Brit. J. Pharmacol. (1954), 9, 498.

IDENTIFICATION OF AN ISOPRENALINE-LIKE SUBSTANCEIN EXTRACTS OF ADRENAL GLANDS

BY

MARY F. LOCKETIFrom the Department of Physiology, Chelsea Polytechnic, London, S.W.3

(RECEIVED AUGUST 16, 1954)

A sympathomimetic amine, not hitherto describedas naturally occurring, was unexpectedly encoun-tered in an experiment in which the adrenalineand noradrenaline, in saline extracts of cat adrenalglands, were being separated chromatographically.

In this experiment a chromatogram, made froman aliquot of extract corresponding to just lessthan one-third of a cat gland, was oxidized, byspraying with a solution of potassium ferricyanide,to convert any primary or secondary catecholamines to the corresponding adrenochromes.Adrenaline and noradrenaline then appeared asred and pink spots respectively, and were identi-fied by means of their RF values. There was,however, an additional very pale pink spot (thirdamine), with an RF value of approximately 0.7,lying ahead of the adrenaline area from which itwas separated by a negative horizontal band ofpaper. Corresponding bands from another, simi-lar, and adjacent chromatogram were thereforeeluted and prepared for biological testing. Theeluate from the negative zone separating theadrenaline area from that of the third amine waspharmacologically inert, whereas that from thezone of the third amine was highly active. lfore-over, the type of activity encountered in the latterzone differed markedly from that obtained fromthe adrenaline and noradrenaline areas. Thethird amine, like adrenaline and unlike nor-adrenaline, strongly antagonized the action ofacetylcholine on the rat uterus. However, whendoses of adrenaline and of third amine eluates,which had proved equally active on the rat uterus,were injected intravenously into a cat underchloralose, the adrenaline eluate raised the meanarterial pressure and caused contraction of thenictitating membrane as expected, but the eluateof third amine lowered the blood pressure, causedtachycardia, and was without action on the nictita-ting membrane.The results of a further investigation of this

third amine are presented here in three sections.The first section is concerned with the great simi-

larity between the third amine and isoprenaline.The second section deals with the presence orabsence of the third amine in the adrenal glandsof various species. Finally, the properties andactions of the third amine have been contrastedwith those of lactyladrenaline and lactylnoradrena-line, because these two compounds are knownsometimes to arise as artifacts in extracts ofadrenal glands. Lactyladrenaline was first isolatedby Kendall (1932) and was studied, together withlactylnoradrenaline, by Crawford (1951). Workby Serlin and Goldenburg (1953) on the instabilityof synthetic noradrenaline in acid ethanol indicatedthat these so-called lactyl derivatives may proveto be ethers. Their hypothesis has not been in-vestigated; the terms lactyladrenaline and lactyl-noradrenaline are here used to refer to compoundswhich are believed identical with those previouslyencountered in extracts of adrenal glands byCrawford.

METHODSCollection of Adrenal Glands.-Adrenal glands

were removed from spinal cats or from cats underanaesthesia (ether, chloroform, chloralose, or pento-barbitone), and from rabbits and guinea-pigs imme-diately after death from a blow on the head. Cat,rabbit, and guinea-pig glands were at once put intoa deep freeze at - 20' C. Human and monkey glandswere removed under ether anaesthesia, and werequickly carried to the laboratory in dry tubes sur-rounded with ice in a thermos flask.

Preparation of Solutions for Chromatography.-The procedure described below is that finally adoptedand used for glands of all species. Frozen glandswere rapidly weighed, cut into small pieces, andground in ice-cold saline (0.9% NaCl, 2 ml./gland)within 2 hr. of excision from the animal. Coarsedebris was removed from the saline extract by centri-fuging at 3,000 rev./min. for 3 min. Usually 0.3 ml.M/15 phosphate buffer, pH 7.4, and 0.2 ml. 0.9%NaCl were added to each 0.5 ml. aliquot of super-natant fluid; sometimes the buffer was replaced byadditional saline. Proteins were then precipitated bythe addition of 7 ml. ethanol and 0.25 ml. 4N-HCI.

ISOPRENALINE IN ADRENALS

After centrifuging, 7.5 ml. of supernatant fluid wasevaporated to dryness in vacuo at a temperature notexceeding 50' C. The residue was extracted with3 ml. of a mixture of equal parts of acetone andethanol; 2.5 ml. of this acetone-alcohol extract wasevaporated to small volume and used for chromato-graphy.Chromatography and Elution.-The methods used

for chromatography, for the development of chromato-grams, for elution, and for the preparation of eluatesfor biological testing have been described in fulldetail elsewhere (Vogt, 1952) and need only be sum-marized here. The concentrated acetone-alcohol ex-tracts were quantitatively transferred to acid-washedWhatman No. 1 filter paper. The area of the paperto be traversed by gland samples destined for use inbioassays had previously been sprayed with a solu-tion of ascorbic acid 50 mg./100 ml. Phenol contain-ing 15% w/v 0.1 N-HCl was used as solvent, andascending chromatograms were run in an atmosphereof C02. Phenol was removed by washing with ben-zene, and the whole process, including drying, wascarried out at room temperature. RF values weredetermined from chromatograms developed by spray-ing with 0.44 g. potassium ferricyanide dissolved in100 ml. 0.2 M-phosphate buffer, pH 7.8.Adrenaline, noradrenaline, isoprenaline, and lactyl-

adrenaline were used as reference compounds,Chromatograms of not less than two samples froma single gland, and of mixed reference compounds,were made in parallel on a single sheet of paper.The solvent front was allowed to advance 14-16 cm.beyond the line of application of the extracts beforethe process was interrupted. Chromatograms fromone sample of each gland, and of the reference com-pounds, were used for determination of RF values;those of other gland samples were eluted and pre-pared for bioassay as described by Vogt (1952).

Pharmacological Methods.-Adrenaline and nor-adrenaline were assayed either on the blood pressureand nictitating membrane of a spinal cat, or on therat uterus and colon as described by Gaddum, Peart,and Vogt (1949). The third amine was comparedwith isoprenaline in parallel quantitative assays inwhich at least three-and usually four, and some-times five-different tissues drawn from two differentspecies were employed. The pharmacological differen-tiation of the new amine from adrenaline was greatlyfacilitated by the use of an atypical strain of ratsobtained, accidentally, from Taylor (London). Theuteri, colons, and ileums of these rats were remark-ably insensitive to adrenaline, but were normally sen-sitive to isoprenaline and to the third amine (Table I).Assays of the third amine on the blood pressure,nictitating membrane, and bronchiolar resistance ofcats were carried out under chloralose anaesthesia.

Drugs.-(-)-Adrenaline tartrate (Burroughs Well-come, Ltd.), (-)-noradrenaline and (±)-isoprenaline(Sterling Winthrop) were obtained commercially.Lactyladrenaline and lactylnoradrenaline were syn-thesized as described by Crawford (1951).

TABLE ITHE RELATIVE SENSITIVITY OF TISSUES FROM DIFFER-ENT STRAINS OF RATS TO (-)-ADRENALINE AND

(±)-ISOPRENALINE

Rats Ratio of Equally Effective Doses,(-)-Adrenaline/(+)-Isoprenaline,

Expressed as a Range

No.Source Exam- Ileum Colon Uterus

inedGaddum, Peart andVogt (1949) .. 1 1-2

Chester Beatty Insti-tute . .. 4 1-50 13 12

London School of 8 (typical)Pharmacy.. .. 8 1-11 135 13

Burroughs WellcomeLtd. .. 4 1-5 1-2 1-3

Old assay rats andTaylor (London).. 25 35-65 20-60 12-50 (atypical)

RESULTSThe Properties of the Third Amine

Pharmacological.-Preliminary experiments hadshown that the pharmacological properties of thethird amine differed from those of adrenaline andnoradrenaline, and resembled those of isoprenaline.Both the third amine and isoprenaline caused a fall

mm.Hg130-

1'

0.3 0.75 0.3 0.5 1.0 2.0X I X I A A

80-

60-

0.5 0.5 0.3 0.75x I X I

FiG. L.-Potentiation of the depressor actions of isoprenaline andthird amine by tolazoline. Cat, under chloralose. Arterialblood pressure before (upper record) and after (lower record)7 mg. tolazoline HC1. All drugs injected i.v. A, (-)-adren-aline, in jg.; I, ()-isoprenaline in pg.; X, an eluate of thirdamine, from cat adrenal, in ml. Time, 10 sec.

499

MARY F. LOCKETT

in blood pressure and tachycardia when injectedintravenously into spinal cats or cats anaesthetizedwith chloralose, and their depressor actions wereequally potentiated by adrenaline antagonists(Fig. 1). This indicated that both amines have aweak but comparable vasQconstrictor action whichis normally masked by their stronger dilator effect,and is abolished by sympatholytic drugs. Theeffect of third amine on the nictitating membranevaried from cat to cat, but always in parallel withthat of isoprenaline. Isoprenaline, 0.5 to 2 jug.,was without effect on the nictitating membraneof 10 animals, caused contraction in 7, and relaxa-tion in 3. Cocaine, injected subcutaneously intocats in doses of 5 to 7 mg./kg., affected the re-sponses of the blood pressure and nictitating mem-branes to the two amines similarly. An initialapparent potentiation of their depressor action wasonly once observed, and was followed by thetyp.cal change seen in each of three other experi-

Moreover, doses of these two amines, which wereequi-effective on the blood pressure and on thenictitating membrane, were equally powerful inthe relief of pilocarpine-induced bronchospasm(Fig. 2).

Isoprenaline and the third amine were so alikepharmacologically that they could not be dis-tinguished from one another by means of parallelquantitative assays. Fig. 3 illustrates one suchassay, in which an eluate of third amine obtainedfrom cat adrenal gland was compared with iso-prenaline. The top records in Fig. 3 show that1 ml. of this eluate was about equal to 1 Mug.(± )-isoprenaline in causing contraction of thenictitating membrane and vasodepression in a catunder chloralose. The centre tracing shows that1 ml. of the eluate equated with 1 ,ug. (+ )-isoprenaline as an antagonist of acetylcholine onthe rat colon. The lowest record demonstratesthat 1 ml. of the eluate had more than the activity

B

Foe~~~~~~~~~~~~~~~~~~~~~.-1,M7A

mmHg90

70

A

2.0 1.0 2.0 2.0 1.5 3.0 2.0l X A X A

FIG. 2.-Comparison of third amine with isoprenaline, before and after pilocarpine. Cat, under chloralose. Records,from above, are of bronchiolar resistance, arterial blood pressure, and contractions of nictitating membrane-A, before, and B, after, 0.85 mg. pilocarpine nitrate s.c. Other drugs injected i.v. in the doses indicated.A, (-)-adrenaline in pg.; I, (±)-isoprenaline in pg.; X, eluate of third amine, from monkey adrenal, in ml.

ments: the depressor actions of these two amineswere converted to pressor activities, and theirpower to cause contraction of the nictitating mem-brane was potentiated, in parallel (Fig. 4). Fur-ther remarkable similarity between the two aminesbecame apparent after the subcutaneous injectionof pilocarpine nitrate, 0.5 mg./kg., into cats. Pilo-carpine caused great and selective potentiation ofthe contractions of the nictitating membrane inresponse to isoprenaline and the third amine (butnot in response to adrenaline and noradrenaline),and converted their depressor to pressor activity.

of 0.7 Mug., and less than that of 1.3 pug., (+)-isoprenaline in inhibiting the action of acetyl-choline on the rat uterus. Since the ratios of theequiactive doses, (-)-adrenaline / ( ± )-isoprenaline,were, for these particular tissues, uterus 40-45and colon 1.0 (see Methods) the activity attributedto the third amine cannot have been due to someadrenaline which had become displaced by anoverloading of the paper during chromatography.Fig. 4 shows another comparison, this time betweenisoprenaline and an eluate of third amine obtainedfrom human adrenal gland. The top records are

500(

ISOPRENALINE IN ADRENALS

mn

80

60

40

0.5 0.25 1 0.5 1 2 4 A B A C A A Cx x I X I I

I . I I I 1

2 1.5 2 1 1.5 IA I I X I I

200.2 5 10 0.2X/lo I I X;/10

20

0.15 50

Xlgo) I100 0.15 50

1 X/1c I

0.2x/10

0.3 2 0.3 0.3 4 0.3

,.' Ii '/ 2f) 2l, A \

Fio. 3.-Comparison of third amine, from cat adrenal, with iso-prenaline, in parallel assays. Top record, from cat underchloralose, shows, from above, arterial blood pressure, nictitatingmembrane, signal, and time in 10 sec. Drugs were injected i.v.Doses of I and of A in pg., and of X in ml. Middle record,isolated rat colon responding to 0.1 pg. acetylcholine added atmin. intervals, and in contact for 15 sec. At each arrow an

inhibitory drug was added 30 sec. before the next addition ofacetylcholine. Doses of I in ng., of X in ml. Bottom record,rat uterus, contracting every 3 min. in response to 3 pg. acetyl-choline acting for 45 sec. Arrows mark additions of inhibitorydrugs, 60 sec. before the next addition of acetylcholine. Dosesof I in ng., of X in ml. In all records, A is (-)-adrenaline,I is (±)-isoprenaline and X is eluate of third amine.

2K

0.15 5 5 0.15 0.1 2.5 5 0.2

x/100 X/101 X/1o0 I x/ 100

FIG. 4.-Comparison of third amine, from human adrenal, with

(+)-isoprenaline, in parallel assays. Top record, arterial blood

pressure of spinal cat. Drum slowed, and 12 mg. cocaine HC1given s.c., between the 2 parts. Other drugs injected i.v. A,1.5 pg.; B, 2.5 pg., and D, 1.0 pg., isoprenaline; C, 0.5 ml.

eluate of third amine. In middle record (rat colon) and bottom

record (rat uterus) the contractions are in response to a fixed

dose of acetylcholine; inhibitory drugs were added as indicated.

I, (+)-isoprenaline in pg. in top record and in ng. in others;X, eluate of third amine in ml.

mm.Hg120_

100-

80 -

501

...............

I

I .-r- p I )

MARY F. LOCKETT

of mean arterial pressure, and were taken froma spinal cat weighing 1.9 kg. The first tracingshows that 0.5 ml. of the eluate had more depres-sor action than 1 jug. and less than that of 2 Mug.(± )-isoprenaline. After the subcutaneous injec-tion of 12 mg. cocaine hydrochloride, 0.5 ml. ofthe eluate had a pressor activity approximatelyequal to that of 1.5 tug. (± )-isoprenaline. Thetwo lower records show that 0.5 ml. of the eluateequated with 1.7 Mtg. of (± )-isoprenaline as an

antagonist of acetylcholine, both on the rat colon(centre) and on the rat uterus (lowest). The ratiosof equally effective doses, (-)-adrenaline/(±)-isoprenaline, were, for these particular rat tissues,uterus 2.0 and colon 56.The great similarity between isoprenaline and

the third amine was not confined to pharmaco-logical activity.

Colour Reactions.-Both third amine and iso-prenaline gave green colours with ferric chlorideand very strong adrenochrome reactions whentreated with oxidizing reagents, and fluorescencewas obtained in the ultraviolet for both compoundsafter treatment with ferricyanide. Adrenochromeformation is typical of catechol amines with pri-mary and secondary amino nitrogen in the sidechain, and fluorescence could not occur were thehydroxyl group in the side chain substituted(Lund, 1949).RF Values.-So great was the similarity between

the RF values obtained for isoprenaline and for thethird amine that isoprenaline has been used suc-cessfully throughout this work as a chromato-graphic indicator for the position of the thirdamine.

Table II summarizes the R F values whichwere obtained simultaneously for synthetic com-pounds and for gland amines. There was no

significant difference between the RF values fornatural and synthetic adrenaline and noradrenaline,or between the values obtained for isoprenalineand the third amine.

The Incidence of the Third Amine in the AdrenalGlands of Various Species

The Criteria Adopted for the Identification ofthe Third Amine.-The existence of the thirdamine in an extract of adrenal gland was not con-sidered to have been proved until each of threeconditions had been satisfied. These criteriawere: (i) An RF value and (ii) a depressor actionsimilar to those of isoprenaline; (iii) an inabilityto distinguish between the amine and isoprenalineby means of parallel quantitative assays carriedout on at least three tissues drawn from twodifferent species. In addition, the tissues selectedincluded either a rat uterus or a rat colon whichwas abnormally insensitive to adrenaline, butwhich had normal sensitivity to isoprenaline (seeTable I and colon, Fig. 5). The latter precautionwas adopted so that no possibility of confusionbetween adrenaline and the third amine shouldexist.The Frequency with which the Third Amine

has been Encountered in the Adrenal Glands ofVarious Species can be read from Table II. Thisshows RF values, simultaneously determined, forreference compounds and for gland amines. Whena gland chromatogram showed no visible spot foran amine, the RF value -for the correspondingreference compound in that particular experimentwas omitted in the compilation of the Table. Sinceadrenaline was present in every gland chromato-gram, the total number of animals in each speciesthat was investigated may be found in bracketsfollowing the mean RF value + the standard errorfor gland adrenaline. The number of animals ofthat species in which third amine was foundfollows the mean RF value+ S.E. for third aminein brackets. It was found in the gland extractsfrom all 4 monkeys, from all 3 human beings,and from three-quarters of the cat populationexamined. It was occasionally encountered inextracts from rabbit glands, but never in thosefrom guinea-pig glands. Third amine is also

TABLE IISIMULTANEOUS DETERMINATIONS OF THE Rp VALUES OF SYNTHETIC AMINES AND THOSE OCCURRING

IN SALINE EXTRACTS OF ADRENAL GLANDS(Solvent, phenol containing 15% w'v 0-IN-HCI. For explanation of numerals in parentheses, see text.)

Source Noradrenaline Adrenaline Isoprenaline LactyladrenalineSynthetic 0-210±0-010 (19) 0-486±0-009 (28) 0-684+0-008 (17) 0-801 ±0021 (5)

Third amineCat .. 0-207±0 007 (12) 0-463±0-019 (12) 0699±0-026 (9) 0-780±0 051 (2)*tMonkey .... 0-203±0-037 (4) 0-459±0-044 (4) 0-672±0 029 (4) 0-820 (M)tRabbit. .. 0-210±0-010 (2) 0-504±0-018 (5) 0-675±0-122 (2)Guinea-pig 0-23040-001 (2) 0-496+0 009 (6) 0-780±0-017 (2)**Man - 0490±0010 (3) 0707-£0-118 (3)

* Gland stored in deep freeze. t Extract stored overnight in refrigerator after precipitation of proteins with ethanol and acid.

502

ISOPRENALINE IN ADRENALS

apparently absent from ox adrenal gland. Bycourtesy of Messrs. Allen and Hanburys Ltd.residues obtained after extraction of the corticoids,and equivalent to a ton of fresh ox adrenal gland,were worked up by Mr. Eastland to an ammo-niacal concentrate of a total volume of 2 1. Thisconcentrate yielded 127 g. of crude adrenaline,but neither the crude adrenaline nor the motherliquors contained demonstrable third amine.

The Quantity of Third Amine Present in Extractsof Adrenal Glands.-Only very small amounts ofthird amine have been encountered. It has beenmeasured as (±)-isoprenaline ÷2, and is enteredin column 5 of Table III as a percentage of the

TABLE HIITHE PERCENTAGE DISTRIBUTION OF SYMPATHOMIMETICAMINES IN SALINE EXTRACTS OF THE ADRENAL GLANDS

OF VARIOUS SPECIESThird amine measured as (±)-isoprenaline + 2. The values in the

Table are the means±their standard errors.

Mg. Amine % Total Extracted AmineSpecies, and Extracted fromNo. Used I g. Fresh (-)-Adren- (-)-Nor- Third

Whole Gland aline adrenaline Amine

Cat (12) 0-967±0-135 74-37±2-89 23-42±3 23 2-21 O051Monkey(4) 1-062±0 350 9493±0-80 3-72±0-96 1I35±0-43Man (3) .. 0-123±0-241 86-33±1-34 13-30±0-82 0-53-0-24Rabbit (5) 0-530+0 111 95-32±0-47 4-44±0-24 0-240-42Guinea-pig

(6)* 0-351±0.098 93-87±3-32 6-13--337 Nil

Groups of animals.

total amine extracted from the glands of thevarious species. The second column of this tableshows the total weight of amine obtained per gramof fresh gland; the yields were low, indicatingthat only a part of the medullary amine went intosolution during mild extraction with ice-cold saline.The very low values obtained for human glandare probably explained by the fact that two of thethree glands were cancerous, and all of them hadbeen cut open and scraped during examination bythe surgeons. Glands from other species werealways obtained in intact form. The last threecolumns of Table III record the relative amountsof (-)-adrenaline, ( -)-noradrenaline, and thirdamine found in extracts of the adrenal glands ofthe different species, expressed as a mean % ofthe total amine + the standard error of the mean.Very small quantities of noradrenaline, which

had not been visible on the developed chromato-grams, were frequently found on elution of theappropriate areas of parallel chromatograms fromhuman, guinea-pig, and rabbit glands. This nor-adrenaline was estimated either on the rat colon,or on the blood pressure of a spinal cat, and theidentity of the amine was always confirmed by

demonstration of a typically weak action as anantagonist of acetylcholine on the rat uterus.Third amine, like isoprenaline, gave a muchstronger adrenochrome reaction than noradrena-line, and was therefore more readily visible thanthe latter amine when present in trace quantities.

The Properties of Lactyladrenaline and Nor-adrenaline Contrasted with those of the Third

AmineBoth lactyladrenaline and lactylnoradrenaline

were found in extracts made from a humanphaeochromocytoma, which had been cut inpieces and stored in 0.1N-HC1 by Crawford in1951. Crawford obtained RF values in phenolof 0.62 (lactylnoradrenaline) and 0.75 (lactyl-adrenaline) both for his synthetic and naturallyoccurring compounds.RF Values.-In my experiments the RF values

for synthetic lactyladrenaline and lactylnoradrena-line in phenol have ranged from 0.74-0.86 and0.55-0.58 respectively, and confirm Crawford'sfindings. Lactylnoradrenaline was not furtherexamined, because the RF values obtained inphenol were too low to permit of its identificationwith the third amine. Lactyladrenaline did, how-ever, require full investigation. The RF valuesobtained for synthetic lactyladrenaline have beenconsistently higher than those for the third amine(Table II), and lactyladrenaline has only beenencountered as an artifact either when the glandswere stored overnight in the deep freeze, or whenextracts from which the proteins had been pre-cipitated with acid alcohol had been stored in therefrigerator. On one occasion both lactyladrena-line and third amine were present in a chromato-gram from a stored monkey gland.

Moreover, when an aqueous eluate of chromato-graphically pure lactyladrenaline (RF value inphenol 0.79) was re-chromatographed, two spotsappeared, one of RF value 0.80, and another whichcorresponded in RF value to the adrenaline reference spot. By contrast, re-chromatography ofan eluate of third amine yielded one spot onlywhich had an RF value identical with that of theoriginal substance.Colour Reactions.-Whereas both lactyladrena-

line and the third amine gave typical adreno-chrome reactions, that from the third aminefluoresced in ultraviolet light, but that fromlactyladrenaline did not. Moreover, when chro-matograms of the two compounds were sprayedfirst with alkaline hydroxylamine solution andthen with acid ferric chloride (Whittaker andWijesundera, 1952) only with lactyladrenaline

503

MARY F. LOCKETT

was there any fugitive purple colour amongst thegeneral medley of colours.

Pharmacological Actions.-Lactyladrenaline hadonly very weak pharmacological actions whichresembled those of adrenaline, and which mayhave been in large part due to partial hydrolysis,of the ester link in aqueous solution (see above).Fig. 5 illustrates the similarity between the effectsof adrenaline and of a solution of chromatograph-ically pure lactyladrenaline 500 tug./ml. The topset of records show that 10 jAg. (- )-adrenaline and0.15 ml. of the latter solution caused similar con-tractions of the nictitating membrane, and had

Hg

..I C

-:;Ii,\ ~)

10 75 15 15A LA A A

75LA

I10A

10A

75LA

0.5 15 1 5 15 3.75 1A A LA LA A

!

0.1A

0.- 1.0 0.05 0.5LA LA A LA

comparable pressor effects, when injected intra-venously into an anaesthetized cat. Whenmeasured as antagonists of acetylcholine, 10 Mtg.(-)-adrenaline equated with 0.1 ml. of the lactyl-adrenaline solution on the rat colon (middlerecord) and with 0.2 ml. on the rat uterus(bottom record). It was also noticed that rattissues which were abnormally insensitive toadrenaline were also insensitive to lactyladrena-line (middle record).

DISCUSSIONThe evidence presented has clearly differentiated

the newly discovered third amine both from themedullary hormones (adrenaline and noradrena-line), and from those artifacts (lactyladrenalineand lactylnoradrenaline) which are known to arisein-extracts of adrenal glands.The evidence also indicates that the third amine

is not an artifact. Firstly, it was found only inthe glands of certain species, although identicalmild extraction and separation processes wereapplied to those of all the species. Secondly, werethird amine produced as an artifact, some corre-lation would be expected either between the totalweight of amine extracted from the gland andsubmitted to chromatography, or between theadrenaline/noradrenaline ratios of the glands, andthe incidence of third amine: no such correlationexisted. Finally, the very great pharmacologicalpotency of this third amine argues against theartifact hypothesis.The colour reactions given by the third amine

indicate that it is a catechol amine, containingeither primary or secondary amino nitrogen and afree hydroxyl group in the side chain. The typeof pharmacological action shown by this amineresembles, in general, that of noradrenaline witha single N-substituent of chain length equal to orexceeding two carbon atoms (Konzett, 1940b).In particular, the great potency of the third amineas an antagonist of pilocarpine-induced broncho-spasm indicates a possible identification with iso-prenaline (Konzett, 1940a). This last hypothesisreceives great support frpm the results of a seriesof parallel quantitative assays which failed todifferentiate the third amine from isoprenaline.The acceptance of this identification reveals a

hitherto unknown biochemical synthesis, whichremains to be investigated.

FiG. 5.-Comparison of adrenaline, lactyladrenaline, and isoprenaline.Top record, arterial blood pressure and contractions of nictitatingmembrane (cat under chloralose). Middle record, rat colon.Bottom record, rat uterus. Contractions of colon and uterus inresponse to a fixed dose of acetylcholine, and inhibited by theaddition of other drugs. A, (-)-adrenaline in pug., LA, lactyl-adrenaline in pg., I, (±)-isoprenaline in ng.

SUMMARY

1. A hitherto unreported sympathomimeticamine has been detected in saline extracts ofadrenal glands from cats, monkeys, and man.

III 111111111fliffullilmill

504

ISOPRENALINE IN ADRENALS 505

2. This amine was separated chromatograph-ically, and was present only in trace quantity.It has been shown to differ in RF value, and inpharmacological activity, from adrenaline, nor-adrenaline, and their lactyl derivatives.

3. The third amine resembles isoprenaline veryclosely in RF value, colour reactions, and pharma-cological activity. It could not be differentiatedfrom isoprenaline in parallel assays.

The expenses incurred in this work were defrayed bya grant from the Medical Research Council. Theequipment was provided by a grant from the WellcomeTrust.

It is a pleasure to thank Miss Ann Hommersham forher skilled assistance.

REFERENCES

Crawford, T. B. B. (1951). Biochem. J., 48, 203.Gaddum, J. H., Peart, W. S., and Vogt, M. (1949).

J. Physiol., 108, 467.Kendall, E. C. (1932). J. biol. Chem., 97, v.Konzett, H. (1940a). Arch. exp. Path. Pharmak., 197,

27.(1940b). Ibid., 197, 41.

Lund, A. (1949). Acta Pharmacol., 5, 121.Serlin, I., and Goldenburg, M. (1953). Biochem. J., 54,

483.Vogt, M. (1952). Brit. J. Pharmacol., 7, 325.Whittaker, V. P., and Wijesundera, S. (1952). Biochem.

J., 51, 348.