circulation - pdfs.semanticscholar.orgpressor amines in normal subjects, patients with various...

Circulation NOVEMBER 1954VOL. X NO. 5

A Journal of the American Heart AssociationChemical Quantitation of Epinephrine and

Norepinephrine in Thirteen Patientswith Pheochromocytoma

By WILLIAM M. MANGER, M.D., EUNICE V. FLOCK, PH.D., JOSEPH BERKSON, M.D.,JESSE L. BOLLMAN, M.D., GRACE M. ROTH, PH.D., EDWARD J. BALDES, PH.D.,

AND MARTIN JACOBS

Fluorescent quantitation of epinephrine and norepinephrine in plasma of patients suspected ofhaving pheochromocytomas is of considerable diagnostic value. The concentrations of thesepressor amines in normal subjects, patients with various diseases with and without hypertension,and 13 patients with pheochromocytomas are reported. In all patients with pheochromocytomasand sustained hypertension the concentrations of pressor amines were significantly elevated. How-ever, in patients with paroxysmal hypertension secondary to pheochromocytomas the pressoramine concentrations may be unelevated when the blood pressures are normal. In this latter groupprovocative tests with intravenous histamine are indicated to induce hypertension prior to quanti-tating pressor amines.

ITH a chemical method available forestimating the concentration of epi-nephrine and norepinephrine in

plasma,, 2 it has been possible to quantitatethese pressor amines in 13 patients withpheochromocytomas. Initially in this study,the method of Weil-Malherbe and Bone,' whichconsists of alumina adsorption of the pressoramines from plasma followed by elution withacetic acid and condensation with ethylene-diamine, was used to obtain a measurablefluorescence. Since this method allowed onlythe estimation of the concentration of totalpressor amines (that is, "adrenalinelike"substance), we devised a modification2 thatconsists in the use of sodium thiosulfate, which

From the Mayo Clinic and Mayo Foundation,Rochester, Minn. The Mayo Foundation is a part ofthe Graduate School of the University of Minnesota.

Abridgment of portion of thesis submitted byDr. Manger to the Faculty of the Graduate School ofthe University of Minnesota in partial fulfillment ofthe requirements for the Degree of Doctor of Phi-losophy in Medicine.

641

affects differentially the fluorescence of epi-nephrine and norepinephrine, thus enablingthe quantitation of each. According to Weil-Malherbe and Bone,3 it has been establishedthat the fluorescent substances estimated inthe blood of normal human subjects are"identical with adrenergic amines in theiraffinity to amine oxidase in RF values and inthe fluorescence spectra of their derivatives."However, it is possible that in some diseasestates, particularly where there is associatedazotemia, there may be a significant retentionof fluorescent substances other than the pressorcatechols which could cause erroneous calcula-tions.

The concentration of epinephrine and norepi-nephrine in venous plasma was estimated unlessotherwise indicated, and it was not required thatpatients be fasting when blood was obtained. Con-centrations are expressed in micrograms per liter ofplasma (1) of "epinephrinelike substance" and (2)of epinephrine and norepinephrine individually. Theestimate of "epinephrinelike substance" refers tothe concentration equivalent of the fluorescence

Circulation, Volume X. November 1964

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QUANTITATION OF EPINEPHRINE AND NOREPINEPHRINE

Concentrations, ug./liter plasma

Epineph-rne-like

substance*

2.02.02.22.51.61.71.31.61.20.31.4

1.620.3-2.5

0.59

Epineph-rinet

00

0.20.60

0.10.1

0.140-0.60.21

Norepi-nephrine

5.26.23.63.34.60.84.0

3.960.8-6.2

1.7

* This is the equivalent of the fluorescence read-ing (before addition of Na2S203) if it were due en-tirely to epinephrine. It is equal to the epinephrineplus 0.316 times the norepinephrine.

t When calculated values (equation 5, ref. 2)were less than zero (due to errors intrinsic in themethod when small quantities are involved) theconcentration of epinephrine was recorded as 0.

reading before addition of sodium thiosulfate were itall due to epinephrine. The fluorescence is usuallydue to both epinephrine and norepinephrine, butsince norepinephrine exhibits only about a third ofthe fluorescence that epinephrine does, the recordedconcentration of "epinephrinelike substance" is lessthen the sum of the recorded epinephrine and nor-epinephrine. This term "epinephrinelike" (or"adrenalinelike") was first introduced by Weil-Malherbe and Bone1 and has been retained, sincemany such determinations were performed before amethod of estimating the individual concentrationsof epinephrine and norepinephrine had been devised.

RESULTS IN NORMAL SUBJECTSThe plasma concentrations of pressor amines

in 11 normal subjects without hypertensionare recorded in table 1. The concentration of"epinephrinelike" substance was never foundto exceed 2.5 ug. per liter, and this was almostentirely norepinephrine with little if anyepinephrine. The mean concentration of"epinephrinelike substance" was 1.6 jg. perliter.* However, from the analysis of large

* Weil-Malherbe and Bone' reported a meanconcentration of approximately 3 sg. per liter.

quantities of plasma by the method of paperchromatography followed by elution andfluorometric quantitation, it appears that asmall percentage of fluorescence in normalplasma may be due to substances other thanepinephrine and norepinephrine.

RESULTS IN PATIENTS WITHOUTPHEOCHROMOCYTOMA

In table 2 are recorded the plasma concen-trations in 25 patients without pheochromo-cytomas. As indicated, most of these patientshad hypertension--either primary or associatedsecondarily with their disease process. Manyof these patients were chosen for study becausesome of their symptoms, signs and laboratoryfindings may simulate those in patients withpheochromocytomas. It is noted that thehighest value for epinephrinelike substance was3.1 jig. per liter and that the sum of epinephrineand norepinephrine was never more than 11.2Mg. per liter. The latter concentration occurredin a patient with renal hypertension and bloodurea of 196 mg. per 100 ml. It is possible thatin this patient, and in two other patients withessential or renal hypertension and blood ureasof 342 and 122 mg. per 100 ml. respectively,retention of catechols other than epinephrineand norepinephrine accounts in part for thehigh values of pressor amines. In all the otherpatients, the blood ureas, when determined,were essentially normal. If the patients withazotemia are excluded from the series, it isnoted that the sums of epinephrine andnorepinephrine for all the subjects are lessthan 8 ig. per liter. The values obtained in oneof the remaining patients are difficult to inter-pret since this patient (case 23) had receivedepinephrine, corticotropin and cortisone fortreatment of asthma prior to quantitation ofher blood. Adrenocorticotropic hormone andcortisone have been reported to decrease theurinary excretion of epinephrine and nor-epinephrine4, 5 and may possibly influence theplasma concentration of the pressor amines.Patient 1 had false positive Regitine testsperhaps because of previous sedation, and anexploratory laparotomy revealed no evidence ofa pheochromocytoma in the abdomen. Therelatively high percentage of epinephrine in

TABLE 1.-Normal Subjects

Subject

1234567891011

Mean ................Range...............Standard deviation....

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WILLIAM M. MANGER ET AL.

TABLE 2.-Patients without Pheochromocytoma

Approximate bloodpressure, mm. Hg

220/156198/138142/100210/150190/120176/126190/100180/105174/114170/110150/90168/116135/105152/104180/115174/110180/82150/95120/85180/110146/60135/72170/110134/94124/84105/75130/95114/74110/80

Diagnosis

Essential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionEssential hypertensionParoxysmal* hypertensionRenal hypertensionRenal hypertensionPregnancy with hypertensionCushing's with hypertensiontCushing's with hypertensionGushing's with hypertensionCushing'sCushing's:Thyrotoxicosis with hypertensionThyrotoxicosisMyxedemaAnxiety and vascular hyperreactorAnxiety and vascular hyperreactorAsthmatic with severe attack§Asthmatic after improvement§Post partumPost partumGastric carcinoma

Concentration, pg./liter plasma

Epinephrine- pineprine

2.73.1; 2.4

0

1.32.80.61.30.41.70.11.11.93.11.80.11.21.81.01.71.31.31.20.70.82.50.51.10.40.7

0

0.6

0.3

0.1

1.10.0

1.20.80.40.11.00.2

0

0.4

Norepi-nephrine

0

6.9

3.2

5.0

2.411.2

1.90.64.03.71.03.2

7.9

2.2

Blood urea,mg. per 100 ml.

NNN48342NN46-56N42-5044122196NNNNN

NN

NNN

* Necropsy revealed no pheochromocytoma.t Patient had undergone 1 adrenalectomy and subtotal adrenalectomy of the other gland, a bilateral infra-

diaphragmatic sympathectomy and celiac ganglionectomy.Receiving cortisone in preparation for subtotal adrenalectomy.

§ Receiving cortisone.

patients with Cushing's disease and one of thepatients with thyrotoxicosis is an interestingfinding which must be further investigated.No correlation was apparent between the

height of the blood pressure and the concentra-tion of pressor amines. But it should be men-

tioned that some of these patients were beingtreated with antihypertensive drugs whichpossibly may decrease the plasma concentra-tion of the pressor amines. Certainly with theuse of a ganglionic blocking agent it would seem

possible that the quantity of norepinephrineliberated into the blood would be decreased.However, this problem requires further in-vestigation.

RESULTS IN PATIENTS WITH

PHEOCHROMOCYTOMA

The major complaints and findings in 13patients with pheochromocytoma are sum-

marized in table 3. Nine of these had sustainedand four had paroxysmal hypertension. Therewere nine men and four women in this series,with an age range of 12 to 51 years. All of thesepatients complained of headaches; six hadvisual complaints; nervousness, excessive sweat-ing, tachycardia, palpitations, sensations ofweakness, and loss of weight were not un-

common. A severe retinopathy was observedin six of the patients, and elevation of thebasal metabolic rate and blood sugar was

Pt.

1

234567891011121314151617181920212223

24

25

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TABLE 3.-Major Complaints and Findings in Patients with Pheochromocytoma

Relevant past histories and major symptoms

Exertional dyspnea 18 mo., headaches14 mo., hypertension known 12 mo.,blurred vision 6 mo.

For 2 yr.: Diminished vision, head-aches, hypertension known, tachy-cardia, nervousness, weight loss

"Goiter" removed 8 yr. ago because ofenlarged thyroid, palpitations andnervousness but BMR-6 preop. Dia-betes mellitus (mod. severe) 3 yr. For8 mo.: Headaches and hypertensionknown, failing vision 6 wk.

For 3½ yr.: Diminished vision, head-aches, hypertension known, "jittery"feelings, easy sweating

Headaches, hypertension known 5 mo.,nose bleeds, easy sweating and weightloss 3 mo. One convulsion with highBP (190/150), tachycardia (172) andtwitching left arm and leg 2 mo. ago

Headaches 5 wk., hypertension known 2wk.

For 5 mo.: Headaches, easy sweating,palpitations, hypertension known

Hypertension known 12 yr., cerebro-vascular accident with hemiplegiaand hemianopsia; pheochromocytomaand metastatic rib lesion removed 3yr. ago. For 1½ yr.: "Weak spells,"excess sweating, headaches, epi-gastric discomfort

For 3 yr.: Epigastric pressure sensationsextending to chest and throat. For 2yr.: Episodes of headaches, shaking,pallor, slowing of pulse and hyper-tension known

Headaches 2 yr. Accompanied for 2-3mo. by: sweats, palpitations, tachy-cardia, scotomas, nausea, vomiting,paroxysmal hypertension, weightloss. Symptoms aggravated by "de-sensitization" treatment with I.V.and subcutaneous histamine EEG re-vealed left temporal abnormality.

For 7 yr.: Hypertensive attacks withpalpitations, dyspnea, cephalgia,nausea, vomiting, pallor, easy sweat-ing, trembling, salivation

Hypertensive attacks with headaches4~ mo. ?Myocardial infarction 11yr. ago. For 4 mo.: Easy sweating,weakness, weight loss

For 1 yr.: Headaches accompaniedsometimes by black spots before eyes,unsteady gait and hypertensionknown

Ophthalmoscopic findings

IVt

IV

IV

III

IV

Normal

IV

II

I

II

I

Retinal hemorrhages? due to malnutrition

Normal

Laboratory data

Blood Blood ureaBMR, % sugar mg./ mg./100

100 ml. mi.

+39

+27

+49

+21+6+6+9

+18

+13+24+36+20

+4

+20

+21

+2

142§

358256238

107109

125

130151111

135

115

125

198226

100

24$

28

26

2438

22

4426304020

22

28

Age,

Age,yrs., sex

38M

43F

39M

28M

12M

36M

26M

21M

39M

44M

48F

51F

44F

Pt.

l-s*

2-s

3-s

4-s

5-s

6-s

7-s

8-s

9-s

10-p*

11-p

12-p

13-p

* s = Sustained hypertension; p = Paroxysmal hypertension. t Grouping according to Keith, Wagener andBarker classification.6 : Normal blood urea range at Mayo Clinic = 10 to 40 mg. per 100 ml. blood. § Normalblood sugar range at Mayo Clinic = 80 to 120 mg. per 100 ml. blood.

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TABLE 4.-Pressor Amines in Patients with Pheochromocytoma before and after Removal of the Tumors

Concentration, pig./liter plasma

Preop.

Epinephrinelike Epinephrine Norepineph-rine

4.717.7; 15.4

6.0

4.26.16.17.811.44.9

Only estimatedtest

8.810.25.8

2.4; 2.61.7

* Receiving cortisone.

(A)

0.8

3.0

0.5

1.62.1

12.4

9.4

11.7

14.112.7

0 14.8after provocative

2.32.9

0.5

20.623.0

3.7

Postop.

Epineph-rinelik

Epephrlneepinephrine1.5 0.1 4.52.1

Not estimated-tumor notremoved1.61.62.43.4

3.11.0

1.2

0.90.00.8

0.00.10.6

0.9

0.2

0.00.00.1

6.34.65.6

7.0

3.3

2.70.02.1

Postop. bloodpressure,mm. Hg.

185/125150/100

186/132130/85150/100120/80

166/100132/96

105/70

100/74105/70130/75

(A) Arterial plasma.

frequently noted, but none of these patients inwhom the blood urea was determined hadazotemia. The results of pharmacologic tests forpheochromocytoma (histamine or Regitine or

both) were positive in all 13 patients.In table 4 and figure 1 are recorded the

preoperative and postoperative (at least threedays after all pressor amine medication forblood pressure stabilization had been dis-continued) plasma concentrations of epi-nephrinelike substance, epinephrine and nor-

epinephrine in the patients with pheochromo-cytoma. In the group with sustained hy-pertension and in two of the patients withparoxysmal hypertension, the amounts ofepinephrinelike substance and the sums ofepinephrine and norepinephrine were in-variably greater than the highest values ob-tained on normal subjects or patients withoutpheochromocytoma. The lowest value inpatients with pheochromocytoma and sustainedhypertension was 4.2 Mug. per liter of epineph-rinelike substance and 12.2 Mug. per liter forthe sum of epinephrine and norepinephrine.Also, if patients with azotemia are excluded,the norepinephrine concentration in thepatients with sustained hypertension and

pheochromocytoma was higher than in normalsubjects or patients without pheochromo-cytoma. In two of the patients with paroxysmalhypertension the preoperative concentrationsof pressor amines were not elevated, but theblood pressures were normal when bloodsamples were obtained. In two of the patientsfollowing removal of the tumors the concentra-tions of epinephrinelike substance were slightlyhigher than in any of the normal subjects or

patients without pheochromocytomas. Oneof these patients (case 8-s) had metastaticpheochromocytoma and had also receivedcortisone just prior to operation, which mayhave influenced the pressor amine concen-

trations. When the latter patient was seen one

year later, the concentrations of epinephrine-like substance and norepinephrine were ele-vated (4.5 and 14.6 Mug. per liter, respectively),and, though the pharmacologic tests forpheochromocytoma were equivocal, this pa-tient very probably has residual functioningtumor tissue.

EFFECT OF PHARMACOLOGIC TESTS

In table 5 are recorded the effects of severaldrugs on the blood pressure and plasma con-

Pt.

1-s

2-s3-s

4-s5-s6-s7-s

8-s*9-s

10-p

11-p12-p13-p

Preop. bloodpressure, mm. Hg

250/170240/150210/140

210/130195/145190/100180/150168/136160/130148/98

150/90134/94

128/90120/80

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q) 300 30

C2j~00 - N200 - c01

1 10

s

It.)~~~~~~.

b

200 Z5

5 l

jo-p li-p

m

ba ababC a b a b a b ab2s 3-z 4-s 5-s 6-z 7-.s 6-s 9-s

Diaztolic]

=Epinephrine-like

I=Epinephrine0 Norepinephrinea= Preoperativeb= Postoperative

s Sustained hypertensionp= Pacroxysmal hgpertension

___ j]---Highest value obtainedL . (epinephrine-like) Patients

CL b c b without pheochromocytomal1-p 13-p

FIG. 1. Pressor amines in patients with pheochromocytoma before and after removal of thctumors.

TABLE 5.-Effect of Pharmacologic Tests on Pressor Amines in Patients with and without Pheochromocytoma

Drug used

Regitine + benodaine IV.Regitine IV.Histamine + Regitine I.V.

Histamine + Regitine IV.Histamine IV.Histamine I.V.

Histamine I.V.Histamine IV.Histamine IV.Histamine I.V.1 yr. postop. histamine IV.Regitine IV. (previous seda-

tion)

Concentration, gg./liter plasma

Before drug given

Approximateblood pressure,

mm. Hg

180/150168/136148/98

134/94190/100128/90

174/114114/74170/110134/94130/90220/156

Epinephrinelikesubstance

7.811.4

Not estimated

10.26.1

2.4; 2.6 (A)

1.70.40.70.80.92.7

After drug given

Approximate Epinephrineblood pressure, like susrtance

mm. Hg lk ac

144/110* 7.1146/120 9.9194/124 4.7144/98 6.0t244/164t 19.7240/138 8.1250/160 11.9

184/118112/72210/110178/118148/110144/124

0.70.62.11.41.63.7

* Blood pressure response to drugs actually blood pressure was 170/136 when blood sample was obtained.

t Estimated 30 minutes after above sample.t Blood pressure response to histamine-actually blood pressure was 114/74 when blood sample was obtained

after regitine.(A) Arterial plasma.

646

Pt.

7-s

9-s

10-p6-s12-p

ControlControlControlControl9-sControl

1-

-

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centration of pressor amines in five patientswith pheochromocytoma and five patients(controls) without pheochromocytoma. Inpatients 6-s, 10-p and 12-p, histamine intra-venously administered caused a rise in bloodpressure and an increase in concentration ofepinephrinelike substance in plasma obtainedseveral minutes after administration of thedrug. In patient 9-s, estimation of the con-centration before administration of drugs wasunfortunately not performed. However, severalminutes after histamine (and Regitine to de-press the marked rise in blood pressure) hadbeen given intravenously, the concentration ofepinephrinelike substance was 4.7 Mg. per liter.Also about 12 hour later the concentration was6.0 Mg. per liter though the blood pressure was144 mm. Hg systolic and 98 diastolic when theblood for this second determination was ob-tained. This latter finding and the fact that inpatient 7-s Regitine alone and Regitinefollowed by Benodaine intravenously ad-ministered caused no appreciable changes inthe pressor amine concentrations, despite a

lowering of the blood pressure, supports theview that neither of these two drugs destroysthe pressor amines. No significant increase inthe epinephrinelike concentration was notedfollowing the intravenous administration ofhistamine to patients without pheochromo-cytoma or to patient 9-s when he returnedabout one year after operation with symptomsslightly suggestive of a recurrent pheochromo-cytoma. The reason for the slight increase inquantity of epinephrinelike substance in one ofthe control patients with hypertension from2.7 to 3.7 Mg. per liter following intravenousadministration of Regitine is not known. Inhigh concentration, Regitine will producefluorescence detectable by the method used fordetermination of the pressor amines but notat the concentrations found in plasma, evenafter very large amounts have been admin-istered intravenously.

EFFECT OF ANESTHESIA

The effects of anesthesia on blood pressureand pressor amine concentrations in five

q) 300

A200io.

" 60

N7060

q50

Z

40

30

20

10

01

m-E =Epinephrine-like O=Before

- .~~~~~ n~~nezthe,3ia.Hi=q|Emprn P=Pentoth:tl

N=Norepinephtine NNitrous oxide~NoeiephineE =Ether

C=CurareSsoi e~iatanesthetic agent

Blood samples drawnsimultcLneously

.s=Suzstcined. htjpertensionp=Parox9.smal hypertension

rv~~~~~~~~~~~r

O(P)NC 0 (E)N 0 (E)NP 0 (P)NC 0 0N 0 (E)NControl l-s 4-.s 6-s -11p 13-p

FIG. 2. The effect of anesthesia on patients with pheochromocytomas.

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m-ho

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patients with pheochromocytoma and onepatient without pheochromocytoma are repre-sented in figure 2. It should be noted that inpatients 4-s, 6-s and 13-p the arterial plasmaand not the venous plasma was analyzed duringanesthesia, which does not permit a strictcomparison with the preanesthetic venousconcentrations. In all the patients withpheochromocytoma except 1-s, the pressoramines during anesthesia were significantlyincreased, and markedly so in patient li-p.In the control patient no appreciable changewas observed in venous or arterial plasmadrawn simultaneously during anesthesia.

EFFECT OF ANESTHESIA AND PALPATIONOF PHEOCHROMOCYTOMA

The effect of anesthesia and anesthesia pluspalpation of the pheochromocytoma in threepatients is represented in figure 3 and also theeffect of anesthesia and anesthesia plus pal-pation of the adrenal glands in a patient inwhom no tumor was found. In patient 3-s theincrease in concentration of epinephrinelike

substance from the effect of anesthesia aloneis noted and then the very marked increaseafter palpation of the tumor. In the latterinstance concentrations were determined onplasma obtained simultaneously from venousand arterial blood of the same arm. It is in-teresting that the arterial sample containedmore than twice the concentration in thevenous plasma, indicating a considerable loss ofpressor amines in the forearm secondary tometabolic destruction or diffusion or both.This patient had a malignant pheochromocy-toma with extensive metastases which were notremoved. In patient 12-p there was no effect ofanesthesia on either the venous or arterialpressor amine concentrations. However, pal-pation of the tumor caused a considerableincrease in the arterial concentration. Only aslight increase in concentration was noted inthe patient without a pheochromocytoma dur-ing anesthesia and adrenal gland palpation.No correlation is apparent between the type

of anesthetic agents employed and the quantityof pressor amines liberated into the circula-

=Epinephrine-like U=BE

*= ¢=Epinephrine P=PEN=Ni

LlNorepinephrine NEt

-PalpcationF=Bloodc samples drawn sim=-5tuztained hypertension

p=Paroxysmal hypertension

0 (E)NP -1 K(NCP)j0-p U-p

* 4 minutes after removal of tumor

FIG. 3. The effect of anesthesia and operative manipulation on patients with pheochromocytomas.

400Q)

z 300

100N

o

01

H

160-

Q; 150-

t120

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tion in these patients with pheochromocytoma.However, the series is too small to permit anydefinite conclusions. Neither Pentothal sodiumnor ether produced any fluorescence with our

analytic procedure.RESWLTS OF PHEOCHROMOCYTOMAS ANALYZED

Finally, alcoholic extracts of the pheo-chromocytomas removed at operation were

prepared in the following manner as suggestedby Dr. E. C. Kendall:As soon as each tumor was removed it was

placed in dry ice and frozen solid to minimizeoxidation and deterioration of the pressoramines. Five to 10 Gm. samples of the frozentumor were crushed, weighed and extractedwith 5 volumes of cold alcohol containing a

small amount of acetic acid and sodium bisul-fite.

These extracts were then diluted 5,000 to10,000 times with glass-distilled water, and 10ml. of this dilution was condensed directly withethylene diamine without initial adsorptionon an alumina column as is necessary withblood. This was deemed advisable since thepercentage loss of pressor amines from thesealcoholic extracts during the adsorption, andelution process, despite the addition of sodiumthiosulfate as an antioxidant, is considerable.A poor recovery is also noted if water solutionsof epinephrine and norepinephrine with thisantioxidant are passed through an aluminacolumn. With addition of water solutions ofpressor amines or tumor extracts to a mixture ofplasma, antioxidant (sodium thiosulfate) andanticoagulant (sodium fluoride) the recovery ofepinephrine or norepinephrine or both is ap-proximately 93 per cent. Thirteen experimentsto determine the recovery percentage were per-formed on specimens of plasma to whichvarious quantities of epinephrine or norep-inephrine or both had been added. Thesewere then analyzed according to the methodof Weil-Malherbe and Bone.1 The mean re-

covery was 92.9 per cent, and the standarddeviation of the recovery percentages was 4.7per cent. The latter adds to the error of 14 percent previously reported2 when quantitation ofepinephrine and norepinephrine is made using

TABLE 6.-Pheochromocytomas

Pt.

1-s

2-s

4-s5-s6-s7-s

8-s9-s

10-p11-p12-p

13-p

Site

L. ad.*L. ad.*

R. ad.tL. ad.*Liver hilusL. ad.*

PeriaorticR. ad.t

R. ad.jR. ad.tL. ad.*

L. ad.*

Wt.Gm.

25165

16203270

25120

2545

200

17.2

Mg./Gm. tumor

Epineph-rine-hlke

1.73I 0.38

II 0.290.361.080.56

I 0.06II 0.27

0.52I 4.5

II 5.781.785.87

I 0.38II 0.70

3.30

Epineph-rine

1.210.040.020.200.020.1

0

0.764.490.200.461.59

Nor-epineph-

rine

1.641.070.850.503.341.45

1.6

3.224.370.570.765.40

Note: Tumor not removed from patient 3-s.* Region of left adrenal gland.t Region of right adrenal gland.

aqueous mixtures with and without sodiumthiosulfate. Considering the errors as inde-pendent, the total error measured as standarddeviation is about 15 per cent (V142 + 4.72).

Extraneous fluorescence from substancesother than epinephrine and norepinephrine inthese diluted extracts is negligible. Conse-quently, quantitation of the epinephrine andnorepinephrine in these tumor extracts wasaccomplished in about two hours, whereas theanalysis of plasma required about four andone-half hours. A number of tumor or plasmaextracts may be analyzed simultaneouslywithout requiring much additional time. Ifkept in the refrigerator, the alcoholic tumor ex-tracts did not appreciably deteriorate for atleast several weeks (one sample showed nochange in the pressor amine concentrationsafter 27 days).The locations, weights and concentrations

expressed in milligrams of pressor amines pergram of tumor are recorded in table 6.

In all of the tumors analyzed for epinephrineand norepinephrine, the amount of norepi-nephrine was greater than that of epinephrineexcept in patient 11-p, in whom the per-

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centages were about equal. Two extracts wereprepared from different portions of four of thetumors, and the difference of pressor amineconcentrations is particularly noticeable inpatients 7-s and 12-p. This latter finding indi-cates that the concentrations are not uniformin some pheochromocytomas. In additionto the more accurate fluorometric quantitationof pressor amines in the tumor extracts, esti-mations were also made by the method ofpaper chromatography.7 Similar values ob-tained by the two methods lend further supportto the specificity of the fluorometric method.Recently in one pheochromocytoma discoveredat necropsy, hydroxytyramine in addition toepinephrine and norepinephrine was found onchromatographic analysis. Addition of knownhydroxytyramine to the sample intensified thisspot when the chromatograms were run intwo different solvents (75 per cent phenol;normal butanol saturated with 1 normalhydrochloric acid). It is possible that the ap-pearance of hydroxytyramine was in some wayrelated to postmortem changes in the em-balmed specimen. This is the first time that wehave observed any catechols other than epi-nephrine and norepinephrine in 35 pheochro-mocytomas analyzed chromatographically. Hy-droxytyramine has been found in sheepadrenalss and human urine.9

COMMENT

Of the 13 patients with pheochromocytomareported, all those with sustained hypertensionand two with paroxysmal hypertension hadelevated plasma concentrations of pressoramines as compared with normal subjects andpatients with hypertension due to othercauses. Of the two remaining patients withparoxysmal hypertension and pheochromo-cytoma, the concentration of pressor aminesbecame elevated in one after a provocativehistamine test and in the other during an-esthesia administration-which often simulatesa provocative test in patients with pheo-chromocytoma. Unfortunately the effect ofhistamine was not observed in this latterpatient. In this series and method of analysis,plasma concentrations greater than 4 ug.

per liter of epinephrinelike substance and 12jig. per liter for the sum of epinephrine andnorepinephrine have without exception beendiagnostic for pheochromocytoma. However,since our series is small, patients with azotemiashould be more carefully evaluated, since in thisinstance there may be retention of fluorescentsubstances other than epinephrine and norep-inephrine which would falsely give high valuesfor the pressor amine concentration. The pos-sibility of the retained fluorescent substancebeing phenol has been fairly well excluded bytreating ethylenediamine with concentrationsof phenol which may occur with severeazotemia. No fluorescence was obtained.

It was found essential to obtain blood duringor shortly after a paroxysm of hypertension-either naturally occurring or induced by adrug such as histamine-to establish a diagnosisin some of the patients with paroxysmal hyper-tension secondary to pheochromocytoma. Inthis series of 13 patients the per cent of norep-inephrine in the preoperative and postoper-ative plasma was invariably higher than thatof the epinephrine. This latter finding is con-sistent with the higher concentrations ofnorepinephrine noted in the tumor extracts andin the plasma of normal subjects. In patient9-s, chromatographic separation of pressoramines in the tumor extract revealed 5 mg. pergram tumor of epinephrine and 1.3 mg. pergram tumor of norepinephrine. Very likely theplasma concentrations in this patient wouldhave also contained a high percentage ofepinephrine, but unfortunately the method2for determination of the individual pressoramines was not available.

Using an entirely different fluorometricmethod of analysis,10 Lund reported data onfive patients in whom the total amounts ofepinephrine and norepinephrine in plasma wereelevated during episodes of paroxysmal hyper-tension." Four of these patients were proved tohave pheochromocytomas, but no tumor wasfound in the fifth patient. Von Euler, Lund andco-workers,12 using Lund's method, also re-ported the total plasma concentrations ofepinephrine and norepinephrine in a patientwith pheochromocytoma. These investigators

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were unable to estimate the concentrations ofpressor amines in the plasma of normalsubjects.

SUMMARYThe results of quantitation of epinephrine

and norepinephrine in the plasma and tumorextracts of 13 patients with pheochromocytomaand in the plasma of small groups of normalsubjects and patients with hypertension andvarious diseases unassociated with pheochro-mocytoma are reported. The preoperativeplasma concentrations of epinephrinelike sub-stance and total epinephrine and norepi-nephrine in all the patients with sustainedhypertension and pheochromocytoma weresignificantly elevated (more than 4 Mug. perliter of plasma of epinephrinelike substanceand more than 12 gzg. per liter of plasma for thesum of epinephrine and norepinephrine).Though only two of the four patients withparoxysmal hypertension secondary to pheo-chromocytoma had an elevated concentrationof pressor amines, these concentrations couldbe significantly increased with a provocativedrug (for example, histamine) in patients withpheochromocytoma but not in patients with-out such a tumor.

It appears thus far in this investigation thatchemical estimation of the pressor amines inplasma--if combined with provocative tests inpatients with the paroxysmal type of hyper-tension-is a valuable test for screening pa-tients suspected of having pheochromocytoma.Except in patients with pheochromocytoma,the magnitude of hypertension appears to bearno relation to the level of pressor amines in theplasma.

Attention is also called to the occurrence ofepinephrine-norepinephrine release in largeamounts in most of these patients duringanesthesia and especially after palpation of thepheochromocytomas.

SUMARIO ESPANOL

Se informan los resultados de la deter-minaci6n de epinefrina y norepinefrina en elplasma y en los extractos de tumores en 13pacientes con feocromocitoma y en el plasma

de un pequefio grupo de sujetos normales yen pacientes con hipertensi6n y varias otrasenfermedades no asociadas con feocromocitoma.Las concentraciones plasmgticas preoperatoriasde substancias similares a la epinefrina y eltotal de epinefrina y norepinefrina en todos lospacientes con hipertensi6n sostenida y feo-cromocitoma estuvieron significativamente ele-vadas (mas de 4 microgramos por litro deplasma de substancias similares a epinefrina ymas de 12 microgramos por litro de plasma dela suma de epinefrina y norepinefrina). Aunquesolamente dos de cuatro pacientes con hiper-tensi6n paroxistica secundaria a feocromoci-toma tuvieron una concentraci6n elevada deaminas presoras, estas concentraciones pu-dieron ser significativamente aumentadas condroga provocativa (por ejemplo, histamina)en pacientes con feocromocitoma pero no enpacientes sin tal tumor.Aparentemente en esta investigaci6n la

estimaci6n quimica de las aminas presoras delplasma-si combinada con pruebas provoca-tivas en pacientes con el tipo paroxistico dehipertension-es una prueba valiosa para en-cubrir pacientes sospechados de tener feocromo-citoma. Excepto en pacientes con feocromoci-toma, la magnitud de la hipertensi6n pareceno tener relacci6n al nivel de aminas presorasen el plasma.Tambi~n se llama la atenci6n a la posibilidad

de liberacidn en grandes cantidades de epine-frina y norepinefrina en muchos de estospacientes durante la anestesia y especialmenteluego de la palpaci6n del feocromocitoma.

ACKNOWLEDGMENTWe are grateful for the kind advice of Dr. C. F.

Code, Dr. E. V. Alien and Dr. A. Faulconer, Jr.

REFERENCES1WEIL-MALHERBE, H., AND BONE, A. D.: The

chemical estimation of adrenaline-like sub-stances in blood. Biochem. J. 51: 311, 1952.

2 MANGER, W. M., BALDES, E. J., FLOCK, E. V.,BOLLMAN, J. L., BERKSON, J., AND JACOBS, M.:A method for quantitative estimation of epi-nephrine and norepinephrine: Preliminary re-port. Proc. Staff Meet., Mayo Clin. 28: 526,1953.

3WEIL-MALHERBE, H., AND BONE, A. D.: The

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QUANTITATION OF.EPINEPHRINE AND NOREPINEPHRINE

adrenergic amines of human blood. Lancet 1:

974, 1953.4 yON EULER, U. S., AND LUFT, R.: The effect of

adrenocorticotrophic hormone (ACTH) andadrenocorticotrophically active peptides(ACTH peptides) on the circulating eosinophilsand urinary excretion of adrenaline and nor-adrenaline in a human subject. Acta Endocrinol.3: 323, 1949.

5 LUFT, R., AND VON EULER, U. S.: Excretion ofcatechol amines during administration ofACTH, cortisone and desoxycorticosterone ace-tate. Metabolism 1: 179, 1952.

6KEITH, N. M., WAGENER, H. P., AND BARKER,N. W.: Some different types of essential hyper-tension: Their course and prognosis. Am. J. M.

Sc. 197: 332, 1939.7ROTH, G. M., AND FLOCK, E. V.: Unpublished

data.

8GOODALL, McC.: Dihydroxyphenylalanine andhydroxytyramine in mammalian suprarenals.Acta chem. scandinav. 4: 550, 1950.

9 VON EULER, U. S., HAMBERG, U., AND HELLNER,S.: f3-(3:4-Dihydroxyphenyl)ethylamine (hy-droxytyramine) in normal human urine. Bio-chem. J. 49: 655, 1951.

'0 LVND, A.: Simultaneous fluorimetric determina-tions of adrenaline and noradrenaline in blood.Acta pharmacol. et toxicol. 6: 137, 1950.

' LUND, A.: Adrenaline and noradrenaline in bloodand urine in cases of pheochromocytoma. Scan-dinav. J. Clin. & Lab. Investigation. 4: 263,1952.

12 VON EULER, U. S., LUND, A., OLSSON, A., ANDSANDBLOM, P.: Noradrenaline and adrenaline inblood and urine in a case of phaeochromocy-toma. Scandinav. J. Clin. & Lab. Investigation5: 122, 1953.

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BOLLMAN, GRACE M. ROTH, EDWARD J. BALDES and MARTIN JACOBSWILLIAM M. MANGER, EUNICE V. FLOCK, JOSEPH BERKSON, JESSE L.

PheochromocytomaChemical Quantitation of Epinephrine and Norepinephrine in Thirteen Patients with

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