catecholamine turnover...

Catecholamine Turnover in Nornotensive and

Hypertensive Man: Effects of Antiadrenergic Drugs

VINcENT DEQuArrRo and ALBERTSJOERDSMA

From the Experimental Therapeutics Branch, National Heart Institute,Bethesda, Maryland 20014

A B S T R A C T Intravenous administration of tri-tium-labeled 3,4-dihydroxyphenylalanine (dopa)to human subjects resulted in the labeling of en-dogenous catecholamines and vanillylmandelic acid(VMA). Determination of the changes in specificactivity of these compounds with time in fractionalcollections of urine and in cardiac biopsies from pa-tients undergoing corrective cardiac surgery per-mitted estimation of apparent turnover rates. Theaverage half-time of the exponential decline inspecific activity of labeled urinary norepinephrinewas about 8 hr and that of VMAwas 11-16 hr infive normal subjects. No significant differencesfrom normal were observed in eight patients withessential hypertension. The average half-life ofnorepinephrine was only 5 hr in cardiac patientsundergoing surgery, and the levels and rate of de-cline of cardiac norepinephrine specific activitycorrelated well with the exponential phase of theurinary disappearance curve. There were sig-nificant effects of treatment with alpha-methylty-rosine, reserpine, and pargyline hydrochloride onthe labeling and apparent turnover rates of nor-epinephrine and VMA; the effects noted wereconsistent with known actions of these three drugs.It is suggested that the technique used is a suitablemeans of assessing "over-all" catecholamine me-tabolism in man, particulary if combined with

This work was presented in part at the 1967 AmericanHeart Association Meeting. Circulation. Supplement II,36: 96.

Dr. DeQuattro's present address is the University ofSouthern California, Los Angeles County Hospital,Department of Medicine, 1200 North State Street,Los Angeles, Calif. 90033.

Received for publication 29 March 1968 and in re-vised form 10 June 1968.

quantitative assay of urinary catecholaminemetabolites.

INTRODUCTION

It has been suspected that dysfunction of the sym-pathetic nervous system is a pathogenetic factor inessential hypertension. This supposition is tenablebecause of: (1) clinical impressions that some hy-pertensive patients exhibit sympathetic "hyper-activity;" (2) the enhanced vascular responsive-ness of hypertensive subjects to injections of nor-epinephrine (1, 2); and (3) the effectiveness oftreatment with antiadrenergic drugs. One wouldexpect that gross abnormality of sympatheticnerve activity would be detected by measurementsof the rate of excretion of norepinephrine and itsmetabolites in the urine. However, numerousstudies (cf. 3) of urinary catecholamine metabo-lites have failed to reveal any consistent abnormali-ties in cases of essential hypertension. Nonethe-less, a few reports of alterations in the metabolismof endogenous or exogenous norepinephrine inhuman (3-6) and experimental (7, 8) hyperten-sion indicate the issue is not yet settled.

Isotopically labeled tyrosine, 3,4-dihydroxy-phenylalanine (dopa), 3,4-dihydroxyphenylethyl-amine (dopamine), and norepinephrine have beenused to determine the turnover rate of norepi-nephrine in tissues of intact animals and in per-fused organs. These studies have contributed toour knowledge of sympathetic nerve function.Attempts to measure norepinephrine turnover ratein man have been rather limited, employing DL-norepinephrine-'H (4, 9, 10) and dopamine-"C(11). For this reason it was decided to carry out

The Journal of Clinical Investigation Volume 47 1968 2359

further studies, particularly in regard to the ques-tion of an abnormality in norepinephrine metabo-lism in essential hypertension.

Dopa-3H was chosen for these studies of cate-cholamine turnover for several reasons. Dopa ismetabolized rapidly after intravenous infusion,allowing for pulse labeling of tissue catecholamines,whereas the dietary precursor, tyrosine, persistsin tissues for long periods after administration(12). Unlike norepinephrine (13) and dopamine(14), L-dopa readily penetrates the blood brainbarrier (14) and nonspecifically enters sympa-thetic neurones (15, 16) where it is converted tothe neurotransmitter substance, L-norepinephrine.Thus, endogenous labeling of norepinephrine fromdopa_3H occurs by a more physiologic route. Ofpractical importance is the fact that dopa-3H ofhigh specific activity has recently become avail-able as the levo isomer, labeled exclusively on thering portion of the molecule.

The feasibility of endogenous labeling of cate-cholamines in man was shown in preliminarystudies in which, after administration of dopa-3H,easily measured quantities of radioactivity wererecovered in urinary dopamine, norepinephrine,and vanillylmandelic acid (VMA) (17). In thepresent work, the rate of exponential decline ofradioactivity in each urinary compound after la-beling with dopa-3H is utilized as an index of

turnover rate. Comparative studies of turnoverrates were made in normal subjects and patientswith essential hypertension, the latter both in con-trol state and during treatment with drugs knownto decrease sympathetic neural activity. In severalpatients undergoing surgery for heart disease, thelevels and rate of decline of specific activity ofcatecholamines in urine after dopa-3H labelingwere correlated directly with those in cardiactissue.

METHODSThe subjects of study were: five normal volunteers (twomales and three females, aged 19-35 yr) ; 10 patients(six women and four men, aged 32-54 yr) with uncom-plicated essential hypertension; and eight patients(four males and four females, aged 16-42 yr) with con-genital or rheumatic heart disease (functional class I-II[American Heart Association criteria], six cases; classIII, one case; class IV, one case) who underwent cor-rective cardiac surgery. All subjects were hospitalized atthe time of study. Blood pressure elevation was sustainedin the hypertensive patients throughout the period of theturnover studies at approximately those values given inTable I. The mean supine blood pressure for the un-treated group was 162/111. Patients with secondarycomplications were excluded in preliminary studies.Blood urea nitrogen and serum creatinines (of volun-teers and patients alike) were normal; all patients hadnormal rapid sequence intravenous pyelography, and inthose instances where confirmation was sought, renalarteriography was normal. The patients were not in

TABLE I

Clinical Features of the Hypertensive Patients

Renal Prestudy therapyllBlood Retinopathy Serum evaluation

Patient Age, Sex pressure* grader creatinine tests§ Initial Repeat

mm/Hg mg/1OO ml

1 A. L. 32 F 134/96 I 0.8 IVP None Reserpine2 E. H. 49 F 152/110 II 0.9 IVP None a-MPT3 D. E. 43 F 140/112 II 1.0 IVP,A None a-MPT4 R. L. 54 M 160/120 II 0.9 IVP,A None5 B. R. 52 M 250/138 II 1.0 R None6 A. T. 34 M 190/120 II 0.9 IVP,A None Pargyline7 M. D. 48 F 140/100 II 1.0 IVP,A None a-MPT8 H. T. 54 F 130/94 II 1.2 IVP,A None9 G. D. 33 F 146/108 II 0.9 IVP,A Pargyline

10 XV. J. 40 M 140/100 II 0.9 IVP Reserpine

* Mean of three measurements, supine, at the time of the initial turnover study.t Keith-Wagner classification. D. E. had grade IV retinopathy 1 yr before the initial turnover study.§ Tests for screening renal arterial disease were negative in all patients: IVP, rapid sequence intravenous pyelogram;A, renal arteriogram; R, renogram in patient with iodide allergy.

Patients designated as None received no medication for 4-6 wk before the study. The duration and dosage of medica-tions of a-MPT, pargyline, and reserpine are given in both Table VI and the legend of Fig. 7.

2360 V. DeQuattro and A. Sjoerdsma

congestive heart failure nor were they receiving digitalisglycosides. Eight patients received no drug therapy for4-6 wk before study. Two were studied only duringdrug treatment, pargyline in one case and reserpine inthe other. Five of these eight patients were studiedagain during drug therapy, three during treatment withalpha-methyl-para-tyrosine 1 (a-MPT) and one eachduring treatment with reserpine and pargyline hydro-chloride (Eutonyl).

Tritium-labeled 3,4-dihydroxyphenylalanine (dopa)was obtained 2 in two forms. These were DL-DOPA-3H(G, 0.256 c/mmole) and L-DOPA-3H (ring 2,5,6-$H, 0.5 c/mmole and 2.0 c/mmole). The purity ofeach batch of labeled compound was ascertained bystrip counting of paper chromatograms of the material,developed in n-butanol saturated with 2 N HC1, and inn-butanol: acetic acid: water (4: 1: 1). In one instanceit was necessary to purify the racemic compound by ad-sorption and elution from alumina (18). Solutions ofcompounds for intravenous use were prepared by dis-solving or diluting in 0.25 N acetic acid or 0.01 N HClcontaining 1% sodium-meta-bisulfite as preservative, fol-lowed by 0.45 g Millipore filtration (filtrate pyrogentested) and storage in dark vials at - 15'C. Before in-fusion, 0.4-1.5 ml of solution was diluted to 100 ml with5% dextrose in water.

Clinical ProceduresIntravenous infusions of dopa-3H were given in the

fasting state, except in some of the patients undergoingcardiac surgery who were given dopa-3H 2-22 hr beforecardiac biopsy (atrial, seven cases; papillary muscle,one case). All dopa-3H infusions were given at a con-stant rate for 30 min. In the first seven studies (fournormals and three hypertensives), varying doses ofdopa-3H were administered to establish the feasibility ofendogenous labeling of catecholamines. These dosesranged from 1.02 to 4.8 mc (0.1-3.7 mg of L or DL-dopa)and were administered at a rate of 1.33-5.32 ,c/kg permin for a total dose of 15-80 juc/kg. In subsequent stud-ies L-dopa-RH was infused at a rate of 0.5 gc/kg per minfor 30 min for a total dose of 0.35-1.35 mc (0.14-0.53mg). These amounts of L-dopa were found to be insuffi-cient to alter detectably the urinary excretion of nor-epinephrine or VMA; the higher doses used in earlystudies did cause a slight increase in urinary dopamineduring the 1st 3 hr after the infusion.

Beginning just before the infusion, timed urine sampleswere collected for 3-7 days in glass bottles containingsufficient 6 N HCl to give a final urinary pH less than3.5 and were stored at 0°C for periods up to 1 month.In surgical patients, urine was collected by indwellingcatheter during and following the operation. The atrialappendage biopsies were performed as a routine partof the surgical procedure for the purpose of cannulationof vena cavae and establishment of cardiopulmonary by-

1 Supplied by Dr. Elmer Alpert, Merck, Sharp &Dohme, West Point, Pa.

2 Nuclear-Chicago Corporation, Chicago, Ill.

pass, and not for the purpose of the present study. Inone instance (C. R.) the tissue obtained was a piece ofpapillary muscle that was taken 10 min after institutionof cardiopulmonary bypass.

RadioassayAliquots of urine, various eluates, and extracts were

dissolved in scintillation fluid (19) and counted in aPackard Tri-carb liquid scintillation spectrometer, series4000. Counting efficiency was in the range of 15-20%with a background of 10-20 cpm. Counts in any samplewere not considered significant unless they were at least40 cpm above background. In all instances, counting timewas adjusted to assure a counting error of less than 5%.

Chemical AssaysA. CATECHOLAMINES

Dopa, dopamine, and norepinephrine were isolated fromaliquots of urine by combinations of column chromatog-raphy on alumina, IRC-50 (Na+) and Dowex-50 (Na+),and paper chromatography (12, 18, 20). Specific separa-tion and assay techniques were as follows.

(1) Dopa. For assay of unchanged dopa-8H, 50 ltg ofcarrier DL-dopa was added to urine aliquots. The aliquotswere then subjected to alumina adsorption and elution,the eluate applied to IRC-50 columns, the effluent there-from reapplied to alumina, and the dopa again waseluted with 0.2 N acetic acid. The final eluate was evapo-rated to dryness under nitrogen, redissolved in a smallamount of 0.1 N HCl in ethanol, and was chromatographedon No. 1 Whatman paper in n-butanol: acetic acid: wa-ter (4:1: 1). Paper strips were cut and the dopa waseluted with 0.01 N HCl. Aliquots of appropriate stripeluates were assayed for dopa fluorometrically (20), anda second aliquot was used for scintillation counting. Inpatients who had received DL-dopa-3H, a third aliquot wasassayed for L-dopa-3H by conversion to dopamine withguinea pig kidney decarboxylase (21). Since dopa is notdetectable in urine normally, the radioactive dopa inurine was assumed to have the same specific activity asthat administered. Accordingly, the excretion of dopa-'Hcould be calculated from the degree of dilution of itsspecific activity by the carrier dopa.

(2) Dopamine and norepinephrine. These catechola-mines were adsorbed on alumina from aliquots of urineand protein-free filtrates of heart homogenates, eluted,and a portion of the eluate assayed for norepinephrine(18). In patients treated with alpha-methyltyrosine, thenorepinephrine was assayed after elution from a Dowex-50 (Na+) column as described below. The recovery ofnorepinephrine from urine averaged 80%. Remainingalumina eluate plus carrier norepinephrine was then ap-plied to an IRC-50 column as for dopa (see above).Norepinephrine and dopamine were eluted with 1 N aceticacid and then chromatographed on Dowex-50 (Na+) andeluted as separate fractions with 0.4 N HCI and 2 N HCI,as described by Bertler, Carlson, and Rosengren (20).The amines were assayed by natural fluorescence (nor-

Effects of Antiadrenergic Drugs on Catecholamine Turnover 2361

epinephrine) or as fluorescent derivative (18, 22). Ali-quots of the eluates from the Dowex-50 columns wereevaporated under a N2 jet, the amines redissolved in0.01 N HCl, and radioassay done. Over-all recovery ofnorepinephrine and dopamine from urine with the "3-column" procedure averaged 70%. The aforementionedprocedures permitted accurate determination of thespecific activities of norepinephrine and dopamine inurine following administration of dopa-3H.

The validity of determinations of the specific activitiesof dopamine and norepinephrine in Dowex-50 eluates wasestablished by paper chromatography (Fig. 1). The spe-cific activities of the amines in the Dowex eluates weresimilar to those of the amines after isolation from thepaper. In separate studies it was also ascertained thatneither the 0.4 N HCl or 2.0 N HCl Dowex eluates (frac-tions A and B, Fig. 1) contained significant amounts ofradioactive epinephrine.

B. VMATl~e specific activity of urinary VMA in patients re-

ceiving L-dopa-3H was determined by conversion tovanillin and subsequent spectrophotometric and radio-assay. A simple modification of the procedure of Pisano,Crout, and Abraham (23) was used in that vanillin

6

.5Ii* ~ ~~~~~~I'

4 t -Froctioni

E ICL3 Fraction A /03AzcpmXI

I ~~~~~52 8 zcpniX/

0 5 10 15 20

DISTANCE FROMORIGIN IN CM

FIGURE 1 Purity of Dowex-50 eluates used for deter-mination of specific activities of norepinephrine and dopa-mine. This is a semischematic presentation of results ob-tained when the radioactive catecholamines were isolated(see text) from the urine of a hypertensive patient(H. T.) who had received DL-dopa-3H. Aliquots of the0.4 N and 2.0 N HC1 Dowex-50 eluates (fractions A andB) containing 7.28 X 10' and 9.8 X 10' cpm were chro-matographed descending in a butanol: HCl system onNo. 1 Whatman paper; 1.25-cm paper strips were cut,eluted (1 ml 0.01 N HCl), and counted for radioactivity.Purity of the two radioactive fractions is demonstratedwith migrations of radioactivity on paper coinciding withthose of cochromatographed authentic norepinephrine(NE) and dopamine (DA). The latter were locatedwith a potassium ferricyanide spray.

was returned to 2 N NH40H instead of 1 M K2CO0 tofacilitate miscibility with scintillation fluid. Specificityof the determination was assured by chromatographingthe thiosemicarbazone derivative (24) of vanillin onpaper in isopropanol: ammonia: water (40: 1: 10).8 Theappropriate region was eluted with ethanol: 0.1 N NH40H(1: 1). One aliquot was assayed fluorometrically in 0.1 Msodium borate, pH 10, and another radioassayed in thescintillation mixture. Specific activities of the derivativeafter chromatography agreed closely with those ofvanillin.

Tritium exchangeInvestigations were made for evidence of tritium ex-

change in vivo and in vitro. When acidified urine sampleswere evaporated under a jet of N2, less than 3% of theradioactivity was volatilized. It was found however thatthe 0.4 N HC1 Dowex eluates of the initial urine speci-mens from a few patients underwent a 30-50% loss ofradioactivity with evaporation. There was no subse-quent loss with repeated acidification and evaporation.Also, it was found that several batches of L-dopa-3Hcould be evaporated from 6 N HCl without occurrenceof tritium exchange. Ring-labeled dopamine prepared bydecarboxylation in vitro of a batch of L-dopa-3H (500 mc/mmole) was carried through the three step procedurewithout evidence of tritium exchange. Therefore, the vola-tile radioactivity found in some Dowex eluates wasprobably tritiated water arising from metabolites otherthan dopamine or norepinephrine. In the case of sucheluates the specific activity values presented are thoseobtained after evaporation. In the experiments withDL-dopa-3H (G), no corrections were made for tritiumdisplacement from the beta-carbon of the side chain re-sulting from enzymatic hydroxylation at this position.

RESULTS

Urinary catecholamine excretion in normal vol-unteers and hypertensive patients

The urinary excretion of the catecholamines,metanephrines, and VMA for both normal vol-unteers and hypertensive patients were withinthe range of normal values established for this lab-oratory (Table II). There were no significantdifferences between the two groups, nor werethere changes after reserpine therapy. The post-therapy values of patients A. L. and W. J., re-spectively, were catecholamines, 29 and 34 ,ug/day;total methylated amines, 0.30 and 0.40 mg/day;and VMA, 4.4 and 4.3 mg/day. Changes in cate-cholamine metabolite excretion were noted, how-ever, after pargyline therapy. The posttherapy

3 Authentic vanillin thioseinicarbazone for referencepurposes was supplied by Dr. Harry. Goldenberg, Bio-Science Laboratories, Los Angeles, Calif.

2362 V. DeQuattro and A. Sjoerds-ma

TABLE I IUrinary Calecholamines and Metabolites in Normotensive

Volunteers and Hypertensive Patients

Norepi- Epineph- TotalPatients nephrine rine metanephrines VMA

jgg/24 hr* mg/24 hr*Normotensive

1 D. B. 26 4 0.30 3.42 M. B. 42 16 0.35 4.03 T. R. 29 6 0.37 3.64 C. K. 22 5 0.27 3.2S L. G. 39 10 0.26 2.9

Means 4 SE 32 ± 4 8 1 2 0.31 4 .02 3.4 4± 0.2

Hypertensive1 A. L. 23 6 0.24 4.42 E. H. 53 5 - 3.93 D. E. 59 6 0.32 3.64 R. L. 17 6 - 3.85 B. R. 65 12 0.54 4.36 A. T. 54 6 0.36 4.47 M. D. 33 11 0.34 4.08 H. T. 34 10 0.25 1.6

Means i SE 42 ± 6 8 ± 1 0.34 4±.04 3.8 ± 0.3

* Each value is the mean of two or three consecutive 24-hr urine values.The unconjugated catecholamines and VMA were determined byfluorometric (18) and photometric assay (23), respectively. See textfor details. Total, conjugated and unconjugated, normetanephrine,and metanephrine were determined by photometric assay (45).

values of patients A. T. and G. D., respectively,were catecholamines, 56 and 30 Jug/day; totalmetanephrines, 1.1 and 0.8 mg/day; and VMA,0.8 and 1.7 mg/day. Alpha-methyltyrosine therapyresulted in decreased excretion of the catechola-mines and VMA in the three patients E. H.,M. D., and D. E., as given in Table VI.

Fate of dopa-5HAs shown in Fig. 2 there was rapid excretion

of radioactivity in the urine after the administra-tion of radioactive dopa intravenously, with ap-proximately 50%o of the administered dose of iso-tope being excreted within 3 hr. The total excre-tion of radioactivity in 24 hr as a percentage ofdose was 92%o for radioactive DL-dopa and 93%ofor L-dopa. The percentages of radioactivity ex-creted as unchanged dopa and as urinary dopa-mine, norepinephrine, and VMA are shown inTable III. Although 0.2%o of the radioactivity fromDL-dopa-3H and 0.6%o of L-dopa-8H could be re-covered as unchanged L-dopa, no radioactiveL-dopa was found after 3 hr. This finding wascritical and indicated that there was not continuousrelabeling of the catecholamines by the precursor.Following infusion of DL-dopa-sH, small amountsof the D-isomer could be detected for 15-20 hr and

the total as per cent of dose was about 11.5%o(Table III). The excretion of radioactivity asdopamine was in the order of 10%o; 1-27o wasrecovered as norepinephrine plus VMA. Countsappearing in these latter compounds were suffi-cient for determination of specific activity, not onlyfor the first 24 hr, but for an additional 24-48 hras well. The ratio of counts in VMAto that innorepinephrine was about 70 to 1 and approxi-mated the ratio of endogenous levels of thesecompounds in the urine.

Turnover rates of norepinephrine, dopamineand VMAin normal and hypertensive subjectsWhile dopa-3H disappeared rapidly from urine,

valid information on the turnover rate of norepi-nephrine also required ideally that the specific ac-

100

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CI)

2 50

LUI

uj

I-4

(i' 25

0

* -D -DOPA-3H0 - L- DOPA-*H

6 12 18 24

HOURS

FIGuRE 2 Cumulative excretion of radioactivity in theurine following intravenous administration of radioactiveL-dopa and DL-dopa. Each point represents the mean ofresults in three subjects for each compound; the bracketsdenote ranges of maximum and minimum values. Indi-vidual values did not deviate from the mean by more than10%.


TABLE IIIPercentage of Administered Dose of Radioactivity Recovered

in Various *Urinary Metabolites during the 1st 24 hrafter Intravenous Dopa - 3H

Norepi-Compound Dopa- neph-

administered* Subject D-Dopa L-Dopa mine rine VMA

DL-Dopa-3H D. B. 10.0 0.2 8.6 0.023 -

M. B. 13.0 0.2 8.0 0.023 -H. T. - - 7.4 0.014 -

Mean 11.5 0.2 8.0 .020

L-Dopa-3H A. L. 0.0 0.4 12.0 0.027 1.5E. H. 0.0 0.5 13.0 0.020 1.1D. E. 0.0 1.0 16.0 0.040 1.9R. L. - - 10.2 0.019 2.0B. R. - - 10.0 0.019 1.6

Mean 0.0 0.6 12.2 0.025 1.6

* Doses administered ranged from 20 to 80 uc/kg for DL-dopa-3Hand were 15 uc/kg for L-dopa-3H.

tivity of the intermediate compound, dopamine,also declines rapidly. A typical example of the ratesof decay in specific activities of dopamine, norepi-nephrine, and VMAis shown in Fig. 3. The spe-cific activity of dopamine first exceeded, then, at

10,000

8

-LE 1000

C1

100

12 hr became less than that of norepinephrine.Thus, the possibility exists of a small degree ofrelabeling of norepinephrine from dopamine dur-ing the 1st day. On the other hand, the decaycurve for norepinephrine-3H generally became ex-ponential before its specific activity exceeded thatof dopamine. The norepinephrine decay curve borea precursor-product relationship with the VMAdecay curve.

The norepinephrine specific activity plottedsemilogarithmically against time from a repre-sentative normal subject is shown in Fig. 4. Inthis instance, and generally, there was an initialrapid fall, and after 6-12 hr, an exponential rateof decline with a half-time of 8 hr. This exponen-tial decline lasted 2-3 days, the period in whichthere was measurable activity. In a few subjects,studies of pooled alumina eluates demonstrated thesame decay rate for 4 days. Extrapolation of thisexponential to zero time (y axis) affords an indexof the size of the "pool."' The height of the y in-tercept is inversely related to the amount of en-dogenous norepinephrine in the pool (dilution ofthe tracer). If the exponential curve is subtracted

10,000

000

8I.0EEE

100

0 1 2 24 36 48 60HOURS

10 I' FIGURE 4 The curve of decline of norepinephrine spe-o 12 24 36 48 cific activities of normal subject T. R. (cf. Fig. 3) and

HOURS its two major components are illustrated. The exponential

FIGURE 3 Comparison of the rate of change of specific phase of decay is extended to its Y intercept and theactivity of urinary dopamine, norepinephrine, and VMA early rapid component is derived graphically by sub-after administration of L-dopa-5H in a normal subject tracting the exponential component from the curve of(T. R.). decline.


from the original curve there remains an earlycomponent indicative of one or more pools ofnorepinephrine having a rapid turnover.

In Fig. 5, the specific activity-time curves forurinary norepinephrine in five normal subjectsare compared with the average -of results in sixpatients with essential hypertension. The curvefor normal subject L. G. who received the samedose of isotope as the hypertensive subjects wasalmost identical to the mean curve of the hyper-tensives. Individual turnover rates for the threemetabolites were calculated graphically from theexponential portions of curves constructed visu-ally, and are listed in Table IV. The latter alsoincludes results from two additional hypertensivepatients (M. D. and H. T.) who received a dose

o0p000

1000

W

EE-E

100

t00 12 24 36 48

HOURS

FIGURE 5 Comparison of the rate of change of specificactivity of urinary norepinephrine in five normal sub-jects and six patients with essential hypertension. Radio-active dopa was infused over a period of 30 min begin-ning at zero time. Doses of precursor in the normal sub-jects were as follows: D. B. and M. B., 20 ,c/kg ofDL-dopa-'H; L. G., 15 ,uc/kg of L-dopa-'H; T. R., 25 ,uc/kg of L-dopa-3H; and C. K., 35 ftc/kg of L-dopa-'H. Allsix hypertensive patients received the same dose ofL-dopa-'H, 15 ,uc/kg; this enabled a single curve to bedrawn utilizing mean values. The brackets indicatestandard errors of the means.

TABLE IVTurnover Rates of Dopamine, Norepinephrine, and VMA

in Normal and Hypertensive Subjects

Half-lives*

Norepi-Dopa- neph-

Group Subject mine rine VMA

Normal 1 D. B. 7 82 M.B. 6 7 -3 T. R. 7 7 154 C.K. 6 8 165 L. G. 5 8 11

Mean 6.2 7.6 14.0

Hyper-tensive 1 A. L. 8 8 11

2 E. H. 9 8 133 D. E. 8 8 124 R. L. 8 7 145 B. R. 8 9 126 A.T. 7 8 137 M. D. 12 8 168 H.T. 8 8 -

Mean 8.5 8.0 13.0

*Dosages of dopa-3H were as per legend, Fig. 5, and text.Values for half-lives were estimated to the nearest hrfrom exponential components of the decay curves.

of isotope (20 P&c/kg of L-dopa-3H and 80 juc/kg ofDL-dopa-3H) different from that given to the otherhypertensive patients (15 uc/kg of L-dopa-83H).Weinterpret the results as showing no significantdifferences between the two groups. This is clearlythe case for norepinephrine with average half-livesbeing 7.6 and 8.0 hr for the normal and hyper-tensive groups, respectively. Possible minor dif-ferences in turnover rates of dopamine and VMAwould require evaluation of a much larger seriesof cases. There was considerable variation in the yintercepts (cf. Fig. 4). With correction for varia-tions in isotope dosage, the means and standarddeviations of y intercept values for the normals andthe hypertensive patients were 4828 + 2407 and3540 + 539 dpm/mpumole, respectively.

The half-time of the rapid component (cf. Fig.4) of the disappearance curve of radioactive nor-epinephrine was 1 hr for both normals and un-treated hypertensives. As a further comparison ofthe fate of norepinephrine-3H in the two groupsof subjects, the ratios of counts excreted in nor-epinephrine within 3-6 hr to the total counts ex-creted in norepinephrine in 48 hr were calculated.


As shown in Table V, 50 and 54%o of the totalnorepinephrine counts were excreted by the twogroups within 3 hr, and both excreted 68%o within6 hr. Thus, there was no evidence of an abnor-mality in the disposition of norepinephrine-3H inpatients with hypertension, regardless of the timeelapsing after dopa-3H administration.

Effects of antiadrenergic drugs

Three drugs were selected for study because oftheir distinctive and fairly well documented ac-tions on catecholamine metabolism. These werealpha-methyltyrosine (a-MPT), reserpine, andpargyline. Alpha-MPT decreases tissue levels ofcatecholamines in animals by inhibiting the hy-droxylation of tyrosine to dopa (25). Adminis-tration of the compound to humans results in amarked decrease in the urinary excretion of cate-cholamine metabolites; this is presumed to be in-dicative of inhibition of catecholamine synthesisat the tyrosine hydroxylase step (26, 27). It iswell known that reserpine depletes tissue storesof norepinephrine (28). It is generally agreed thatreserpine interferes with mechanisms of storage ofthe amine in sympathetic neurones (29). Pargylinehydrochloride is a monoamine oxidase inhibitor.Monoamine oxidase inhibitors interfere with ex-tra- and intraneuronal metabolism of monoamines,and in some species increase tissue levels of nor-epinephrine (30, 31.).

A. Alpha-methyltyrosine. It was of interest todetermine whether, in the presence of a decreasedrate of catecholamine synthesis produced by alpha-

TABLE V

Cumulative Norepinephrine-3H Excretion at 3, 6, and 48 hrafter Dopa-3H Infusion in Normal and

Hypertensive Subjects

Fraction of 48 hrnorepinephrine-sH

Norepinephrine-1H recovered

Hr... 0-3 0-6 0-48 0-3 0-6

(dpm X 103)Normals

1 L. G. 328 414 575 0.57 0.722 D. B. 414 535 821 0.51 0.663 M. B. 297 518 823 0.36 0.634 C. K. 403 586 915 0.44 0.645 T. R. 558 677 910 0.61 0.74

Mean 0.50 0.68

Hypertensives1 A. T. 705 890 1,140 0.62 0.782 H. T. 646 842 1,520 0.56 0.733 M. D. 358 415 703 0.51 0.594 R. L. 287 391 551 0.52 0.71S A. L. 294 336 473 0.62 0.716 B. R. 287 405 653 0.44 0.627 D. E. 396 535 775 0.51 0.698 E. H. 260 310 500 0.52 0.62

Mean 0.54 0.68

MPT, the formation of norepinephrine and VMAfrom dopa-3H was intact and, if so, to observe theapparent turnover rate of norepinephrine. Threepatients were studied both before and during treat-ment with the inhibitor. The effect of alpha-MPTon the urinary excretion of norepinephrine and

TABLE VIEffect of Alpha-Methyltyrosine on the Urinary Excretion of Norepinephrine and VMAand on the

Formation of These Compounds from L-Dopa-3H.

Norepinephrine* VMA* Norepinephrine-gHt VMA-'HHypertensivepatients Control Drug§ Control Drug§ Control Drug§ Control Drug§

(jug/24 hr) (mg/24 hr) (dpm X 10') (apm X 104)M. D. 32.9 11.1 4.0 1.4 703 1235 3732 2762

E. H. 52.8 15.3 3.9 2.1 500 801 2330 2920

D. E. 59.3 46.1 3.6 2.1 775 1360 2335 2355

* Values shown are means of results of determinations on three consecutive 24-hr urine collections.Values shown are dpm in 1st 48 hr after administration of L-dopa-3H, 20 pc/kg in patient M. D. during control and

15 pc/kg during drug treatment, and 15 pc/kg in patients E. H. and D. E. during both control and drug treatment periods.§ Alpha-methyltyrosine treatment was: 2.0 g/day for 14 days in patient M. D., 3.0 g for 7 days in E. H., and 2.0 g for7 days in D. E.; equal doses (0.5 or 0.75 g) of drug were given at 6-hr intervals and treatment was continued throughoutthe period of study.


VMAand on the formation of these compoundsfrom dopa-3H is shown in Table VI. The decreaseof norepinephrine synthesis produced by the drugwas apparent from reductions in urinary excre-tion of the two compounds. That the pathway ofsynthesis from dopa to norepinephrine was intact,however, is indicated by the fact that total incor-poration of radioactivity from dopa-3H into uri-nary norepinephrine was not inhibited but in-creased slightly (Table VI). The values for spe-cific activity of urinary norepinephrine afterL-dopa-3H and the half-life times of norepineph-rine-3H disappearance in the three patients aregiven in Table VII; specific activity-time curvesfor one of these patients are depicted in Fig. 6.The specific activity of norepinephrine at varioustimes after dopa-3H was increased during alpha-MPT treatment to 3-10 times that found in thecontrol state. The turnover rate was decreased,as indicated by a 1-4 hr increase in half-life. Turn-over rates of VMAwere unchanged from an av-erage half-life of about 14 hr.

B. Reserpine and pargyline. A comparison ofnorepinephrine-8H disappearance rates in hyper-tensive patients receiving each of these drugs withthat of the six untreated hypertensive patients isshown in Fig. 7. It may be seen that the decaycurves for two patients receiving long-term treat-ment with 0.25 mg/day of reserpine appeared to

TABLE VIIEffect of Alpha-Methyltyrosine on the Specific Actitity and

Disappearance Rate of Urinary Norepinephrine-3Hin Three Patients with Hypertension

Specific activity of norepinephrineHours M. D. E. H. D. E.

afterinfusion Control Drug* Control Drug* Control Drug*

dpm/m~umole0-3 8,000 51,000 7,100 20,000 5,400 17,0003-6 3,930 19,200 1,450 8,800 2,100 3,4806-12 1,350 5,100 1,900 5,600 1,430 1,600

12-18 540 3,000 730 2,120 308 57518-24 570 2,030 570 1,500 360 40024-30 250 1,100 - 980 - 22230-36 185 870 125 - 90 16036-42 120 700 - 7136-48 450 7342-48 60 650 82 5248-60 10 250 37 - 12 -

Half-life (t4)of norepineph-rine-AH in hr 8 11 8 12 8 9

* Dosage of drug and dopa-H as per footnote to Table IV.

0 12 24 36 48

HOURS

FIGURE 6 Specific activity-time curves of urinary nor-epinephrine in a hypertensive patient (M. D.) before andduring treatment with alpha-methyltyrosine. Doses ofdrug and isotope were as given in Table VI.

have a single component with an elevated initialspecific activity (y intercept) and a markedlyshortened half-life of 3 hr. The latter was muchless the average half-life of 8 hr in the six un-treated patients and in one of the patients (A. L.)who served as his own control. The percentage ofdopa incorporated into labeled norepinephrine andVMAwas unaltered in these patients. VMAhalf-lives were 9.5 and 10 hr compared with a mean of13 hr in untreated hypertensive patients, and 11hr in the patient serving as his own control.

In contrast to the effects of reserpine, treatmentof two patients with pargyline resulted in a con-siderable slowing of norepinephrine turnover rate,as indicated by half-life values of 15 and 20 hr(Fig. 7). A control study on the patient (A. T.)with the latter value had yielded a half-time valueof 8 hr, identical to the average of untreated pa-tients. Zero time specific activity was elevated inpatient A. T. and the curve in this case appearedas a single component. The VMAhalf-life timeswere 17 and 20 hr in the pargyline-treated patients.

Effects of Antiadrenergic Drugs on Catecholamine Turnover 2367.

00EEEa.

\ \A.L. and WA

16 24 32 40 48HOURS

FIGURE 7 Alterations of norepinephrine turnover rateproduced by reserpine and pargyline. The biological de-cay curves during pargyline above, and the curves dur-ing reserpine, below, are compared with the averagecurve of six untreated hypertensive patients (cf. Fig. 5).L-dopa-'H dose in each case was 15 uc/kg. The isotopewas administered to G. D. and A. T. during the 4th wkof pargyline therapy (75 mg/day orally) and to A. L.and W. J. after 5 months and 1 yr, respectively, of re-serpine (0.25 mg/day orally).

Comparison of cardiac and urinary norepi-nephrine-3HVariotus stressful stimuli in animals as well as

sympathetic nerve stimulation have been shown toincrease greatly the rate of synthesis and turnoverof norepinephrine in sympathetically innervatedtissues (32, 33). It is reasonable to suppose thatcardiac surgery is a considerable stimulus to thesympathetic nervous system. Also, the same com-bination, "stress" and overactivity of the sympa-thetic nerves, is frequently alluded to in relationto the pathogenesis of essential hypertension.

The patients undergoing cardiac surgery hadan increased rate of turnover of norepinephrineand VMA, which was accompanied by an increased

4level of excretion of these compounds in the urine.4 DeQuattro, V., and R. Kramer. Norepinephrine syn-

thesis and turnover in man during cardiac surgery. Inpreparation.

The half-life values as estimated from urinary dis-appearance curves are listed in Table VIII. Withtwo exceptions (W. M. and D. S.), all the nor-epinephrine half-lives were less than those en-countered in normals or hypertensives (see alsoTable IV); with one exception (W. M.) this wasalso true of VMA. Dopamine half-lives were inthe same range as in the combined normal-hyper-tensive group.

Mean values for the specific activities of urinarynorepinephrine for the eight cardiac patients andthe individual cardiac norepinephrine values atvarious times after L-dopa-3H (15 tic/kg) areshown in Fig. 8. Initially, the specific activity of

10,000

0 Cardiac NE

0 * * * Ut/nor; NE

1000 0°0

0

EO A

E

.0.

100 -

10o0 8 16 24 32 40 48

HOURS

FIGURE 8 Comparison of the specific activities of nor-epinephrine in the-urine and cardiac muscle of eight pa-tients undergoing cardiac surgery following infusion ofL-dopa-'H (15 /Ac/kg). Each muscle point represents onevalue in a single patient, whereas the urinary points aremeans of several patients' values. Atrial muscle was ob-tained from 2 to 22 hr after infusion of labeled dopa.The tissue biopsy at 12 hr was 'specimen of papillarymuscle from a patient with class IV rheumatic heartdisease with mitral and tricuspid insufficiency.


TABI

Turnover Rates of Dopaminin Patients Underg

with Cardiopt

Cardiac FuPatient lesion* c

1 B. S.2 D. S.3 W. M.4 D. B.5 C. R.6 J. T.7 C. H.8 D.S.

ASDSASASDVSDMI,TIIHSSASDASD

* ASD, atrial septal defect;mitral insufficiency; TI,idiopathic hypertrophic sLtricular septal defect.I Estimated from biologicalfollowing 15 ,c/kg of L-dop

urinary norepinephrinenorepinephrine, but afsurprisingly similar inrate of decline. In theat 2 and 6 hr after dcatecholamine radioactiepinephrine and the renof the latter was foundor 24 hr after infusion. Iin any of the cardiac bi

DISCUSSIOI'

Labeling technique.ministration of dopa-31labeling endogenous dolin man. Evidence of erepinephrine was also pdioactive urinary VMAendogenous catecholamcated by: (1 ) the radopa-3H; (2) satisfactcisotope in endogenous pmum specific activityabout 5 mc/mmole (

mc/mmole for the precincrement in urinary nc

,E VIII cretion following dopa-8H; and (4) absence ofte, Norepinephrine, and VMA any pharmacologic effect with the small amounts

,oing Cardiac Surgery (0.05-4.2 mg) of dopa infused. The possibility ofmrnonary Bypass significant relabeling of norepiniephrinie from

Half-lives* dopamine exists since specific activity of thelatter compound generally exceeded that of nor-

nctional Dopa- Norepi- epinephrine for the 1st 10-12 hr after injection oflass mine rine VMA dopa-3H. If this occurred, the specific activities

hr of urinary norepinephrine and apparent turnoverI-11 10 6 10 times represent maximal values. We doubt thatI-II 7 5 8 there was significant relabeling, since the studies1-11 7 7 12 on heart tissue showed that dopamine disappeared1-11 7 6 9 rapidly from this key organ. Further, the decay of

III 5 4 8 urinary norepinephrine-3 H became exponentialI-II 6 4 9 within 12 hr. However, the latter does not provideI-II 5 7 10 complete reassurance, since large deviations from

Mean 6.9 5.2 9.5 a decay curve may not be apparent on a semi-logarithmic plot.

SAS, subaortic stenosis; MI, Turiover rates and pools. The assumption thattricuspid insufficiency; IHSS, changes in the specific activities of urinary dopa-ubaortic stenosis; VSD, yen- mine, norepinephrine, and VMAmirror chemical

decaycurvesof specificactivity events in sympathetically innervated organs seemsa-3H. most valid in the case of norepinephrine. The sym-

pathetic nervous system is the major source ofexceeded that of cardiac plasma norepinephrine and norepinephrine in

ter 10 hr the two were plasma is rapidly excreted in urine independentmagnitude and apparent of renal innervation (34). On the other hand,

tissue specimens obtained dopamine has not been detected in plasma and thelopa-3H infusion, 80% of origins of urinary dopamine are uncertain. Proba-ivity was isolated in nor- bly the changes in specific activity of urinary dopa-nainder as dopamine. None mine represent a composite of extraneuronal me-in cardiac tissue at 12, 17, tabolism of dopa-3H in such organs as liver andNo dopa-3H could be found kidney, as well as turnover in sympathetic neuraliopsies. structures and the basal ganglia of brain (35).

It is now thought that a major portion of intra-neuronal norepinephrine is metabolized by mono-

It is apparent that ad- amine oxidase and excreted predominantly asI is an effective means of VMA (30). Thus, urinary VMA-3H curvespamine and norepinephrine might be representative of degraded intraneuronalidogenous labeling of nor- norepinephrine. However, VMAmay also ariserovided by isolation of ra- via O-methylation followed by deamination. Thus,i. That suitable labeling of in discussing the present work, we prefer to avoidLines was achieved is indi- precise interpretation of VMA specific activity.pid metabolic disposal of curves; however, they tended to vary in the same

ry dilution of incorporated direction as those of norepinephrine.)ools, e.g., calculated maxi- There is no direct experimental evidence to de-

of norepinephrine wascompared with 256-2000.ursor; (3) absence of any)repinephrine or VMAex-

termine which tissue pools contribute to urinarynorepinephrine and VMA. It appears that a por-tion of the early phase of norepinephrine-3H dis-appearance is related to rapid turnover of the


amine in some tissues. From the results obtainedin cardiac patients, synthesis and metabolism ofnorepinephrine in the heart appears to contributea major share to urinary norepinephrine andVMA specific activities. In these patients, thespecific activity and rate of disappearance of nor-epinephrine-3H in the heart were similar to thosein urine. If one assumes that the normal heartweighing about 300 g contains from 0.3 to 0.6mg of norepinephrine (1-2 jug/g [28, and footnote4] ), which, has a half-life of about 8 hr, then ap-proximately 1 mg of norepinephrine or its metabo-lites may be released from this organ daily. Thisrepresents a considerable contribution to total uri-nary metabolites.

VMApool sizes may be calculated from valuesfor estimated zero time specific activities of thismetabolite and the total amount of radioactivityincorporated from dopa-3H into this end-product.In the hypertensive subjects in whomVMAturn-over rates were determined, the extrapolated valueat zero time averaged 2.0 x 103 dpm/mptmole.On the average in these studies 1.6% of the in-fused dose of dopa-3H was converted to VMA,i.e., 3.7 x 107 dpm. Dividing the latter figure bythe former ([3.7 X 107]/[2.0 X 103]) yields aVMApool size of 18.5 Mimoles or 3.7 mg. Basedon a turnover half-time of 14 hr, about 4.4 mg ofVMAshould be excreted per 24 hr, which is inagreement with actual excretion values.

Catecholamine metabolism in essential hyperten-sion. In reviewing studies on this subject before1966, Crout (36) concluded that, if abnormalitiesof sympathetic function or catecholamine metabo-lism are present in essential hypertension, moresophisticated experimental design must be appliedto find them. Wewould agree with this conclusion,and add that careful studies of norepinephrineand VMA turnover following dopa-8H labelingalso fail to reveal any abnormality in hypertension.In view of these findings and the fact that asignificant degree of inhibition (up to 50%) ofcatecholamine synthesis may be produced by alpha-methyltyrosine in patients with essential hyper-tension without lowering of blood pressure, evena minor abnormality in catecholamine metabolismis unlikely to account for maintenance of essen-tial hypertension (37).

A considerable refueling of the controversy wasaccomplished recently by de Champlain, Krakoff,

and Axelrod (7, 8) who have demonstrated a sig-nificant reduction in tissue concentrations of nor-epinephrine in uninephrectomized rats -made hy-pertensive with desoxycorticosterone and NaCl.They demonstrated also a defect in the storageand retention of norepinephrine in sympatheticnerve granules of such animals; this might accountfor the hypertension through access of increasedamounts of active catecholamine to receptor. It isunknown at the moment whether these findingsare unique to the experimental model. No suchabnormalities are apparent in the present studies,either from the extrapolated y intercepts of theexponential portions of the norepinephrine-3H dis-appearance or from norepinephrine-3H disappear-ance rates (turnover). Support for relating thefindings in the rat to the clinical situation is of-fered, however, by the work of Gitlow et al. (9)who reported a slightly increased rate of dis-appearance from plasma of exogenously adminis-tered DL-norepinephrine-8H and interpreted thisas possibly representing a defect in tissue-bindingmechanisms. Also, favoring this hypothesis arerecent reports of a statistically significant increasein the urinary excretion of norepinephrine (6)and normetanephrine (5) in hypertensive as com-pared with control subjects.

Induced alterations of labeling and turnover.Although the techniques employed here failed todetect any abnormality of catecholamine metabo-lism in patients with essential hypertension, devia-tions from normal were easily shown in patientstreated with alpha-methyltyrosine, reserpine, andpargyline, as well as during operative "stress."The mechanism of the alterations observed areinterpretable if the findings are related to previousstudies in animals and in man.

It is well documented in animal studies thatalpha-methyltyrosine inhibits catecholamine bio-synthesis at the first biosynthetic step, i.e., thethe hydroxylation of tyrosine to form dopa. Thus,synthesis of norepinephrine from tyrosine-'4C isinhibited by the drug, whereas when dopa-3H isadministered to bypass tyrosine hydroxylase, in-corporation of counts into norepinephrine remainsintact and is actually increased in the presence ofdrug (25). In man, it is not considered feasibleto study norepinephrine biosynthesis from tyro-sine-14C. However, that alpha-methyltyrosine in-hibits catecholamine synthesis in man is apparent


from the marked reductions in urinary catechola-mine metabolites occurring during drug treatment(26, 27), as was also observed here in threepatients. The findings with dopa-3H indicate thatin man also, the site of inhibition by drug is atthe first step. Thus, total incorporation of dopa-3Hinto urinary norepinephrine was normal or in-creased. The higher specific activity of norepineph-rine after dopa-3H during treatment with alpha-methyltyrosine was probably due to the fact thatunder tyrosine hydroxylase blockade the dopa-3His not diluted as much by endogenously formeddopa (38). There was also a reduction in the rateof turnover of norepinephrine during alpha-methyltyrosine treatment, with a 1-4 hr prolonga-tion of half-life. Apparently, relative to the alreadyreduced tissue catecholamine stores, there waseven less norepinephrine synthesized and releasedthan under normal circumstances.

Reserpine, in small daily doses (0.25. mg),produced a marked increase in rate of norepineph-rine turnover. This low level of dosage does notappear to alter catecholamine production rates inman (39). In the two patients studied here, uri-nary catecholamines and VMA in one patient(A. L.) during therapy were unchanged fromcontrol values of 29 jug and 4.4 mg/24 hr, respec-tively, while the other patient (W. J.) had levelsduring treatment (34 pg and 4.3 mg) which werenormal by our methods. Although the rate ofdecline in specific activity of norepinephrine inthese two reserpine-treated patients was rapid, thezero time specific activity was considerably in-creased. These results are consistent with thewell-known inhibitory effects of reserpine on tis-sue catecholamine storage mechanisms (28, 29).In the reserpine-treated state, with synthesis in-tact, dopa-3H is readily incorporated into a re-duced catecholamine store that is therefore morehighly labeled, but which is also more rapidlydiluted by newly formed endogenous amine. Theslightly increased rate of VMA turnover afterreserpine is in agreement with the findings in ani-mals by Kopin and Gordon (40) that, followingthe administration of reserpine, norepinephrine isreleased preferentially onto oxidative enzymes.

Pargyline hydrochloride produced a marked re-duction in norepinephrine turnover. Although theeffects of monoamine oxidase inhibitors on tissueconcentrations of norepinephrine vary considerably

with species, and even organ (30), it is reasonableto assume that the two patients studied had ele-vated tissue levels of norepinephrine. However,the degree of labeling (zero time) was increasedin one patient and was unchanged in the other.Also, the percentage of label excreted as nor-epinephrine was increased. These results suggestenhanced incorporation from dopamine, an excel-lent substrate for monoamine oxidase, as seen insimilar studies in animals (38, 41). Neff andCosta also demonstrated a considerable slowingof norepinephrine turnover in brain and heart ofpargyline-treated rat, an animal whose tissue con-centrations of norepinephrine increase after ad-ministration of the drug (42). They suggestedthat their results might be due to end-productinhibition of tyrosine hydroxylase with a conse-quent decrease in synthesis and turnover rate. Anumber of observations in our laboratory, includ-ing studies of incorporation of label from tyrosineand dopa into norepinephrine in animals treatedwith pargyline, also lend support to the hypothesisthat norepinephrine synthesis is regulated by amechanism of end-product inhibition at the tyro-sine hydroxylase step (cf. 38). If this also occursin man, then the total of urinary catecholaminemetabolites should be reduced during pargylinetreatment, even as during treatment with alpha-methyltyrosine. In one of the patients (A. T.)studied here, measurements of the sum of urinarycatecholamines plus metanephrines plus VMAaveraged 4.8 mg/24 hr during the control periodand was reduced to 1.9 mg during pargyline treat-ment. Reduced catecholamine synthesis as a factorin the hypotensive effects of monoamine oxidaseinhibitors has been considered only recently (37,42) and will require further evaluation.

The fourth alteration of catecholamine turn-over observed here was the rapid turnover ofnorepinephrine and VMAin patients undergoingsurgery. It is well known that during a variety ofstressful stimuli, of which cardiac surgery mustbe among the more severe, there is an outpouringof the catecholamines and their metabolites in theurine. A reduced pool size due to rapid release inthe presence of a normal synthesis rate might beadvanced to explain the increased turnover rate.However, Cooper et al. demonstrated that humanatrial norepinephrine is not decreased during car-


diopulmonary bypass (43). Thus, our findingsseem best interpreted as evidence of acceleratedcateclholamine biosynthesis. They are also consist-ent with studies in animals showing clearly that in-creased sympathetic activity occurring during se-vere stress (exercise and exposure to cold), aswell as that produced by direct sympathetic nervestimulation, is associated with increased rate ofsynthesis (32, 33). Thus, tissue concentrations ofnorepinephrine are maintained in the face of con-comitant rapid release of the amine.

Assessment of catecholamine metabolism invivo. It is doubtful that the excretion of a singlesubstance, e.g. norepinephrine, normetanephrine,or VMA, is an adequate chemical index of sympa-thetic nerve dynamics. The urinary excretion ofnormetanephrine has been offered for this purpose,since animal data suggest that norepinephrinewhich leaks away from "receptors" is preferen-tially metabolized by O-methylation (44). How-ever, no studies in man have been made todocument this. Urinary norepinephrine might beacceptable, except the extent to which plasma nor-epinephrine represents neurohormone that hasacted on receptor is unknown. Difficulties in em-ploying a single chemical index are illustrated inthe effects of treatment with a monoamine oxidaseinhibitor which results in lowering of blood pres-sure and, therefore, a presumed decrease in releaseof active catecholamine. Urinary norepinephrineexcretion is little changed during such treatment,whereas normetanephrine is increased and VMAis reduced. Similarly, although small doses ofreserpine exert an obvious effect on norepineph-rine turnover rate, and probably on sympatheticnerve function, this is not apparent from the levelsof excretion of norepinephrine and its majormetabolite, VMA. Until such time as direct tissueassays of the neurohormones and their biosyn-thetic enzymes become feasible, combined studiesof turnover, as reported here, and the measure-ments of norepinephrine plus normetanephrineplus VMAmay afford the best chemical means ofdetecting altered sympathetic function in man.

ACKNOWLEDGMENTSWe gratefully acknowledge the cooperation of DoctorsAndrew G. Morrow and Richard Kramer, CardiacSurgery Branch, National Institutes of Health, in thestudies of their cardiac patients and the technical as-

sistance of Mrs. Diane Warren.

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