems likely that the renín in renal r¡nnph gains aecess to this compartnent through d.ireet seeretå.on into the renal ånterstitium

from the juxtaglomes"ular region" To explain the d.issoeÍatÍon between plasma and- lynph values of renån and. angiotensån Tr forlowång mereurÍe chlori"d.e Ínfusåon i.n one animal and. on one ooeåsÍon in another, åt is neoessery to postulate a d.ifferential effeet on the seeretíon

of renin Í.nto the rer¡a1 in'terstitial tissue and. the route of

seoretåon into plasma. ThÍs d.issocåation can be partly accounted.

for by the increa,sed. l¡rnrph flow seen after the mercuríc ehlori.d.e i.nfusion, fi,nw rates íncreasíng up to 1g tímes. This ís not the whole expl.anatíon, holuevere as si"mÍrar j-nereases in flow ra.tes were

seen ín all animals d"espite the varåa'bion in restil.ts" fhe meraurie chl-oråd"e led" to eonsÍ.ðerable ð"å,sruptÍoy: erf the renal arehi.teoture, wÍth partå.cular3.y severÕ effeets on'the renal tubur-es, and ít is possÍble that a seleetÍve impaírment of renin seeretion into .the renal interetitÍ,um could. have oeeurred"" An alternatíve explanatton would. be that the severe 'ùoxi.c ehanges eoulå lead. to oed.ema and ischaemia of the kååney; if the renal blood. flow vrere J-ow enough, a greater pereentage of the renÍn seoreted. could. gaån aecess to the lynph, 128. This would l"eail to lr:igher lymph vaLues und.er eonclitÍons of greater

renal d"arnage and. lower values when the d.amage was less severe. However, the higher val-ues ån l¡nmph than in plasma under norr¡al cond.itíons Ls evid.ence agaÍnst thi"s h¡¡pothesis. Ïn view of the demonstration that plasna renin eoncentration can rise to irigh levels while the renån eoncentration in renal lJttnph Ís very low, a sågnifteant eontribution to the plasma renån from thts sol¡res seems unlikel-y. Further e:rperiments using smaLler d.oses of mercuric chl"oråd.e mÍght help to elarÍfy some of these poÍ.nts, 129.

CTTAPTM. 8

DTSOUSSTON

The j"nåtial respc,:rse 'to t?:e åemonstnetj"ein of a potent pressor

subs'tanee of renal orågÍn and. the e:eperimental prcd.ue'tion of h¡rper-

tensåon rese¡¡rbli"ng eJ"ånÍ.cal h¡pertensÍv'e d.Ísease ån man was optímåsm

that f'urther investigatå.,en ,of the rentn-angÍotensÍn system wourd provÍd.e anslrer,$ to many of the questi.orrs regard"f"ng the elånieal sltuatåon. Further i"nvesti"gatf,onu however, revealed" that, altheiugh the renån:angiotensÍn system was probably åmportant in the aetåo]"ogy of renovascÈ;rlar h¡rpertensåsn and in nost eases of malignanb or aecelerated. h¡rpertension, åts role ån the a,etiology

of essentåal h¡pertensj-ein was by no meå,ns olear" Meas¡.:rements of aettvtty of the renåsl-angÍotensÍ,n system using methoð.s avaålabf.e up to the present have not consåstently shown any Ìr,ereaseå aetívíty in subjeets with essentåaL. h¡pertension when eompared. wåth normal sub jeets.

A nole for the rer:in-angiotensin system in the aetåology of essentåa1 h¡rpentensåcn c¿nn,at be eompletely ði.seounted", h,owever, for i"t may be possåb1.e that tkre rrnormâ}r l{.e.we].s fou¡:d. j.n essentåal h¡rpertension are Ínappr,epriately high fn the fae,e ef ir:c¡r'eased. avj"d.åty of sodiun reten*i.en Õr ÊÕffie sther faotor whi,oh woulð. no::n,alJ"y suppresõ the ar:tj"vity ef, the system. Tn .th_is eontext, "ùhe substanti.al group (appr.-erxåmat.el.y zúrt) ú pa,tients with essential- h¡rpertension and" suppr"e*qsed prasrna ren.{.c.r ae.tivlty are of interes,t 13Q " (cnar¡¡rt"etc st-gå, rg6g)1 9nð å't has been sugges,üerl tlrat son¡e as yet unknow:n nlr¡eroloeortåe.ef.å n:T"ght be pree,ent ån exoe,ss in sueh patåents (Vloods sË_el, 1969)"

rhere is also a possåbÍlity that plasma revels of renín and angiotensin d.o not reflect the coneentrations prenent at the reeeptor sÍtes of tnportanee for the aetåein of thes,e substances, anil stu¿Íes

of the tissue btnd"Íng ot' angiotensin may provide some of the &nswers to this prerblem (ßoodfrienå and Lín, ..1g70)"

ïnvestågatiens of the role of the renån*angiotensin system in normal sod.Íum and. water homeostasin have shown that angiotensin aets

ao a trophåe Liormor¡e for the release of ald.e¡steror¡e from the ad.renal eorfex, as ïreL,l as prod.ueing vaso*t:nstråetton and havÍ.ng a probably rninor d"Íreet effeet on renar tu.bul-ax" f.t;notiori, anå Ín ad.d.ition has a

conplex but possibly important effeet er¿ the autonorn-i* nervous system" The control of aLðesterone seeretåon d.epend.s not only on the angio* tensi"n TI eoncentration in plasma but also on the coReentratÍons of ACTH, po*a.ssf,um, sodium, and. probabL)' cther as yet un:identÍfied. fai:tors

(ltair:west €t-éå¡ {gzo)" lhere ås th¡;¿s a e,s:np.tex homeostatie meehanism for sod"åt-¿m ard water, íu.volvång the above faetors as well ae the Antid.iuretåe Hormer.r¡,e meehar:åsin"

Me'thod.s of assesså.r:g the aetå.vå'ty e¡f the rentn*engiertensín system up t,e the present ti.me ha've not been eomplet*ly sra.tåsfaetory.

Measurements af alðos'terone exerr*'Licn or aïðosterene seeretion ra.tes ean be maðe wi"th eonsid.erablc ae.luråoy using d.ouble S"sotopc method.s but, as the seoreti.on of af.d.e,sterono i"s affe,r.ted. by ether faeters, these 131 , measurements d-o not neeessarily refl-ect the actåvity of the renln-angÍo* tensín system, Method.s previously avaíl-able for the assay of angic* tensl"n in blood. were not satj"sfaotory beoa'use of the large volumes of blood. required. and. the laek of sensitivíty, which meant that values in no:ma1 subjeets eouId. often not be measurecl. Plasma renin activity and reni"n eoncentration method.s enabled. much useful Ínformatíon to be obtaLned but both are relatívely nor:-speeifås and both have the dis- ad.vantage of a final bioassay step to measure the amount of angiotensj-n generated., thus íntrod.ueing the inaeeuracies inherent in thís t¡pe of estÍmatåon"

FollowÍng the introð.uetåen of the radí*immurnoa"ssay teehntque for measurement of peptåd"e hormos¡es (Yat,ow and" Berson u "t)J6) and. the demonstration of antibodåes to angÍotensån (Deodhar, ng6!), the applieatåon of thås method. to the assay of angiotensin in blood. seemeð a 1eg5"ca1 d.eveS.opment, in the hope of aclvåeving Í"ncreasecl sensÍtivity and. speeifictty, the rad.ioimmu¿noassay proeeðure prcmiseð. these ad.vantages as wel.l" as ease anrl" så,mpIÍ.eåty of hand"l-Íng of large numbers of samples. Applicatåon ofl the radi"r¡åmmurroassay proeedure to the measurement of pS"asma angiotensån TT conoentratiilr¡s has certainly provid.ed. i"nereased. sensitåvå'by, so that angi.oter:sin ïT eoneeætratiens ean be assayed. with relatåve ease in normal subje,+ts" There are, however, potential d-åsad,vantages whieh necessi.tate earefuS teohnique and. striet a'ttentåon to eontrcl prooed.ures iin order to avcåd. erroneous results, 132"

The firet major problem ås that ef specifícÍty. Cain et_gl Ugeg) demonstratecl that antibodies to angl,otensi,n II díffer in their ability to reaet with biologically inactÍve fragments of angiotensin fT, thus raisÍng the possibi.lity of d.isparity betwaen bioassay and. radioimmuno- assay resul-ts" These authors found. that the largest fraetion of the angiotensín rr measureil in venous plasma was in the form of the biologically inactÍ"ve hexapeptid.e, but that the levels correlated reasonably wel3- with arteråa1 levels and. eould. be used. for routine elinicaL purposes" lhe antibod,y usecl in the present stud"y has been shown to cross-reaet wíth biol-ogÍcally inactive fragments but the extent of the cross-reactåon has not been charaoterized. exaetly,

Although part of the angiotensin IT ¡neasurecl in venous plasma clurång these stud.ies may have been ín the form of inaetíve fragments, it was felt that the assay gave e val-td. measure of the aetivíty of the renin-angiotensin system" lhe results obtained. usíng thi.s antibod.y were eomparable with those reported. by other groups (Boyd e-!__ê1, 196Tt

Catt et al, 1967; Page e3_ê1., 1969; Sunðsfjorð,, 1g7O). Non-speetfie effects of pl"asma extraets on the bind.ing of labelled angiotensi.n rr by antåbody presen't a problem" Tt is neeessary for the samples to be assayed. and. the stenð.ard- ou1"¡re reaetj.on nixtures to have as nearly identieal eonståtution as possible, sÍnoe non- specifie offe,ots on antit¡od.y bind"ing rnÍght be ånterpreteð as d.ísplaeement of l"abelled. peptid.e by angiotensin II present in .thre samples.

Ïfihen pl"asma sampJ-e,s are assayeð d.ireeibly, the problem can be partÍally overcome by making up the standard.s in anephrie plasma so that berth 133 " stand.ard. eumle sampS.es and. sa"mpl"es to be assayeå eontaÍn the sa^me ameun.t

of pl.asma" Thís s,sl"'u;tåon is not entåt"ely satåsfaetory as the anephrto

plasma and. the san'ç]"e to be assayed. may d.iffer" :i"r¡ cons'ti.tutícn other

than Ín the cantent of angS.otensin" The methods reported. whi"eh assey

angiotensin Ín plasma d"íreetly (Goeke _9.t__&1, 1g6gi Good.fråend. e_Ë_eÅ, 1969) have yíe3,ded values hågher than those obtaine,l when a prior extraetÍon proeedure ås empJ-oyed", and. some of thÍ.s apparent loss of speeÍ"f5"cÍ.ty may be relateð to non*speeå.fåe effeets of other eonstÍtuents in plasma"

ïn the ease of extracted" plasma sampres the situatj_on is agai.n

not completely satisfaetox"y" Tt ís perssíble to use as bl-anks

extraeted. anephx"åc plasma, wa'ber, en to re*extraot the p)-asma samples

from which the angi"otensin has alread"y been remove,å by the extraetion

prooed-ure" Nöne of these is enti"rely sa'üåsfaeteiry, harÍever, for none ensures the same eonstj"tution, apart fron an6j"ot,ensin eontent, of sampS"es anå blanks. rf non*speeåfÍc effeets on anttbody þånðÍng ere

d.ue to small peptíd.es prese;:r't Í.r: ex*raeted. plaæma, the same ¡:eptíd.es may neit be presen't ån the re*extra.-oted. blank sam¡:J-e" Tp praetiee, Ìt was found. that some rariatåon in t,he peneen'ti,ege bounð dåd. oee.ur Ín t?re

easo of the various bl"arics but the magnítud.e of thås varåati.on was sueh that åt eorrespûndecl with a eoneer¡tr."aüion of less than J pe/n\ in the origÍnal sampJ.e. 125r:tu,bulred. rt was f ounð tira't ,¡a15*angiotensin ïï amÍåe

("H¡rpertensån't-üåba) was mere s,rraeept"åble to non-speeåJ'åe d.amage .çran labelled^ i'scleuS-sngiotensi-.u: Iï. fhe d.egrr:e of d.amage Fra-e found. t<¡ 13Ì+"

have a linear ælations?rip with the time of j-ncubatÍon in the rad.ioirnmuno-

assay proeedure and" proðueecL a substanti"al error at 24 hours. The labelled. isoleu5-angíotensin ïï was not affeeted by prolonged incubation under these cond.itions and was, therefore, used. routíne}y ín the rad.io-

Ímmunoassay method..

Because of the very srnal"l coneentrati.ons of peptid.e being assayecL, errors d.ue to absorption of the hormone to glassware are very likely to oceur and" tiris was the reason for the high values for plasma angÍotonsin

ïï ínitÍa11y reported. by valLotton et al (1967). rn the present assay, in spite of the use of pol¡rpropylene equipment exclusively, absorptíon of hormone to tubes was troublesome unless the buffers used. containecl camÍer protein. ft was found. that a eoneentration of human serum albumin of 1/Ã was neeessary in order to eliminate this probJ-em. The method. of separation of bound. and. free hormone after incubation in the assay proced.ure was found to require careful attention to teehnique as significant d.íssoeiation of the antigen-antibod.y complexes occurred. if there rvå.s eny d.elay after the add.ition of the d.extran- coated. charcoal príor to centrifugation" This effeet was greatLy exaggeratecl at room temperature.

fn spi.te of efforts to maintain constant eondÍtl-ons from one batch of assays to ar¡other, occasional batches yield.etl less satisfaetory results for reasons whÍeh were not always apparent, and. this is presumabJ"y an lndJ"catlon that all the factons affeeting the reaetíon are not presently reeognfzed" Although this varl"atåon from assay to assay did. occur, f"t was found. that when the same antibod.y and. the same 135.

l.abe1.l"ed. hormone were r.¿sed." 'the pereentage ehange in bånd.ing of traeer with the ad.d.iti.on of st,â.nd.arils 'was relativcL,y eonstant irrespeeti.ve of

the Ínitial bånding fi.gure. lflhen the pereentage eha.:rge differed. from that expeeted., there was usuall-y some teehnioal problem with that

parüieular assåy and. al"l saripS"es were reassayed. in another batch,

trTi"thår¡ the låmtts outli"ned. above, the rad.j.oÍmnunoaËsay method.

afforded" a precise, spec$"fÍe measuÌ:e cf the aetåv{ty of the renårr- angiotens5"n system and. the prneed-ure was used. to ínvestÍgate the role of thås system i"n a varieiby of normal arid abnormal situatíons.

Plasma angi,otensån rr eonerentratÍon in normal subjeets on unrestri.eted" sod.Íun intake was four¡cl to range from t+ pg per mI to

63 pg per mle ïv"i.th a mean value at zï,z *. 1J+"5 (s.0"), These x"esults are comparable with those reporteå by several other grôups (Boyð. .g!.3l, t967; Catt gþ*eå u '1967i Page gb_el , 1969; Sund"sfjord", 1g7o)" lhe effeet of posture on pl,asma angiotensin TT eoneer:Lration was found. to be sÍmil"ar to that for plasma renín aetivåty, the values in the

upright positíon being consåstently hågher than those rneasured" fol,J"owÍng overnS"ghlh ri*eumbeney. lhe paral.lel- behav'Íour of plasma angÍotenså.n rr and. plasma renån acltå.våty was alsÕ seerl fell.owing d.ietary soðium restrietir:n i.n the pirysåoJ-ogieal. studíes Ín normaL subjeets. Although the valr:es of these twe parame.ters of actåvity of the renån*angi"otensin system tend.ed to paraJ.s"eL eaeh other, the agreement was not perfeot, Thi,s i.e not s'r::prisJ"r:g when eine eonsíders the d.ifferent teerhni.q.ues by whi,eh these valt¡.es are ,ebtaåned.; .the plasma angiotensin TT ecvloentratton represents the ecnoentration of 136"

hormone consequent upon the in vfvo aetion of renín and. convertÍng enuJnne on the one hand" and. angiotensånases cn the other, whil-e the plasma renin activity represents the ab5.J"åty of plasma samples to generate angiotensin in vÍtro und.er unphysJ"ol-,ogiea1 cond"itj.ens foi-lowing treatnent to ånhibít angiotensinases. Tt seems IÍkery that the plasma angio*ensin rr

eoneentration wou1d. be the betber meesure of aetívity of the system

sl"nee ít is móre representative of the ån vivo situatåon, Howerf,er, th-is must be bal.an.oed agaånst the ðemonstr"ation by Caån et al ?g1g) that the substanee assayed. in venous bl"ood. may be largely Ínaotive fragments of angiotensÍ.n Tr and., wh:Lle thÍs nay be ad.equate for puxposes of cIånÍea1 sereening of patåents, arteråal eoneentrations of angÍotensÍn rr shoul,d- be used for preeise physíological stud.Íes. It may be that even arteri"al- levels d,o not represent the eoneentrations necessaqr for physioS"ogåcal aeti"on and. tissue leveL.s may be more crÍtical in (Gooð.fx"Íend 'i g7a) this regard and Lin, " This probl.em ís currently not resol"ved., but, in the meantime, p]"asma angiotensÍn rr coneentration almcst eerta.ínI"y prescnts a more preeåse assessment of aetivity of the renin*angioteneån system than prasma renin aetÍvity"

The applieation of the rad.icåmml-znoassay t,o measurement of p1-asnra angiotensÍ.r¡ Tf eonoentrations in situatåons of see ondar¡r h¡ryerald.osteron* i"sm has eonfirmed. high l.evels ån patåen&s wÍth eor:gestÍve card"åae failure, nephrotie s¡rnd.rome, and" hepatåo oírrhosi.s wåth aseÍtee" The rol"e of the renån*angiotensin system ån these cond.j.tj.ons ís d.åffieult'to assess, &ð thås i"s cempLieated" by the faet that the najority of these patients were being treated. vyíth d-iuretåcs anð./ør 137 " sod.iìrm restri"otåon at thè tåme the sampS-es were taken, so that the el-evated. angiotensin fI level"s may have been a seeondary effeet of the treatment rather than a¡l íntegra} part of the d.isease prooess.

Suppor"t for this vi"ew was obtained" by the finding of some values withín the normal range in patåen'ts wÍth untreated. congestive card.iae faåIure"

In one ea.se of prlmary aL.dosterorrîsm associated. with bílateral adrenal cortj.eal h¡rperp)-asia, low levels of plasma angiotensin rr, at the lower limit of the normal rangee were for¡nd, and" these correlated. welJ" with the plasma renin aetivity estÍmations whåch were also of this ord.er. Both parameters fai"leð to ånerease in response to the normaL ståm'r¡1i of d.ietary seðium restråetå.on and upr*"ght posture" Plasna angiotensin TT levels eould. be used. as an alterna*Íve to plasma renin acttvity meaõurement Ín the sereening of patÍents with h¡4ger- tension for eases of primary al-d.oe,teroûåsm"

fn the group of patåents with l:.¡rpertension i,n whom plasma angio* tensin II coneentrations were åssayeð, values were for,ir¡d to range frorn withín the normal range to quùte h:igh levetr-s" Many of these patients were referreð for ånves'tÍgati.on cf possåble renÕvassu1-ar h¡¡pertension and, Ín the grotip in whorn the diagnosi.s was corrfírrned", the p1asma angiotensin II was unålrersall-y eLevateð. Tftien thås grÕutr: was separated. frorn the remaånd,er, there were stil-l many ¡:at5"ents who had eïevated. angiotensin rr leve3.s" some ef these patÍ.ents haå s*rong evidenee of renar parench¡rmal ðåsease anf, this nay have beerr the eau-,se of t,he eleva'hion (catt É-g}., 1969), rlx the group lvsth unccmplÍcated. 138 " rressentíalrr h¡rpertension, many of the angiotensÍn II vaLues were wj-thin the normal range, br¡t a strbstantial number were elevated. so that the mean value rvas signifíeantly above normaL" a possùbre role for the

renin-angiotensln system in benÍgn essential h¡pertensíon has not been conpletely exclud.ed. but the nature of sueh a role Ís not evÍd.ent at

present. In the case of malS"gnant or aeeelerated. h¡4pertension, however,

the plasma angÍotensin IT coneentration was alnost invari.¿bl-y elevated.

whatever the aetiology of the h¡rpertension, and. here it nay represent an Ímportant contråbutory faetor to the cli"nical sítuatíon. Tï¡hen the angiotensin fI radåoimmunoassay proeed.ure was applied. to

the measurement of angíotensÍn generated. d-uring the in vitro method.

for pl-asma renÍn activÍty (skånner, 1967), the values obtained were

found. to be much less than those obtainetl by bioassayc Ï[hen assayed. in cloubling d.ilutiono the curve obtained. was found. to coíneíd.e with the

stand.ard. curve, confirm:ing the id.entity of the substance measured. as angiotensin rr. The reason for the lower values obtained. by rad.io- immunoassay is the inbibition of converting enz¡rme by the EDTA ad-d.ed- to inhibåt angiotensinases. As the i"mnunoassay ås speeific for angiotensin

Ïf , only the hormcne present in the form of the octapeptÍ.d.e is d.etee ted. by this means and. the values would. be mueh lower, d.epend"íng on the d"egree of inhibítion of converting enøJrÆe. Tf the d.egree of i.nh-ibition were constant from sample to sampl.e, the angiotensÍn TT rad.ioi"mmunoassay could be usecl to assess renin aetívity in this ïyay, even though the absolute values would. be mueh lower" This, however, was not the case when comparj"sons were mað.e of renin aetivity values obtained. by the two 139. nethod.s and" the eorrelatå.ion nås net ei"ose enough to enable thi.,s use of

the radioimmui'roassay teehniqu;e. the enly exeeptÍon to thÍs r¡cas found.

to oeeur in the case of símultaneousS-y d"rawn and. åd.entíeaT"ly proeessed.

sampJ,es from the tw,o renal- veins, coJ-J.ee ted. to ånvestågate the presenee of slgnifåean't renal" artery s'tenosås. Tn this case, the r"ati.o of renån aetlvltåes (rrígh såd.e:low sÍde) was fo¡;nd t¡ eorrelate erosely by the two methods. This presumably cÕmes about beeaulse the two samples are eolleeted. s:i-multaneously f'rom the two sid"es and prooessed. Íd.entically, so that the cond.itÍons d.eterminirrg the frae'tÍon of angiotensin present

l"n the form of the oetapeptåd.e are relatively eonstant and- the ratåo gÍves a valid. ind.ieatåon of d.ifferenees i.n renin aetivÍ.ty"

Attempts to reverse the ånhåbitÍor¡ of converting enzJrme by the

ad.d.Íti.on of varåor:s metal ions were not suçeessful beeause c¡f

sÍmul"taneous aetívatì.on of angíotensinases a^trd this approach to the

problem was not praetieable" Tt would" seem more d-esirable to ensure complete Ínhibitíon of convertå.ng enøJrme and. t,o use ân angictensín r raðioímmunoassay to measure the generatcd angÍ-otensin and- this approaeh has been aÕ,opted by a ncimber of groups (Boyð e!_"1, 1969; Haber et_g&, 1969).

A survey of the level"s of angiotensån TT in the plasma of women d"uring normal pregnâ,rrßy re'veal-eå hågh mean valucs throughor-lt, wíth no sågnifieemt d.ifferenoes between the vari.ous stages of pregnaney, values were eL.evateð as eanL.y a,e the seventh week of pregnaney and. remained hågh råght up to term" lhis få¡:td"Ír:g is in agreement with the previousry repor"ted ht6h plasma renå:r,, açäåvity valuee throughout 1¿+0.

pregnanoy (Genest -g!--e1r x965¡ ïrliner, ^,965) and. i.s prestrnably rerated

to th.e high levels of renin subs'Lrate reperted. ín pregnaney (Helner and Jud-son, 1967i Påokens gt_gl, xg6ü. lhe inereaseð actívíty of the renån*angiotensin system and- the inereased seer'etåon of ald.osterone durÍng pregnancy may be reJ-ated. to the antagonÍstic effeet of proges-

teronc on the peri"pheral aetion of ar-d.osterone (Landau el*e¿, 1965) so

that Lnereased. Levels of mineralceonti.coi.d" are requined. to ensure ad"eql"la'te sod"ium homeostasis" Measuremen* of plasma angiotensin IT

coneentratÍons in a smaLl group of women with establåshed- pre-eclamptf.c

toxaemÍa ¡niel.d.ed. values which wene not signlfåcantly d.Ífferent from those found. d.uring normal pregnanry, rbr-is find.ing wouLå suggest that the renån*angíotensín system is not of major ímportanee ín thís situation.

The sheep s'tud.åes have eonfirneð tbie pnevÍous fìnd.ing of inereased.

eoneentrations of renÍn i'n rena1 l¡nnph eermpared. wï"th plasma (Merntosh and. Morris, 1)J1), They have aLso d.emonstrated. inereased. values for renin aetåvi.ty and. angioter¡sin rr concentratíon and. slightly lower values of sub,strate i"n lymph conrpareå wåth plasma, Renin

cone entratåon, renj"cl aotÍvåty, anð angtotenein fT eon,eeyrtratåon î¡ere all found. to ineinease in parallel v¡åth the plasma valuea followÍng

sod.iun depletion" The origåin af the renin and. angiotensi-n TI Ín rer¡al l¡mph were i.nvestigated. by Ínfr.r"såo:n,of isotopí.ca11y-1abe11ed" angiotensin

II ånto the renal antery, fol":Lrwed by isr:T-atíon and" id-entifieatÍon of rad.ioaetivÍ.ty ån renal .t;mph and. urine, ancl by ad.mi-nåstrat,i.on of a tubular bl.ock5"ng agent to ðetermine the effeet on renal- l.ynph leve1s of 141. renin and. engio'Èensån" The evådense obtained from these erçeriments suggested that the renÍn f"n tymph was seoreted" dÍreetl.y ånto thås eompartment fron the juxtagS-omerurlar rogåon of the kidney a:rd. that any contråbution from renin reabecrbed. from the renal- tubules after glonerular filtratÍon was of mj.nor importanee" Sim:i1ar1y, a signåficant contributi"on to the angiotensin II in renal l¡nnph frorn plasma or urtnary sources seems unlike1.y. À d.5.sparåty between the renån and. angiotensin levels irr renal lyinph a^r:d. p1-asma following admilråstratíon of mersurÍc chl"cråd"e to thr.ee sheep was thought to represent either a speeifie effeet on the moehanism by whÍ.eh renin gains aceess to the renal- lymph or *he oonsequence of a markeð red.uction or ðisorganåzatio¡r of renal blor:d" flow. Either effeet could follow the severe d.erangement of renal arctriteoture proðueed. by thfs agent. t+¿"

CH¿,PT R E

ÞWRT

The d.evelopment of a sensÍtåve and. speeifåe rad"åof"mmunoassay for angictensÍn rr ås d.eseri"bed.. The raðíoimmunoessey provld.ed. a preeÍse, speeifi"e mes,sure ofl the aetåvity of the ren$.n-angiotensin system ín the investågatÍe:n of tiie role c¡f this system in a variety of nor¡nal and. abnormal såtuatåons"

Plasma angi.otensín rr eonec'¡itr."atÍons in normal subjeets on &n unrestri"eted" sod.å¡:rn intalçe were found" to range from )+ t,a 6j pg/nl, wåth a mean val-ue af 2þ"2 * 1&"5 (s"D.). The values ín the uprÍ.ght posåtion urere found to be oonsåsten'tly hågherthan those taken after overnåght reeumbeney" P1asma angiotensin II eoneentrations were found. to åncrease in respönse to dietary sod-i"um res'brj-etion Ín no:rnal st¡b jer;ts.

Pl"asma angíotensÍn Tr values were fo¡:nd. to be elev'ated. in eongeståve eard.iae failure, nepLrrr:tå.e s¡mårome, arrd- hepatie címhosi"s, although thås ån,ereå,se may have heen partJ-y d.ue to tÏ:e ðiuretåo therapy employed. ín these eonrT"i,tåor¡s, Elevated. levels of angåotensin fT were ånvaríably found. 5.n patÍects wåth uralignant or aecelerate¿ h¡rpertensÍon or h¡rpertensÍon seeonri"ary to renovå,s.;ulax" dåsease,

ïn patåents wå.th "essentiaS-1' h¡4:ertrerrsÌ.Õn, many af t,he angíoteneån rr values were founð te fal.l wi-thi"n the normal range but the nrean level was såg::å.fåeantly eJ-evated", suggesting that a p,ossib]-e role of the renån*engiotensån system ån thi"s oo:nd"itåon eannet be eernp,l.etely exeluåed a'c preser:t" 143 " The applS"eation of the angÍotensin TT rad^ioimmunoassay to measure- ment of angiotensin generated. in vitro ðurÍng renin activity method.s has been prevented. by the faet that the h,¡rmone generated unðer these eond'itions Í.s largely in the form of angiotensin I whÍch reacts to only a mÍnimal extent Ín the angiotensin TT å,ssay" lhe percentage of the generated. hormone in the form of the octapeptid.e was found to be varÍable and. the only situatÍon where the present assay eorrld be used. to measure renin aetåvity ån this way rvas in, the case of renal vein renin activity ratios. Tn ttrís situatíon, the samples a¡ere d.rairyn simultaneously from the same patienL anå processeð ld.entically so that the cond'itíor¡s d.eterminÍng the percentage of the generated. angiotensín in the forn cf the octapeptíd.e were relatively constant and the ratj.os obtained" eorrelated. closely with those obtained. by bioassay, suggesting a elínåcal applíeatíon of the method. for th:ts puryoseo Levels of plasma angiotensin rI were found to be elevated. throughout normal pregnåney, a find.ing consistent with the previously reported. håglr values of plasrna renÍn actÍ.viLy" Pl.asna angfotensin II leveJ.s in a small group of patåents wlth establisheå pre*eelamptie toxaenr-i"a were not signÍ.ficantly d.ífferent from those founcl at a simil.ar stage of nornal pregnanoyo

stud.ies in sheep have eonfi.rmed. the previously reported. hígh coneentrations of renin Ín renal lymph and. have, ín ad.ðition, d"emonstrated" incr.eased. renín aetj"vÍty and. angiotensj.n rr level-s compared- with pl"asna. Renin eoneen*rati-on, renir: aetivity, and. angÍotensån TI levels in l¡nnph inereased, in paral1.e1 with the plasma tU+, tz5b values folJ.owing sodÍun d.epletion. Infusion experj.ments using l-abelLed. angiotensin Iï suggested. that plasma and urinary sources for the angiotensin Iï in renal lymph were un1ikely. Furthermore, errperfments using a tubular blocking agent suggested. that renin reabsorption from the renal tubules fo1LowÍng glomerular fÍltration d"ið not contribute significantly to the renin in renal lymph.

Thus the renin and angiotensin If measured Ín renal lymph are probably a consequence of d.irect secretion of renin into the lymph compartment from the juxtaglomerular region of the kidney.

lhe angiotensin ïf rad.ioimmunoassay procedure thus represents a precise means of assessÍng the activity of the renin-angiotensi.n system in man and. sheep and has a number of useful applicatíons, includ.Íng physiologicaL studies in man and sheep and. clinical studies in man. 145"

BTBITOGRAPHT

SAILIE M.D., N¿,SH F.D, , end R0SÎORFER. H.H. (1966)å Renal sodÍum netabolåsn and renån se{oretå.on. Fed"Proe. þ z 328. BAITTE MnDo¡ RECïOR Jro F.Co, and SET,DIN D.ïr. (tgZO): Elevateð angiotensån IT ån renal lymph Índ.ícatíng the presenoe of angio* tensín I eonverti"ng enz¡rme" ClÍn.Res. l3 , f+93, BARffm. F"C,, MILTS IoHu, BIGT,Imï EoG" , and DELEA C. (lggg)r Studies r¡n tkre e,or:trol arid" physÍol.ogic action of ald.osterone. Recent Progr"Hormone Res" fi : 311.

Bffi.SON, S.A. Ugil): Cited in Levíne R" and And"erson E" ',Resune of Conferenee oR ÏRsulín ActÍvity Ín Blood and. Tissue FluÍds, May !th*10th1' " BERSON s.A" and YALOTI R.s" (lg1a)z rsotopic traeers ín the study of d.iabetes, ån Tobías c.A" and- Lawrence J.H" (eðs) ¡ Advanc,biol. med.Phys. (New York : Aead.emic Press)

Bffi.SON S.4., TAL014I R.S., AIIRBACH G.D, and pOTTS Jro J.ln (lg6j)z Immunoassay of bovine and. human parath¡rr"oid. hormone" proe.

nat,Acad.,Sci. (fiIash. ) À2 t 6a".3 .

3m.S0N S.4", TAt0Tr¡ R"S", BAUMAN 4", ROTHSCHILD M.A" and NtSiTRLy K.

Ugf6)t Tnsulån*T131 metabolåsm ån human subjects : Densnstration of inst¡li-n bind.ing globulín ån the eå.rculatíon of irisulin treated

subjeetso JnClin,fnvest" þ3. : 1 70. BrNg J. and. FA-aLuP P" ßgas): T,oeation of renin (or a renån-1åke substance) tn tire .qubmaxil-l-ary gland.s of albÍno mieÐ. Aeta"path, Mierobiol..Seanå. @ : 203. 1t+6, BÏNG J" and. FAARIIP P" \çeø)c Qualitative and. quantitative study of renÍn in the d-ifferent layers of the rabbit uterus. Aeta Path" MÍerobÍol,Seaud, 67 : 169.

BING J. and. I(AZIMIffi.CZAK J" (lgøZ)s Renin content of d.ifferent parts of the juxtaglonerular apparatus. Aeta Path"Mi-crobío1"Seanð. l[: 80. BI,AIR*VIESI J.R., C0GHLAN J"P,, DETVTON D.A. , G0DTNG J"Ro, MUNRO J.Ao, PETERSON R"B" and WINT0IIR M" (t962)t Humoral stimulation of ad.renal cortical seereti-on. J,Clin.Tnvest. !! : 1606"

BLAÏR-WEST J"R., COGHLAN Jupn, DE¡TToN D.A., GoDTNG J"R", WTNTOUR M.

and. 1i[RTGHT R.D. (1963)r The eontrol of ald"osterone seeretion

Reeent Progr.Hormone Res, 12 311 z "

BüArR-irTrEST J"R., O0GHLAN JnP., DENT0N D.4., scoGGrNS B"A. and trflRTGHT R.D. (lgll)z Ts there a fifth faetor in ald.osterone eontrol? (Abstrae*) Proc,t¡th Asia & 0ceania Congress of Enðocrinology,

Auckland, N.2., Feb. 1 971 , 31. BLIDDAL J. and NIELSEN I. (tgZO): RenÍn, aldosterone and, eleetrol¡rtes

in ídiopatiii"c orthostatåe h¡potension" Dan.med."BulJ_, :LZ i 15j. B0LLMAN JuL., CAIN J.C"e and. GRïNDLAY J.H" (fggg): teohnåque for

eol,leeti"on of l¡nnph from lå.ver, sma1l" íntestine, or thoraeie d.uet

of the rat, J,Lab"C1ån"Með. þå" t 131+9. BOUOHER R. and GEiVEST J" Ugge): rmprovement of method.ology for: measurement of plasma reni"n aetivíty. canad.,J"Physiol"pharmaeol. !þ: 181 . BOUCIüR R" e VEYnAf R", d.eCHAMPLAIN J" and. GENEST J" ?ge!ù: New proeedures for measurement of human plasma angiotensån and renin

actÍvity leveL.s, Canaû"lVled""Ass.J" æ | 191+. 1l+7 " 30YD G.U/., ADAMSON 4.R., FITZ A.E, and- PEART Vf.S. (1g69): Rad.ioinmuno-

assay d.eterrninatÍon of plasma-renin activity" Laneet 1 : 213.

BOYD G.ïr., LANDON J. and PEARî, W.S. (gel): RaðÍoÍmmunoassay for

determÍning plasma-levels of angiotensin IT in man. Lancet 2 : 1OO2. BOYDEIS S.V. (lgy)t The ad.sorption of protelns on er¡rthroc¡rtes treated

with tannÍc acÍd. and. subsequent haemaggl-utination by antíprotein sera. J.exp.Med." 22 t 1A7. BRAUIï*ME0íENÐEZ 8., FASCToL0 J.C., T,ELOIR L.F. and. Ilfl.ÑOz J.M. (tglg),

La substaneia hipertensora de la sangre d.e1 rifi6n isquemiad.o. Rev.Soc.Argent.Bi"ol. !l r l+2O, BRAUN.-Mm{fivDEz 8., FAsCrOl,o J"c., LEL0IR L.F. and li,fuffioz J.M. (rg¿*O), The substance causing renal h¡ryertension. J.Physiol. 29 , 283, BROWN JnJ., DAVIES D.L", DOAK P.8., LÛ\IEL A.F. and ROBXRîSON J.f.S.

?gel$))z Plasma renin Ín normal pregnancy" Lancet 2 z 900. BROliÍN J.J., DAVTES D.L.r DOA.K p.8., LE\IER 4.F., R0Bffi.ÎSON J.I.S. and TRUST P, (tO65h Plasna renin concentratíon in h¡¡pertensive d.ísease of pregnancy. Lancet 2 : 1219,

BROWN J.Jn, DAVIES 0"L", tEVm. A.Fu, MePHER.SON D. and- ROBERTSON J.I.S. (tg66)c Plasma renin eoncentration in relatÍon to changes in posture" Clin.Scí. ff z 279.

BROl¡fN J.J., DAVIES D"f,N' LEYEA. A"F' ANd. ROBERTSON J"T.S" (Ig6S(A))Z

ïnfluenee of sod.l"um load.Íng and. sod.ium depletion on plasna reni-n in mano Laneet 2 s 278.

BRoTnV J.J. ¡ DAVTES DoLo, IE\¡ER A.Fn,and RoBERTSON J"I,S. (lg6l+)z Variations 5"n plasma renin conoentratíon in several physiological

and. pathologieal states, Canad,med.,Ass.J. .æ : 201 . 1 ¿+9.

BROïN JoJ., DAVIES DoLn, IJE\Im.4.F., RoBm.TSON Jnfo$. and. IREE M. (tg6¿¡): lhe estl"mation of renin in hunan plasma. Siochem.J. 93 t ,94, BROIVN l.Co, DAVIS J.0. and JOHNSI0N C.f. (gee): Acute response in plasma renin and. ald-osterone secretion to d.iuretícs. Am.J.Physiol" & t I+37. BUMPUS FnM., sclÍW.ÂRz H, and PAGE r.H. (lgSl): synthesis and pharmacology of the octapeptide angiotonin. Science 125 : 886.

BUMPITS F.M., SMEtsY R"R. and PAGE I.H. (196t)z AngÍotensin, the renal pressor hormone" Circulat.Res" I t 762. CAIN M.D., CATT K.J. and" COGHLAN J.P. (lg6g)t Effect of circulating

fragments of angiotensin If on rad.ioímnunoassay ín arterial and. venous blood. J"ClLn,Endocr. p z 1639. CANNON P.J., AMES R.P. and" LARAGH J.H. ?gee): Relation between potassium balance and. aldosterone secretíon in normal subjects and. Ín patíents with hypertensíve or renal tubular d.ísease. J.Clin.fnvest. !l : 865. CASTENFORS J. (1167): Effect of ethacr¡mic acid. on plasma renÍn activÍty d.urång supíne exercise Ín nonnal subjects. Aeta physiol. Scand. fQ: 215"

CAll K.J., CATN M.D. and. COGHL.A,N J.P. (lO6l): Measurement of angio- tensin ïï in blood" Lancet 2 : 1005.

CATT K.J., CAIN M.Dn, COGHLAN J.P., ZIMMEÎ P.2", CRAN E. and. BEST, J.B. (lglO): Metabolism and blood. levels of angiotensin fI in normal subjects, renal d.isease, anð essentÍal h¡pertension. Circulat.Res. 26 Suppl" TT, p"|fJ. 1l+9.

CATI K.J., çAIN M.D., ZIMMET PuZ" an¿ CRAN E, (tl6/): Bl.ood- angiotensín

ïI levels of normal and. h¡¡pertensíve subjeets, Britrmed..J" 1 : 819" CATI K. and COêHLAN J.P" .(t167): Generation and use of antibodi-es to angiotensi.n II, Aust.J"exp.Bío1.ned..Sci " !ú" t 269. CATT K., NIALL H.D, and TREAGER. C.T{" (1g66): SolÍd-phase radioimmuno- å,ssay of hunan growth hormone" Biochem,J. llg : 31a. CHANNICK 8.J., ADLIN E.V. and trlARKS A.D. (1g69): Suppressed- plasna

renin activity 1n h¡pertension" Arch.Tnt.Med" 123" , 131.

The effects of posÍ-tion, exercise and. sod.ium íntake on plasma renLn

activity in normal people. (Abstract) Ctin"Res" 12 t 362.

COHE$T 8.L., ROVNER D.R. and CONN J.W. (1966): Postural augmentation

of plasma renin aetÍvf"ty. J"Amer.med..Ass " EJ z 973" CONN J.try., C0HEN E.L, anð ROVI\ER D.R. (lg6l+): Suppression of plasma

renin aetivåty in prÍmary aldosteronism. J.Amer.med..Ass. l_29 z 213. COOK ï¡.F" anð LEE M.R. (1165)s lhe preparatíon of rabbit renin- substrate for the assay of mÍnute amounts of renÍn. Bíochem.J. fi t t+13" COOK Iï.F" and PTCKIRING g.W. (lgf9): lhe loeation of renÍn within

the kid.ney. J.Physiol.(London) 142 t 7Bp"

DAVIS Jo0n¡ AYffi.S C.R" and" CARPÐ{IER C.C.J. (tg6t(¡): Renal orígÍ"n

of an aldosterone*stimul-ating hormone in d"ogs wíth thoracie caval constrietåon and in sodium*depleted dogs. J,Cli.n.Invest" !Q : 1466" DAVIS JoO", BINNI0N PoF., BR01¡/N f.C. and JOIINSTON C.I, (t966)z MeehanÍsms lnvolved" in the h¡4perseeretion of ald.osterone d.uring sodium ð.epletion. Círculat.Res. ![ Suppl, 1 z 143. 1 50.

DAVIS J"0", CARPIßITER C,C., AYERS C.R., HOLMAN J.E" and. BI}IN R.C" (tget), Evåd.enee for seeretion of an aldosterone*stímulating

hormone by the kidney. J.Clín.Invest" !Q : 6Bt+. DAVIS J"0., URQUHART J. and HIGGTNS Jr. J.T. (tl6[)r Renin, angiotensín and ald.osterone in experimental secondary h¡per- alðosteronism. Canad..med".Ass,J. p! : ZLj. DEIVTON DoAo, G0DTNG J,R" and ïllRïGHl R.D. (lgfg): control of adrenal

seeretion of electrolyte-actíve steroid.s. Brít.med..J, Z, 522" DEODHAR s.D. Ugeo): rmmunor.ogic prod.uctíon of anti-angiotensín. J.exp.Med. 1l-l: l+19.

DEODHAR s.D. , F{aAs E. and GOLDBT,ATT H. (lg6t+): productíon of anti- renin to homologous renin and its effect on experimental renal

h¡pertensíon" J.exp.Med.. L1g z UZ5. DTETRT0H F.M. (1167): rmmunochemical analysis of rabbit antÍbodies against angiotensín II" fmmunochemistry l+ : 65.

ELLTOTT D"F. and PEART lcl.s. (lgSz): The amino acÍd" sequence in a

h¡rpertensin" Bi.oehem.J. É2 z 2116"

FASCfOLO JnC., DE VTTO 8., ROMERO J.C. å,nd CUCCHI J.N. (lg6L): The

renin eontent of the blood. of humans and d.ogs und.er several

eondÍtions, Canad."med."Ass.J, 2A6 9Q : " FEtBnR. J.P" ( 1963): ACTH antibod.íes and their use for a radioimmuno- assay for ACTH. Experíentår z pZ7. l;f ) FERRïS f"F. and MULROïI P.J. Ug6Ð: The ,-ít"*,r" 1* u source of renån, Clin,Res" 3å : 206. 15'1 .

GEIVEST J., 3rR0N P. e KorïtI 8., N0ïrAczrNSKT ïr., BOucHm R" and

cm.ErrHv M. (lg6l)z Newer ad.vances in the pathogenesis of human

h¡pertension: the ad.renal cortex and" renal pressor mechanísms. Ann.Tnt.Med, lþ : 103U" GnvESr J., BOUOHER R., de 0IIAMPLATN J., VEYRAT R. , cHRrrrEN M., BïR0N P. , IREMBLAY G., ROY p. and cARTrm. p. ?geu): studies on the renin-angiotensin system in h¡¡pertensive patients. canail. med.Ass.J. gg z 263"

GENESI d'e CIIAMPLATN J., J., VEÏ&A.î R., B0UcHm. R., TRE¡ißLAY 0.y. , STRONO C.G., KOII4I E. and MARC-Affnì¡,n ,f. (geС Role of the

renÍn-angi"otensin system in varj.ous physiologÍcal and. pathological states. H¡4pertension !! : 97. GEI\TEST J., NCITTACZYNSKT TiT., KOTi4r 8., SANDOR [. and. BTRON P. (1960): Adrenal cortÍcal function in essential h¡ryertension. Tn: Essential H¡pertension" An rnternatj.onal symposium sponsored^ by ciba (Berne, June 7*1or 196o) edåted by K.D. Bock and p.T. cottier, BerlLnn SprÍnger-Ver1ag, p,1 26.

Gï,ïcK s., ROTH J,, YAt0w R"s. and BERsoN s.A. (1g6j): rmmunoassay human growth of hormone Ín plasma" Nature JlA z Tg4. GoCKE D.Jo, Gm.TEN J., SHERW00D L"M. and TARAGH J"H. (lg6g)t PhysiologÍcal and. pathological variations of plasma angiotensin Tf

in man. CirculatoRes. 21a Suppl" í."131 " cOcKr DoJ., sHml¡1000 L,Mu, OPPENHOFF T., GERTEN J" and. LARAGH J"H. (tggg)c Measurement of p.lasma angiotensÍn rr and coryelation with renin aetivity" J.elån,End.oer. 28 : 1675. 152"

G0LDBLATT H. (*y:g): Stu&ies on experímentaL l:¡4pertension :

Prod.uetion of the malågnant phase of h¡pertension" J,Exp.Med". 6Z z 809.

G0LDBLATr H", LTNCH J. , HANZAL R.F. and SUMMERVILLE ïV.tr1r" (lgStu)t Stud.íes on experimental h¡pertension: Prod.uction of persistent

elevati.on of systolie blood pressure by means of renal åschaemÍa.

J"Xxp.Med" ll I 3l+7. GOODFRIEND T.L", BALL D"L. and FARLEY D.B. (tg68): Radåoimmunr*

as,såy of angíotensÍno J.Lab.Clin.Med. " k" z 6t+8. GOCIDFRTEND T. , FASMAN G" e KEMP D" and LEVINE L. ?gee): Trrnuno- shemioal stud"i"es of angiotensi.n, fmmunochemistry I : 223. G0ODFRImID tolo, LEVINE L. anð FAS]\4AN Ç,0" (1964): AntÍ'bod.ies to brad.ykinín and, angiotensin: å use of earboðiimi-d.ee ín immunology.

G0ODFRTEND T.L. and T.,IN S.Y, (lg'lA): Re*eptors for angÍotensin I anô II, CircuL.at"Res, !Q Suppl. i2163" G0CIRMAGHTIGH N. (lgSg): Existen*e of an end,:erine glarrd in the metlåa of the renal arteriole. Proe.Soe"Exp,Biol.Med. þl r 688. e0I)0N R,Dn, KUCITEI 0.e ISLAND D.P" and LIDDLE G.lrV. (t966(a)):

Rol.e af s¡nnpathetåe r:e¿'rror;s system Ín mediatÍng renal and ad.reno* c:orbíeal seeretory response,s to upright postureo J.Cli.n.Invest. þ! : 10x6. GORDON R.Dn, KUCIITSL 0.e L,IDDLE G"Yri. anð ISLAND Ð.p. Ugel): Rol-e of the s¡rnpathetÍ-e nervous systern in r"egulating reruin and. alðosterone

produetåon ån ma:'¡" i.Clin.Invest" $1ij : 59g. 153 " G0RÐ0N R.Do, pARs0NS s, and. syMONDs E,M" (1969): A prôspeetÍve study

of plasma renin activity Ín normal and. toxaemic pregnancy"

Lancet 1 : 1+7,

GORDON R.D,, ÏIOIFE L.K., ISLAND D.p. and LIDDLE G"l[i. (ty6g(¡)): A di.urnar rhythm in plasma renin acti"vity Ín ma^n. J.clin,rnvest. þþ r 1587"

GREFI\TWOOD F.C", HUNTER. ïr"M. and Gl0vffi. J.s, (t163): The preparation 131Ï-tabelled of human growth hormone of high specific radioaetåvity.

Biochem"J. pl : 11)+.

GRODSKT G.M", HAYASHTDA r., PEIVG c.T, and GESCHTüIND T.r. (t96t): Prod'uction of glueagon antí.bod.Íes and. their role in the metabolism and. immunoessay proc.Soc.exp.Biol,Med.. of glucagon. fgz : 4-91 .

GROss F. (tp¡g): Reni"n und. h¡rpertensin, physiologiseh,e od"er pathologische wirkstoffe? Klin,Il¡schr, fi 2 693. GROss F. (lgrg): Díe steuerung de alcrosteronsekretÍon.

Schweizerisch Meö,'lVschr. .Q! : 1 . GROss F., BRUNNEI H. and zrEGLm M. (lg6f): RerrÍn.*angÍotensån system, ald.osterone anú sod.Í,um balance. Reeent progr.Hormone Res. & z 119. ÊROss F., SOHAEOHTELTN G., zrEGLffi. M. and BERGER M. (lv6Ð: A renÍn- like substanee in the placenta and uterus of the rabbit. I¡ancêt f : 914. HABER 1., PAGE T,,3, snd JACOBT G"A. (lg6r): s¡mthesis of antÍgerrie

braneh-ehain copplJrmers of angiotensin and. poly-L-1ysine" BÍcrehemistry À z 693.

HA13ER 8", PAGE L"B. and" RTOHARDS F.F" (ges): RadioÍnununoåssa,y emplo¡ring gel fj.ltratÍon" AnalyLÍcal Biochernistry 12 : 16i. 154.

HET,MER. 0"M. (ll6a): Renin aetivÍty Ín bloorl from patients wíth

h¡pertension. Canad..med..Ass.J. l! : 221 . HELMER 0"M" and" JUDS0N If.E. (lç63)z The quantitative determinatÍon of renin in the plasrna of patients with arteríal h¡pertension. Cireulation il-: 1050. HELMER. 0.M. and JIJDSON Tf.E. (lg6l): Influence of high renin substrate

levels on renin*angiotensín system in pregnancy" Am.J,Obst.& G¡mec" qo.o .U- " /. HERBERÎ V", LAU K", GOTTLTffi C"lf" and. BLETCHffi. S.J. (ggf): Coateð charcoal i,mmunoassay of insulin" J.Clj.n.Endoer, å5 : 1375.

HERNAND0 L., CRA33¡ J.r ROSS 8.J", REDDY ]il.J., RENOLD AoEn, NALSON

D.H, and" TI'IORN G.ïr. (1g57)r clinical experience with a physico- chemical method. for estimation of al.d"osterone in uri-ne.

Metabolism 6 : 518.

HTOKLER. R.3", LAUT,ER D.P. and IHORN G.TII. (1963)z plasma angiotensín-

ase aetivÍ"ty in patients with hypertensíon and. oedema, J.clj.n. Invest, W" , 635. HrGGrNs Jr, J"T", DAVrs J"0" and, URQUHAÏT J" (tg6z): Tnereased. angiotensi.n*like activÍty in thoracic d"uct lymph of dogs with experåmental seeond"ary h¡rperal"ðosterontsm. physiologÍst J "" 157. HTGGTNS Jr. J.1., DAvrs J,0" and" uRQ.uHARl J. (lg6¡ò: Demonstration

by press,æ and stereiåd.ogenic assays of increased renin in 1¡nnph of d-ogs with seconclary h¡peraJ"dosterr:nÍ.sm. cåreulat"Res, l:L : ziB" HïLL R,I¡r,e OHESTER J.E. and TfrsH'fBAUGH p"E. (tgzo): The effeet of

i"ntravenous antÍ.rerrÍ.n injeetÍon on chrorric experimental and" acute renin*Índ.ueed. h¡pertension in dogs, Lab.Tnvest " & z l+0A. 155,

HODGE Rotr., L01¡IE R.D" and VANE J.R. (1166)c The effects of alteration

of blood.-volume on the coneentration of efreulating angiotensín i-n

anaesthetízed dogs" J.PhysÍol,(Lond.) l-95 t 613.

H0LL,$IIANS H.J.G., VN DgR MEER J. a¡rd. TOUBER J.L. (tçgg): Raðioi¡nmuno- assay of angiotensin. Nature LZ z 277.

IÍUNIER. W.M. and GREETVIIf0OD F"C . (1g64): .4, radioimmunoeleetrophoretic assay for human growth hormone. Biochen.J. 2_ z l+3. IMAI M. and" SOKABE H" ?ge}): Plasma renin and angiotensínogen levels ín pathological states associated with oeilemao Arch.Dis. Childh" {f : U7j.

JOIINSTON C.T., HLI'ICHTNSON J"S. and MEil{DELSOHN F.A. (lglO): Bíological

signifieance of renin angiotensj.n Ímmuni zation. CÍrculat.Res. 26 : Suppl" ü2215"

JONES K,M., IIJOYD-JONES R., RI0NDEL 4., TAIÎ J.F., TAII S.A.S.,

BUIBROOK R.D. and GREEI\WOOD F.C. (lgfg): Aldosterone secretion and.

metabolísm in normal men and. women and. in pregnancyo Acta.endoer" lp : 341.

K3.PLAN N,M, and c00K R. (1965): The bios¡mthesås of adrenal steroids :

effects of angÍotensin II, ad.renocorticotropin, and- potassiumo J"eIín"ïnvest. ![ : 2A29. L,ANDAII R"Lo, BERGnVSTAL D.M., T,UGIBII{L K. and }(ASCHI M.E. (lgSf): lhe netabol"Íc effects of progesterone in man. J,c1Ín.Enôocr. fi s 1191+.

TANDIS E.Mn, MONTGCIMERY H. and SPARKI\rIAN D. (lgSA): The effects of

pressor d.rugs and" of salÍne kid-ney extraets on blood. pressure and. skin temperature. J"cLån.Tnvest, lf : 189. 156.

LANDSTEINffi. K. and" VAN DnA. SCHffi. J. (lgSZ): 0n the serological speelficity of peptÍdes. J.exp"Med. ll : 781. LARAGH J.H., ANGffi.S M"e I{3LLT 1¡f.G. and LIEBffiMAN S. (tggO): H¡rpo-

tensive agents and. pressor substances. lhe effect of epinephrÍne,

norepinephrine, angÍ-otensín TI, and. others on the secretory rate

of ald.osterone in ma,n" JoAmer.rned..Ass . J.fA, | 231+. LARAçH J.H" and. STOERK H"C. (lgfl), A study of the mechanism of

secretíon of the sod"ium-retainíng ho¡mone (ald.osterone). J.clin.Tnvest. fi, , 383. LASCET,LES A.K. and MORRIS 3. (lg6l), Surgieal techniques for the collection of l¡nnph from unanaesthetized. sheep. Quart.J.e:rp.Physiol. þþ z 199"

Lfi\ITZ K.8., SKDGGS Jr. L.î., VIIOODS K.R., I(A[I{ J.R. and SHUMIIIAY N.P. (lgS6): The amino aciå composition of h¡pertensin II and its biochernícal relationship to h¡rpertensin I. J.exp.Med. ll[ : 183. LEIIER A.F, and PEART W.S. (geZ): Renin and" angiotensÍn*lÍke

activity ín renal lymph" J.Physiol.f@ , 5l+8. LE\IER A,F" and ROBIÏ.TSON J.I.S. (lg6¡+): Renín in the plasma of normal and. h¡pertensive rabbits" J"Physiol" lJg z 212,

LEIIER.4.F., ROBERTSON J.I.S. and" lRffi M. (1g63): "Hormones and the Kíd.ney" (P.C" WÍlliams, od.) p"285, Mem.Soe"End.ocr. No 13.

Aead.emic Press, London and. New York"

LTDDLE G.]ffn, DIjN0AN Jro r.E" and BARTT¡R. F"c. (lgSe): Dual mechanÍsm regulating adrenoeortÍcal functåon in man" Amer.J.Med." 21 : jBO. 157 " LUEîSCHER Jr" JoA. and AXELRAD B,J. (lgfl*)t Tncreased al.dosterone

output d.uríng sodÍ.¡rm d.eprlvation in normal man" Proe.Soc.E]æ.

Båo1"(N.y,)92* 650.

LUETS0HER Jro JnA" and CURTTS R.H. (Wil: Ald.osterone¡ observati.ons

on the regulation of sod.Íum and. potassiun balanee. Ann.Tnt.Med." þþ z 658" LUMBIR.S E.R" and SKINNER S.L, ?geg): Observatj-ons on the origin of renin i.n human urine, Ciroulat.Res. 2l¡ z 689.

MACDONALD G.J.r LOIIIS WoJ"u RENZINI V., B0YD G.ï¡. and. PEART T1I.S.

(lglO)¡ Renal cl1p h¡pertension in rabbÍts immunÍzed against angíotensin II. Cireulat.Res. 28 t 197.

MoGARRY 8.8., BATTANTYNE A. and" BECK J.C. (lg6Z)t Studies with antisera to adrenocorticotropin (ACÏH), in ÛIBA Foundation

ColloquÍ"a on Enðoerinology: Immunoassay of Hormones. (Boston: Little, Brown and Co.).

MoKENZTE J.Kn, l,ÉB M.R, and co0K !T.F. UgeA): Effeat of haernorrhage on arterial plasma renÍn activity in the rabbit. clnculatoResn lp z 269" MARTEts N.J" and. MULR0lv P"J. Ugarh RoLe of the renin-angiotensin system ån the regulation of ald.osterone secretíon j-n the rat.

Endoerinof,oey z 657 .re, " MASSANT Z"Mo, FINKIELMAN S., lf0RCEL M", AGREST A. and. PALADINT A.C.

(lggø): Angiotensin blood 1eve1s in hypertensive and" non- h¡pertensS.ve díseases. Clin.Sci. å9 t 473.

MENARD J., BOUCHffi. R. and. GEI'IEST J, (lg6l)z Applieation of a new

måero-nethod- for measurement of plasma renin activity" Canad"med." Ass"J" t( : 356. 158 " MORAN Jr" Tf.H,e ROSENBffi.G J.t" anð zTMMERMANN E" (lgfg)i rhe regulatton

of ald,osterone outputs significanee of potassåum ion, Surg.Forum

p : 12O.

MORRrs Jr. RoEo and ROBINSON P.R" (ll6t+): A method. for d.etermÍnation of angÍotensín Tr in human blood. 3ul-l"Johns Hopkins Hosp. 1lL z 127. MUrROvr P"J" (tl64h lhe rore of the renin*angi.otensÍn system in the

h¡4pertension associated" with renal vascular d.ísease. canad."Med. Ass.J. !Q : 277" MULROïr P,J. and GAN0NG 1¡¡.F, (gA("))r stimulation of aldosterone seeretÍon by angiotensin fT. Yale J.3ío1.Med, l] : 386. MULROTIT P,J. a^trd, GANONG Tf.F. (lg6l (¡))r The meehanism of aldosterone

sti"roulation following haemorrha8e in the h¡rpophysectomized. d.n6. J"elin"Tnvest" àC : 1065.

MUIROïII P"J. and, G.A,NONG 1{,F" (lg6Z): Role of the kid.ney and the renÍn*angiotensj"n system in the response of ald.osterone secretion to haemorrhage. Circulation 25 z 213.

ODELL T[oD., ïrrtBm. J,F" and" PAUL ]v.8. (1965)r Rad"ioåmmun.oassay of th¡motropin i"n human ser*dmo J.c1i"n,Endocr. !l : 117g. 00M0R0H0E c,c.0oe 0rM0RcH0E P.J. and" HENEY N.M. (lgl0): Renal htlar

lymph. Effeets of d,í.uresi.s on flow anð compasition in d.ogs"

Cireulat.Res. l+69 36 : " PAGE I"H. (lglg): 0n the nature of the pressor aetion of renin"

J"exp"Meå. JQ : 521 ,

PAGE I"H" and. HELMER 0"M" (tg¿uO): A erystallåne pressÕr substanee . (angiotonån) resultÍng from the reaotion between renin and. renin- aativatero Joexp"Med-" f! : 29. 159.

PAGE L.8,, HABffi. I" e KIMURA A.Y. and. PURNODE A, Ugeg)s Studies with the rad,íoimmu,ncassay for angi,otensín IT, anð its application to measurement of reni"n activi"ty" J"e1Ín.Endoer" !l : 200.

PEART 1["S. (lgSS): lhe å,solation of a h¡¡pertensin" Biochem.J. 62 z 52O"

PTOKEI\IS P.T., BUMPUS FuM", LL0YD A.Mu , SMEBY R,R" and PAGE f "H. (lg6f)z Measurement of renin aetivity in human plasma, Circulat"Res. fl z 438, PICKERING G.ïI, anð PRINZMEIAL M. (lgSg): Some observations on renin,

a pressor substanee eontaÍneå in normal kÌ"dney, together with method.

for åts biolog5"c assay, Cl"Ín,Scí , 211 " 2 " PTÎCOCK J"4., HARIR0FT P.M" and NETIW¡IAÏK L.N. ?gfg): Inereased renal pressor ac'tåvity (renån) in sodiurn ðeficient rats anå eorrelation

wåth juxtaglomerular ee1.1 gr"anulatÍ.on" Proc.Soc (m.y. " exp.Biol. ) JS : 868. R3'PPELLI A, anð PEART 1{.S. (tgg8): Renal excretion of renín in the rat" eåreul.at"Res. !f, : 531 " REA.D C.H. anå BRIAN G.T" UgeA): The Í-mmunological assay of human growth hormone. Reerent Progr"Hormone Res. 16 : 187"

REGOLT D. anð VANE J"R. (tgeÀ.)s A sensÍtive method for the assay of

angÌ"otensín" Bri.t"J"Pharmaco7., Æ- | 351 . REçOLI D. and VANE J.R" (lgøe): fhe continuous estimation of angio* 'bensin formed. ån the círeulatåon of the d-og. J.Physiol,(f,ond.")

-1-Êå : 153" 1 60.

R0SSEIIN G. e ASSAN R" e YAl,Ol[ R.S" and SER.SON S..4. (gAe) I Separation of antibody-bound. and. r.¡nbound peptid.e hormones labellecL with íod.åne- 131 by talcum powden and precÍpitated silica. Nature & t 35j. SCI{R0EDEA. Eo?,, EICH R.H", SMULTAN H. e GOITLD A. and GABUZDA G" (lgl0)z Plasma renÍn level Ín hepatic cj.rrhosis" Amer,J.Med" AZ : 186. scHWrzER R" e TSELTN 8., KAppELm. H", RINIKER B. , RIITEL Tf. and ZIIBffi. H. (lg¡g): S¡mthese hockwirksamer oktapeptid.e m:it der verrnutlichen amÍnosnduresequenz des noch unbekannten hypertensins II aus rinderserum (var5-r,ypertensin rr und val5*h¡rpertensin rr-asp-p- amid )u Hetvo0htn"Acta L1 : 1287. scOaNTK 0,4. and" PAl,ADrNr A.c. (t96t ): Angiotensín blood revels in

d.ogs wÍth experimental renal hSpertension. .A,mer.J.Physiol. 2U z

526 "

SHffiÏTOOD L"M., C.ARE 4.0,, MAYER G.D., ATTRBACH G,D" a¡rd" POTTS Jr. J.T. Ugeg): Evaluation by rad"åoimmunoassay of factors controllÍng the seeretion of parath¡rroíð. hormone. Nature ZW- z jZ,

SKIGGS Jr. L.T", LENIZ K.8., KAITN J"R., sHlIMl¡¿aY N.P, and nvOODS K"R" (lgf6): Amino aeåd eequence of hypertensin II. J'.exp.Med.

l-9& : 193. SKEGGS Jr. L"f. e MARSH W.H. , KAHN J.R. ancl SHUMIIAY N.p" (lgSÐt

The existenee of two forms of h¡pertensj"n. J"errp.Med. pJ : Z7S,

SKILIMAN JuJoo LAULER Dop., HrOKLm. RnB., ty0Ns JnH", 0LS0N J,E" anô M00RE F"D. ?peø): Haemorrhage in normal man: effeet on renin, al-d"osterone and eortÍsol, J.elin,Invest. þf, : 1A73, 161.

SKINNffi. S. (1967); Improved assay nethod"s for renin 'teoneentratíonrf tractivityfi and. in human plasma, Circulat,Res, 20 z 391

SKOM J"H. and TALI¡IA.GE D"lTe (gSA): Nonprecipitating insulin antibodies. J"cl-ín.Invest" l/ z 783. SLAT0N Jro P"E. and BIGLIERf E.G. (1967): Red.uced aLdosterone excretion in patíents with autonomic insuffieiency" J.clÍn,Encloer, fl z 37" srAK¡'¡{aNN G. (t960): A renin-lÍke pressor substance found" in the placenta of the cat, Acta path"microbiol.scand.. lQ : j\O. SUNDSFJORD J.A. (tgZo): Rad.ioimmunoassay of angiotensin Tr in plasma" Aeta endoer. fu: 181, IIGIR.SIEDT R. and BERGIIIAN P"G. (tggg)¡ Niere und. krieslauf " Skand.Arch.PhystoJ". B : 223. fOBrAN L" e JANEOTK J, and rOMBOtrf,rAN A. (lgfg): correlatj.on between granulation of juxtaglomerular cells and. extraetable renin in rats with experimentaL h¡4pertension. Proc,Soc.exp.Biol.(N.y. ) tOO z gt+. TRAUTSCHOLD I", II¡R.LE E., SCHMAL A. and HETVDRIK0FF N.G. ?gee): Die horrnonelle beeinflussung d.es isorenÍn-spiegels der submand.Íbular-

- a, isdr'use" cler weissen må,us und- zur lokalisierung ðes enzJ¡rns in d.er dr{fse" Z,PhysÍol.Chemo 3Æ, z 232.

UNGER RnH., EISI$IÎR.AII] 4"M., MoCALI M.S", KEttER. S., LANZ H"C" and.

MADÏSON L"t. (lgfg): slueagon antLbodÍes and their use for immuno- assay for glueagon" Proo"Soc,exp.Bíol" 1OZ z 6Zl" urrGER R.D" (1965): Rad.ioåmïnunoessay of human plasma th¡motropin" J.elån"fnvest, &L : 1277. 162.

urrGTR R.D., ODELL Tf.D. and" CONDLTFFE P.G, (1163): rmmunologi.c stud.ies of purÍfied" human and bovine th¡rrotropin. Endocrinology þ z 359.

IlTIem. R.D., PARKER M"L. and DA.UGITADAY If.H. (1962): Stud.ies on

human growth hormoneo A rad.ioimmunoassay for HGH. J.clin,Invest. ä! : zfl+. vArtOrr0N M.B. ¡ PAGE L.B. and HABER E. (lg67h Rad.ioimmunoassay of angiotensÍn in human plasma. Nature 215 z 714. vANDm. A"J. and. LUCTANO J.R. (tggZ(a)): Effects of mercurial diuresís and. aeute sod.Íum d.epletion on renin release in dog. Amer.J.Physiol. 6tt W, " vÀNDxR A"J. and" LUCTANO J.R. (1967(b)): Neural and. humoral eontrol of renin release in salt d.epletion. Circulat.Res. Z! Suppl.ií:69. VAND0NGEN R. and GORDON R.D. (tgzo)r Plasma renin in congestive heart failure in manu Med..J,Aust. 1 : 215. VEIINTNG E.H. anrl DYRENFIIRTH r. (lgf6): ALdosterone excretion in pregnancy. Joclin.End.ocî" & z 426. VEfAAf R., BRUNNER. H.R., MANNING E.L. and MIILLER A.F. (lg6e)z fnterrelati-ons entre le potassÍum, 1a r6nine et ltaldosterone"

Helv.med" Aeta jl Supp1"46:1i+1 .

\¡EyRAT R", cl.e C¡IAMPLAIN J", B01ICHER R. and GENEST J. (lg6tç)z

Measurement of human arterial renin actívity in sorne physiologicaL and. pathological states" Canad.med..Ass.J. !Q : Zl5" V!¡ATANABE M.e MEll(ER. C"f., GRAY M.J., SIMS.A."H. and" S0L0M0N S. (tg6l)z secretion rate of ald.osterone in normal pregnancy, J.clin.rnvest. þ/ z 1619, 163.

'!ïEISIR. R,4., JOHNSON A.G, and HO0BLER S.Jli¡o (lgSg): The effeet of anti"reni"n on the blood. pressure of the rat with experimental renal

h¡pertensÍon. Lab.Invest" 20 : 326.

ïr&.rE E" e VOGEL R, and. G0LDE"L F.L. (lgsil, ifi** ein blutdrueksteig-

ernd.es prinzÍ"p in extrakten aus d.er gland.ula submaxillaris d.er

weissen mauso Naur¡m Schmied.eberg Arch"E:p.Path. 23_Q ; 236, ITÏDE L" and GEIi{zELL c.A" (lgøo): An inmunological pregnaney test.

Aeta Endoar, þ3. z 261. ÌyrDE L. and. GEUZELL c" ?gez): Tmmunological detersrinatíon of pituÍtary luteinizång hormone in the uríne of fertile and post- menopausal" women and ad.ult men. Acta Endocr, âZ z 5jg. 'nrDa L. and P0R-A.TH J" ?gea): Radioimmunoassay of proteins with the use of Sephad"ex*coupl-ed. antibod.ies. Biochl"m"biophys"Acta (emst") 139 z 257.

1¡IIIDE L", R00S P. and GIMZELL C" (lg6l)z Immunological determination of human pítuitary luteinizing hormone (lH). Acta End"oer. fl : L45. ïfflÍ,ïAills G,H., ROSE L"I., DLUIIY R.G. MoCÂUGIIN 0., JAGGER p.I", HÏOKT,ER R.B. and" LAtrr,ER D.P" (lglo): Abnormal responsi"veness of 'fhe renin ald-osterone system to acute stímulatíon ín patients with essential h¡pertension" Ann.Int.Med.. @ : 317. wrls0N c. and PTCICERTNç G.Tli', (lg1g): Acute arteríal lesions in rabbits wåth experímental h¡pertension. Cl_in"Sei " þ z 3U3. T{INffi. B,M. (%f)¿ Renin in pregnaney and the nenstrual cyele" J.elÍn.Invest. I¡l¡ z 1112.

IIOLFE I,"K", GORDON RnDu, ISLAND D"P" and. LIDDT,S G.TII. (lgøA): An ana- lysls of faotors d.eterminÍng the ej-rcadian pattern of ald.osterone

exeretåo::" J"elin.End.oer" 26 : tr261 . 161+.

1¡{000S J.llfo p LïDDLE G"W., STÀNÏ Jro 8,G., MïCHELÄKïS A"M" and BRILL A.B" (1969): Effeet of an aðrenal inhibÍtor in h¡rpertensive patients wíth suppressed rentn" Arch"int.Med, llJ z 366" I¡IORCEL M. e MEYER P,, ANGLES d.lAtl'RIAC G", PAPANICOI¿AOU N, and. MILLffi. P. (lgl}): The role of angiotensin. Ind-írect studies with antiangío- tensin plasma" CircuLat.Reso 26 Suppl.Lí2223.

YA.LOTIÍ R.S. and Bnf.SON S"A. (lgfg): Assay of plasma ínsulin in human

subjects by ímmunologícal methods" Nature $å : 16¿18"

YAT,OIY R.S", GLICK S.M., ROTH J. and BXR.S0N S.A. (tl6l+): RadioÍmmuno- assay of human plasma ACTH. J.clin,End,oer, @ : 1219"

ADDIlÏONAI BIBTTOGRAPITY

AT,TCIltrK A. (t76t+): Renal biopsy and its clinlcal comelatÍon in toxaemia of pregnancy. Círculatíon åg (Suppl,ii):[J 0AIN M.D., Cj.TT K,i,, COGHLAN J.p" and" SLAIR-TIESf J.R. (lglo),

Evaluation of angiotensín TI metabolÍsm in sheep by rad,íoimmunoassay.

Endoorinofoey gá z 955" DEïcïo{aN T[oJo, P0TTER E.L. and" MecARTNEy c,p, ?gn): Renal biopsies from patients with frtoxaemi"a of pregnaïlclrro Amer.J"0bstet"G¡mec. lJr 1. çORDON R.D" and. PAïfsEY c"G,K. (lgll)z Relative effeets of serum sodium eoneentratÍon and the state of bod.y fluid balanee on renÍn

seeretiono J.o1in.End.oc'r. 12. z 117. 165.

HABIfi Ë., KOERNER T., PAGE L.8., KIIMAN B. ånd- PURNODE A. (lg6g)z ApplÍcation of a radioimmunoassay for angiotensi.n ï to the

physiologic measurements of plasrna renín activity in norrnal human sub jects. J.c1in.End.ocr. !l : 1349, MoCARTNEY C.P., SPARçO 8., LORINCZ A.8., T,IF'EBVRE Y. and NEllllON R.E.

(t g64): Renal structure and. funetion in pregnant patients with

acute h¡pertension. Amer. J.0bstet.G¡rnec . 9,9 : 579 .

McfNlOSH G.H. and MORX.IS B. (lgll)z fhe lymphatics of the kidney

and. the formation of renal lymph. J.Physiol. (ft Press).

IIIAUÏNER W., CHIIRG J., GRISHMAN E. and DACHS S. (geÐ: Pre-eclamptic

nephropathy: An electron mÍcroscopÍc study. Lab.Invest. 11 : 518.

PAWSEY C.G., VANDONGU\I R. and GORDON R.D. (1971)z Renal venous renin ratio Ín the d.iagnosis of renovascular h¡pertensÍon and measurement d.urång acute secretion of renin. Med..J.Aust. 1 r 121 . POLLACK V.E. and" NE*ITLES J.B. (1960): fhe kid.ney in toxaemia of pregnancy: A cl:inícal and pathologÍca1 study based. on renaL blopsÍes. Meilicine ll : l+69. SPAR0O 8., MoCARTNEY C.P. and. ïtITNfivlILI,m. R. (lgfg): Glomorular capillary endothel-iosis in toxaemia of pregnancy. Arch.Path. (cnicaso) @: 593. THURAU K. (lg6t*): Renal haemod.¡mamf.cs. Amer.J.Með,. & z 698.