Studies on diamine oxidase

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U n p u b l i s h e d t h e s e s s u b m i t t e d f o r t h e M a s t e r 1s and D o c t o r 1 s d e g r e e s and d e p o s i t e d i n t h e N o r t h w e s t e r n U n i v e r s i t y L i b r a r y a r e o p e n f o r i n s p e c t i o n , b u t a r e t o be u s e d o n l y w i t h due r e g a r d t o t h e r i g h t s o f t h e a u t h o r s * Bibliographical r e f e r e n c e s may be n o t e d , b u t p a s s a g e s may be c o p i e d o n l y w i t h t h e p e r m i s s i o n o f t h e a u t h o r , and p r o p e r c r e d i t mus t be g i v e n i n s u b s e q u e n t w r i t t e n o r p u b l i s h e d wor k. E x t e n s i v e copying or p u b l i c a t i o n o f t h e t h e s e s i n whol e o r i n p a r t r e q u i r e s a l s o t h e c o n s e n t o f t h e Dean o f t h e G r a d u a t e S c h o o l o f N o r t h w e s t e r n University. T h i s t h e s i s by h a s b e e n u s e d by t h e f o l l o w i n g a t t e s t t h e i r acceptance of the patrons

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DATE

NORTHWESTERN UNIVERSITY

studies oh diamine oxidase

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OP THE REQUIREMENTS fo r the degree DOCTOR OP PHILOSOPHY

DEPARTMENT OP PHYSIOLOGY AID) PHARl'LACOLOGY

BY FRANZ RUDOLF GOTZL

CHICAGO, ILLINOIS AUGUST, 1942

ProQuest Number: 10101453

All rights re se rv e d INFORMATION TO ALL USERS The quality o f this re p ro d u c tio n is d e p e n d e n t u p o n t h e quality o f t h e c o p y s u b m itte d . In t h e unlikely e v e n t t h a t t h e a u th o r did n o t s e n d a c o m p l e t e m a n u sc rip t a n d th e r e a r e missing p a g e s , t h e s e will b e n o te d . Also, if m a te ria l h a d to b e r e m o v e d , a n o t e will in d ic a te th e d e letio n .

uest. P ro Q u e st 10101453 Published by P ro Q u e st LLC (2016). C opyright of th e Dissertation is held by t h e Author. All rights reserved. This work is p r o t e c t e d a g a in s t u n a u th o riz e d c o p y in g u n d e r Title 17, United S tates C o d e Microform Edition © P ro Q u est LLC. P ro Q u e st LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

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TABLE OF CONTENTS

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

4

HISTORICAL NOTE............................................................................................. .

5

INTRODUCTION .

.

THE CHEMISTRY OR THE HISTAMINE INACTIVATING- PRINCIPLE

.

12

a) General Remarks .

.

12

.

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

b) The S p e c if ic ity of the E n z y m e ..............................................

13

c) The Diamine - Diamine Oxidase R eaction

17

d) The Q u a n tita tiv e E stim ation e) On the A ffin ity

*

.

.

of DiamineOxidase.

.

.

23

Between the DiamineOxidase and I t s 32

S u b s tra te s ................................................................. ....... f) On the I n h ib ito r s of Diamine Oxidase .

.

.

.

.

.

35

g) On the A c tiv a to rs of Diamine Oxidase .

.

.

.

.

.

51

h) Nature and Systemic P o s itio n of Diamine Oxidase

.

.

54

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

56

i) F u nctional R elatio n sh ip s Between Diamine Oxidase and Other Enzymes.

THE OCCURRENCE AND PREPARATION OF DIAMINE OXIDASE.

.

70

DIAMINE OXIDASE AND PREGNANCY..........................................................

74

A WORKING HYPOTHESIS OF ALLERGY.....................................................

85

a) S e n s itiz a tio n and Antigen-Antibody Reaction.

85

b) Histamine and Diamine Oxidase

.

. .

.

.

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

88

c) The A lle rg ic R eactions.........................................................................

99

Paee V II.

DIAMINE OXIDASE AND PRIMARY GLAUCOMA......................................................... 102

V III.

THE BIOLOGICALSIGNIPICANC3 OPDIAMINE OXIDASE......................................I l l

; IX.

BIBLIOGRAPHY..................................................................................................................116

X. VISA........................................................................................................................................124 XI. FUBLICASIONS..................................................................................................................I 26

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I

INTRODUCTION By studying and. reviewing the l i t e r a t u r e on "h ista rain a se", or "diamine oxidase", we found i n te r e s tin g and valuable inform ation, Those who l o s t t h e i r o rig in a l i n t e r e s t in t h i s enzyme be­ cause i t proved to be of no p a r t i c u l a r th e ra p e u tic value obviously overlooked experim ental observations and c o n sid era tio n s which clearer in d ic a te the g re a t p h y sio lo g ic a l sig n ific a n c e of diamine oxidase* In the p resen t ^ d isse rta tio n we endeavor, th e re fo re , to re ­ new t h i s o rig in a l i n t e r e s t , f o r which purpose a lso our own ex peri­ ments were designed.

I t seems im possible to give a d e ta ile d account

of everything known, thought, or suggested in connection w ith dia~ mine oxidase a c t i v i t y . At the end of t h is paper we s h a ll summarise the v ariou s reasons c a llin g f o r r e i n t e n s if i e d stu d ie s on diamine oxidase*

II HISTOBICAL NOTE The ex iste n ce of a histam ine in a c tiv a tin g p r in c ip le has been assumed since 1911#

In th a t y e ar Dale and Lai&law ( l ) attem pted to

in a c tiv a te histam ine by adding i t to f l u id , which was p erfu sed through liv e r*

I t was im possible to o b tain an in a c tiv a tio n of more than 10*0

ragm* of the amine*

The authors concluded th a t the i d e n t i f i c a t i o n of

the endproduct was hopeless under these circumstances* In 1913 Oehme (2) , obviously stim u la te d by the work of Ewins and Laidlaw (3) as w ell as Dale and Laidlaw ( l ) on tyramine and h i s t a ­ mine, re p o rte d a comprehensive s e rie s of experiments on the in a c tiv a ­ tio n of histam ine*

Oehme demonstrated by h is experim ental work a de­

pendency of the histam ine to x ic ity upon the s i t e of a d m in istra tio n in ­ s o fa r as a dose of histam ine f a t a l to ra b b its upon i n je c tio n in to the e a r v ein did not k i l l the animal when in je c te d in to a v e in belonging to the p o r ta l system.

Oehme a lso showed th a t twenty tim es the amount

of histam ine f a t a l to ra b b its upon ra p id intravenous i n je c tio n d id not harm the animal a t a l l when slowly in fu se d in to a p e rip h e ra l or the p o r ta l vein .

Chemical and b io lo g ic a l analyses rev ealed th a t n e ith e r

during nor a f t e r such in fu sio n did renal e x c re tio n of histam ine occur and, furtherm ore, th a t histam ine in fu se d in to the bloodstream rap id ly disappeared from the c ir c u la tio n .

Oehme1s f i n a l conclusion was th a t

histam ine i s e it h e r in a c tiv a te d by the tis s u e s , e s p e c ia lly by the l i v e r ,

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o r re ta in e d and s to re d by t h i s organ. A lso, E u s tis (4) b e lie v e d th a t the l i v e r i s the organ i n a c t i ­ v a tin g h istam in e.

He could demonstrate the in a c tiv a tio n of 10.0 mgm.

of histam ine when incubated w ith 10.0 gm. of l i v e r of c a th a rte s aura. Guggenheim and Lft ffl er (5) i n 1916 continued and extended the work of ffwins and Laidlaw (3) on tyramine by in v e s tig a tin g the changes produced in fiv e amines by animal tis s u e s .

Four of these amines —

parahydroxyphenylethylam ine, phenylethylam ine, indolethylam ine, is o amylethylamine — were in a c tiv a te d by o x id atio n to the corresponding a c id .

Several p e rfu sio n experiments were c a r r ie d out w ith histam ine,

bu t never was an endproduct is o la te d ; Guggenheim and Lt t f f l er (5) did not f in d any tra c e of im inazole a c e tic a c id .

The r e s u l ts of Dale and

Laidlaw ( l ) and of Oehme , (2) were confirmed.

50.0 mgm. of histam ine

could be in fu se d in to a ra b b it without harm, i f the in fu s io n was s u f f i ­ c ie n tly slow.

Following such slow in fu sio n no histam ine could be de­

te c te d in the u rin e ; only when u rin e was obtained continuously by cath­ e t e r during the in fu sio n , minute amounts of histam ine were found. Blood samples taken during such slow in fu sio n were fre e from histam ine. I f , however, histam ine was ra p id ly in je c te d death r e s u lte d and hista^mine was d e te c te d in the blood.

Guggenheim and Lt t f f l er (5) concluded

from t h e i r experim ents th a t the body possessed a mechanism f o r i n a c t i ­ v a tin g histam ine as well as the o th er amines.

P e rfu sio n experiments

were a lso c a rr ie d out w ith i s o la te d r a b b i t 's l i v e r ; the r e s u lts con­ firm ed the fin d in g s of Dale and Laidlaw ( l ) , who a lso observed an in ac—

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ti v a t i o n of only about 10 mgm* of histam ine i n t h is manner. In 1924 K oessler and Hanke ( 6) found th a t histam ine i s | formed by i n t e s t i n a l m icro-organism s; these authors suggested sevi

1 e r a l ex p lan atio n s f o r the f a c t t h a t , in s p ite of the presence of g re a t amounts of t h i s

poisonous substance w ith in the i n t e s t i n a l

; lumen, no in to x ic a tio n occurs:

histam ine might be destroyed w ithin

I the i n t e s t i n a l lumen by b a c t e r i a l or c e l lu l a r enzymes; thus the h is ­ tamine d e te c ta b le in i n t e s t i n a l contents would re p re se n t only a t r a n s it i o n a l stage in ;

p r o te in metabolism and not an endproduct*

Or,

f o r in sta n c e , by s u f f i c i e n tl y firm ad so rp tio n to s o lid c o n s titu e n ts of the i n t e s t i n a l co n te n ts, histam ine could be prevented from passing through the i n t e s t i n a l w all.

F in a lly , histam ine might be absorbed,

bu t d e to x ifie d by the tis s u e s during ab so rp tio n .

This d e to x ific a tio n

could be a f a c u lty of the l i v e r or of the i n t e s t i n a l w all i t s e l f . K oessler and Hankie ( 6) adm inistered o r a lly enormous amounts of h i s t a ­ mine to dogs and Guinea p ig s and determined, twelve hours a f t e r t h is feedin g, the amount of histam ine p resen t i n i n t e s t i n a l co n ten ts, in the i n t e s t i n a l w a ll, and in the l i v e r .

In s p ite of feeding such

la rg e amounts of histam ine no harmful e f f e c ts were observed in the ex­ perim ental anim als.

The q u a n tity of histam ine which could be recovered

from the m a te ria l mentioned amounted only to about 25$ of the dose em­ ployed, in d ic a tin g the disappearance of approxim ately 75$ from the in ­ t e s ti n e s .

Only minute amounts of histam ine were considered p o ssib le

to have e n te red the blood stream since Guinea p ig s of the size used

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would have been k i l l e d w ith in a few minutes by an in travenou s in je c ­ t io n of 0 .5 mgm.

There was fa r-re a c h in g s im ila r ity i n the behaviors

of dogs and Guinea p ig s.

K oessler and Kanke ( 6) a rriv e d a t the con­

c lu sio n th a t d e to x if ic a tio n of histam ine took place w ith in the in ­ t e s t i n a l w a ll; the l i v e r was considered to play a minor ro le in th is process of in a c tiv a tio n . L e ite r (7) in 1925 stu d ie d the metabolism of v ario u s iminaz o le s, f a i l e d , however, to include histamine*

K eith er the blood of

the dog nor of man was found to co ntain any iminazole s.

Following .

intravenous i n je c tio n of an im inazole compound, the p a r t i c u l a r com­ pound was d e te c ta b le in the blood f o r a very short time only.

L e ite r

(7) concluded th a t the animal organism has a w ell-developed capacity f o r d estroy ing the iminazole rin g , e s p e c ia lly when a side chain i s a tta c h e d . Ivy and Ja v o is ( 8) in 1924 gave histam ine by stomach tube to Fck f i s t u l a dogs* normal dogs.

The animals were found to be as to le r a n t as

These o bserv ation s were considered to in d ic a te th a t

the i n t e s t i n a l mucosa e it h e r destroys or renders i n e r t la rg e quan­ t i t i e s of histam ine and th a t the l i v e r does not in a c tiv a te histamine* Kendall and h is a s s o c ia te s (9) i n 1927 demonstrated the in v i tr o in a c tiv a tio n of histam ine by formaldehyde.

The form ation of a

condensation product of histam ine and formaldehyde was suggested to render histam ine b io lo g ic a lly in a c tiv e .

Acetaldehyde, paraldehyde,

alco h o l and acetophenone were found to be as e ffe c tiv e as fonnalde-

hyde.

The e ffe c tiv e n e s s was in v e rs e ly p ro p o rtio n a l to the size of

the molecule. Best and McHenry (10) in 1931, i n t h e i r stu d ie s on the e f­ f e c t of formaldehyde on histam ine, confirmed the observation s of Kendall and a s s o c ia te s (9) i n the main p o in ts .

In a d d itio n , i t was

found th a t formaldehyde produces an immediate e f f e c t on a s o lu tio n of h istam in e, causing a lo s s of approxim ately on e-h alf of the phys­ io lo g ic a l a c t i v i t y , and a slow secondary re a c tio n r e s u ltin g in the lo s s of the rem aining a c tiv ity *

Best and McHenry (10) considered

the immediate e f f e c t as being due to the form ation of a condensation product of histam ine and formaldehyde.

I t was p o ssib le to re sto re

alm ost completely the p h y sio lo g ic al a c t i v i t y l o s t in t h is immediate re a c tio n by b o ilin g the m ixture w ith hy droch loric a c id . F llin g e r ( l l ) in 1930 s ta te d th a t u l t r a v i o l e t energy des­ troyed the p h y sio lo g ic a l a c t i v i t y of a 1: 10,000 s o lu tio n of histam ine. Best (12) in 1929 rep o rte d on the disappearance of histam ine from au to ly zin g lung tis s u e .

N atu rally occurring or added histam ine

was rendered p h y sio lo g ic a lly i n e r t when incubated in a s a lin e suspen­ sio n of minced beef* s lung.

The lo s s of a c t iv i t y could be prevented

by h e atin g the suspension to 90° C. f o r fo u r m inutes.

Best (12) con­

cluded th a t the in a c tiv a tio n of histam ine was due to the a c t iv i t y of an enzyme system p resen t i n the lung and c a lle d t h is system “h i s t a m in ase.M Best and h is coworkerg ( i 2j 13j 10f 14 f 15 # 28) stu d ied h i s -

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taminase thoroughly during the years 1929 to 1935. Z e lle r (16) i n 1938 demonstrated the enzymatic "breakdown of diamines a s, f o r in sta n c e , p u tre s c in e , cadaverine, agmatine, and o th e rs .

This au th o r (17, 18) in the same y e ar was a lso able to

show the i d e n t i ty of B e st1s histam inasen& th h is own diamine oxidase. F e lix and Zorn (19) in 1939, independently from the workers mentioned b e fo re , obtained s im ila r r e s u l ts in t h e i r experim ental work and a r r iv e d a t s im ila r conclusions as did Z e lle r . With B e st1s discovery th a t the histam ine in a c tio n repre­ se n ts an enzymatic a c t i v i t y ,

the system atic study of the chemical

mechanisms in volved i n t h is in a c tiv a tio n was made p o ssib le as well as the study of the chem istry of the enzyme i t s e l f .

A ll t h is w ill

be d iscu ssed in d e ta il in the follow ing c h ap ters. B ig le r (20) in 1936, assuming th a t histam ine i s the causa­ tiv e f a c to r in many a l l e r g i c d ise a se s, introduced the “h istam in ase11 f o r th e ra p e u tic u se. in g .

Such an undertaking seemed lo g ic a l and promis­

Successful a d m in istra tio n s of T o ra n til, which i s the commercial

name of the histam inase p re p a ra tio n , were and s t i l l are rep o rted in cases of c o l i t i s u lc e ro sa , co ld a lle rg y , r h i n i t i s vasom otorica, serum sick n ess, e tc .

However, the number of re p o rts on f a i l u r e s of h i s t a ­

minase therapy in c re a se s ra p id ly , e s p e c ia lly so i n asthma b ro n ch iale. Ahlmark and Kornerun (21) in 1940 published a very thorough review on the th e ra p e u tic u se fu ln ess of histam in ase. The re p o rts on the experim ental examination as to the in flu en ce

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which ,,h istam in aseu p re p a ra tio n s may have on events and pro cesses, presumably caused o r accompanied by a re le a s e of h istam in e, are f u l l of disagreem ents, as i s the im pression one gains in reviewing the c l i n i c a l reports* The p re p a ra tio n s which have been i n use so f a r are of such complex n a tu re , th a t i t i s d i f f i c u l t to prove whether a c tu a lly the histam inase or any o th er c o n s titu e n t i s to be blamed f o r the favor­ able e f f e c ts sometimes observed*

Only then, when a complete par­

a lle lis m between a c tio n and a c tu a l histam inase content of various p re p a ra tio n s can be demonstrated independent from the degree of p u r if ic a tio n , an exact e v a lu a tio n of the e ffe c tiv e n e ss of such pre­ p a ra tio n s w ill be possible*

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III THE CHEMISTRY OF THE HISTAMIKE IISACTIVATIm

PRINCIPLE

a) General Remarks As seen from the proceeding ch ap ter, “h istam in ase11 came in to c l i n i c a l use a t a time when i t s chemical p ro p e rtie s and behavior were not completely understood.

Only more recen t work demonstrated th a t

the enzyme in q u estion has a f a r g re a te r p h y sio lo g ic al sig n ific an c e than i s u su a lly assumed* A thorough knowledge of the chem istry of t h i s enzyme would c o n trib u te a good deal to a b e t t e r understanding of v arious problems and p o ssib ly a ls o to a f i n a l success in i t s th e ra p e u tic a l employment. I t seems j u s t i f i e d ,

th e re fo re , to review b r i e f l y the chemis­

tr y of “histam in ase. 11 We in te n tio n a lly d isre g a rd the customary s tru c tu re of s t r i c t ­ ly chemical reviews to be fre e to p o in t e s p e c ia lly to those f a c ts and observations which are of obvious importance to normal and pathologic physiology.

b) The S p e c if ic ity of the Enzyme The e a r l i e r workers (Best and a s s o c ia te s : 10, 12, 13, 14, 15, 28) concluded from t h e i r experiments th a t “histaminase*’ was an enzyme s p e c if ic f o r histam ine*

N either any o th er iminazole d e ri­

v a tiv e , tyram ine, nor tryptam ine were found to be a c te d upon by the enzyme p re p a ra tio n in use*

These observations found confirma­

tio n in l a t e r experiments ( Z e lle r : 16, 17, 18)* I t was shown a ls o th a t enzyme p re p a ra tio n s very a c tiv e in breaking down histam ine e x e rte d no in flu en ce upon any monamine a s, f o r in sta n c e , amylamine, a d re n a lin e , phenyl oxye thy laraine and ephedrine ( Z e lle r : 16, 17, 18; Gebauer-Fuelnegg and A l t : 22)* I t can be assumed, th e re fo re , th a t the enzyme under d is­ cussion has no re la tio n s h ip to the im inazole rin g or to any one sin ­ gle amino group. In a d d itio n , proper enzyme so lu tio n s are w ithout influence upon d-amino a c id s (Frank: 23), upon diamine monocarbonic acids ( Z e lle r : 1? ) , upon choline ( Z e lle r : 24), and upon hypoxanthine ( Frank: 23)* I t seems c e rta in , th e re fo re , th a t the enzyme i s d i s t in c t ly defined from the d-amino a c id oxidase, monamine oxidase, choline and xanthine oxidase* Besides histam ine, however, the follow ing bases are broken down by “h istam in ase” ( Z e lle r ; 16, 17, 25, 26; Z e lle r. Sch&r and S ta e h lin : 27):

1) Ethylene diamine . . .

2^2

2) Trimethylene diamine •

3^2

3) P u tre sc in e ♦ ....................

4h h 2

4) Cadaverine . . . . . .

5^2

5) Spermidine . . . . . .

h 2h (ch 2)3b h ch 2)4h h 2

. ) h h 2(c h 2)3h h os2)3m s

6)

. ) KH2(0Hg)3BH as2)5m s

7) Spermidine

. ) h h 2(ch 2)4h h css)4m 2

3) homologues

9)

. ) h h 3(ch 2)4m h 0H3)5m 8

10)

. ) k h 2(ch 2)5h h as2)5m 2

11) Spermine . . . . . . .

h h 2(c h 2)3b h c h 2)4h h (ch 2)3h h

12) Agmatine . . . . . . .

h h 2o (u h )h h (cs 2)4m h 2

A ll th ese substances are c h a ra c te riz e d by the presence of two amino groups. As c le a r ly i l l u s t r a t e d by experim ental data ( Z e lle r : 25), the d istance between these two b a s ic groups determines the r a te of enzyma^t i c decomposition; the in c re a se in ra te i s almost lin e a r as t h is d is­ tance in c re a se s. Be s u i t s of com petition experiments ( Z e lle r : 25) show th a t r a te of deam inization and a f f i n i t y do not go p a r a l l e l ; th u s, f o r in ­ stan ce, ethylene diamine i n h ib i t s the decomposition of cadaverine more than does trim ethylene diamine.

A sim ila r re la tio n s h ip seems to e x is t

between cadaverine and histam ine. Histamine can be considered as a d e riv a tiv e of p u tre s c in e , in

which one of the two amino groups has been s u b s titu te d .

Apparently

the a c tio n of the enzyme i s not in te r f e r r e d w ith by s u b s titu tio n of one amino group only.

I f , however, both amino groups a,re s u b s titu ­

te d a s, f o r in s ta n c e , in a rc a in e ( Z e lle r : 25), the enzyme i s preven­ te d from a c tin g any longer upon such compound. As mentioned b efo re, spermine and spermidine are deaminized by the enzyme.

This ob serv atio n n a tu r a lly leads to the question whe­

th e r substances having more than two b a sic groups are a c te d upon by the same enzyme as are the diamines. The enzymatic breakdown of spermine and spermidine, as well as the breakdown of simple diamines, i s in h ib ite d by semicarbazide i n exceedingly low co n ce n tra tio n s.

Data of com petition experiments

( Z e lle r : 25) in d ic a te th a t spermine and spermidine a c t as com petitive in h ib ito r s when diamines serve as su b strates* Thus i t seems j u s t i f i e d to assume th a t i t i s the same enzyme causing deam inization of diamines and polyamines. In numerous com petition experiments between simple diamines and histam ine ( Z e lle r : 25), a com petitive in h ib itio n i s found.

Re­

p o rts on the d is tr ib u tio n of ,,h istam inase,, as revealed by b io lo g ic a l methods agree w ith those on the d is tr ib u tio n of th a t enzyme capable of deaminizing h istam in e, as w ell as diamines and polyamines.

The

l a t t e r re p o rts are based on chemical determ inations ex clu siv ely .

The

o x id atio n of histam ine, as w ell as of cadaverine by human serun i s ac­ c e le ra te d during pregnancy (Sffkemann and Werle: 29, 30; Werle and

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Bffkemann: 31, 32; Z e lle r : 33, 34, 35, 36; Z e lle r and Birkhftuser: 37).

B a c illu s pyocyaneus co n tain s 11h istam in ase11, the a c t i v i t y of

which i s shown to he in h ib ite d by p u trescin e a c tin g as a competi­ tiv e in h ib i t o r (Werle: 38, 39).

,fH istam inaseH and the enzyme dea­

m inizing diamine show the same behavior towards c e r ta in in h ib ito r s . A ll these o b servatio ns stro n g ly in d ic a te the id e n tity of "histam ina se H and diamine oxidase, which l a t t e r name was suggested by Z e lle r (17); th ere i s not a sin g le observation or claim in disagreement on the assumption of such id e n t i ty . Summarizing, we can s ta te th a t we deal with an enzyme well d efin ed from o th er known enzymes; i t i s s p e c ific f o r diamines and polyamines having c e r ta in chemical c h a r a c te r is tic s .

A ll these sub­

stances are known to be of p h y sio lo g ic al s ig n ific a n c e .

-1 7 -

c) The Diamine - Diamine Oxidase Reaction .)

The follow ing equ ation ( Z e lle r : 17; Z e lle r. S tern and Wenk: '

40) re p re se n ts the diamine - diamine oxidase rea ctio n : bch 2eh 2

+ o 2 4- H2 0 = BCHO + m 3 + h 2 o 2

In c e r ta in enzyme p re p a ra tio n s the consumption of oxygen ; proceeds a t a co nstant r a te u n t i l almost two atoms of oxygen are used (Werle: 38).

I f , however, such p rep a ra tio n s were p r e c ip ita te d

w ith (DH^gSO^,, or i f the o rig in a l organ was tr e a te d w ith acetone, the oxygen consumption i s lim ite d to only one atom ( Z e lle r. Sch&r and S ta e h lin : 27).

I t seems th a t hy the procedures mentioned an­

o th e r enzyme was excluded, which caused the use of a second atom of oxygen.

This l a t t e r process — second stage of ox id atio n — ob­

v io u sly has nothing to do w ith the a c t iv i t y of diamine oxidase pro­ p e r. The f i r s t stage of o x id atio n rep re se n ts a monomolecular re a c tio n (McHenry and Gavin: 28; Z e lle r. Sch&r and S ta e h lin : 27). Experimental d ata in d ic a te th a t the consumption of one atom of oxy­ gen corresponds w ith the lib e r a t i o n of one molecule of ammonia; t h i s lib e r a tio n of ammonia tak es place no m atter which su b stra te i s used; h istam in e, by being deaminized, lo se s i t s b io lo g ic a l a c t i ­ v ity (McHenry and Gavin: 28; Z e lle r. Sch&r and S ta e h lin : 27; K iese: 41 ).

This o bservation seems a lso to answer the question whether

histam ine i t s e l f , or only a s p l i t product of histam ine, rep resen ts

the pharm acologically a c tiv e agent in animal experiments: agent i s histam ine i t s e l f .

the activ e

I f one imagines th a t the second atom of

oxygen were to oxidize a h ith e r to unchanged histam ine molecule, no in a c tiv a tio n or, as i n case of p u trescin e or cadaverine, no deamini­ z a tio n would take p la c e .

I t seems safe to assume th a t th is second

oxygen atom serves e n tir e ly the oxidation of the f i r s t re a c tio n pro­ duct of the diamine oxidase. The l a t t e r c o n sid e ra tio n may a ls o ex plain the f a c t th a t, in some re p o rts on the r a te s of in a c tiv a tio n and oxid atio n of histam ine, the curve of in a c tiv a tio n la g s behind the one of ox idation; th is de­ v ia tio n a p p aren tly in c re a se s with in c re a sin g duration of the rea ctio n . While in e a rly experiments (McHenry and Gavin: 28) the form ation of ammonia was slow during the f i r s t hour and only l a t e r e x h ib ited a sharp r i s e , more recen t experim ents, in which thoroughly dialyzed ex­ t r a c t s are used ( Z e lle r. S tern and Wenk: 40), demonstrate a steady in cre ase in the form ation of ammonia from the very beginning. The c o n cen tratio n of the su b s tra te i s of marked influence upon the ra te a t which ammonia i s formed.

T herefore, a su b stra te

optimum e x is ts as in the case of xanthine oxidase. As seen from the above formula, an aldehyde i s formed dur­ ing the diamine oxidase re a c tio n which can be d etected q u a lita tiv e ly and determined q u a n tita tiv e ly .

Reports on such q u a n tita tiv e analyses

(McHenry and G-avin: 15) always show a discrepancy between the amount of aldehyde a c tu a lly found and th a t to be expected.

We b eliev e th a t

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t h i s o bserv atio n i s b e s t explained by the assumption th a t the alde­ hyde formed was alread y p a r tly oxidized which process n a tu ra lly could not be prevented*

Since the pH of enzyme s o lu tio n s , even i f well

b u ffere d , s h i f t s to the a c id side a f t e r a longer d u ratio n of the re­ a c tio n ( Z e lle r : 17) , we can conclude th a t the subsequent oxidation of the aldehyde lead s to the form ation of the corresponding acicu During the enzymatic breakdown of histam ine by diamine oxi­ dase adsorbed by F u lle r s e a rth , a f u r th e r o x idation product contain­ ing a carbonyl group i s found.

I t can be c r y s ta lliz e d and seems to

be dinitrophenylhydrazone (Robert and Z e lle r : 42).

The endproduct

of the histam ine breakdown i s apparently a black pigment, the forma­ tio n of which can be prevented by sulfonamide. The equation i l l u s t r a t i n g the diamine - diamine oxidase re­ a c tio n , furtherm ore, in d ic a te s the form ation of peroxide.

This per­

oxide form ation can be shown simply by adding hemoglobin and h i s t a ­ mine to an enzyme so lu tio n ; a brown d e c o lo riz a tio n appears as soon as oxygen i s bubbled through t h is mixture ( Z e lle r : 17).

Upon spec­

t r a l a n a ly sis i t i s seen th a t methemoglobin replaces the oxyhemo­ globin, which ra p id ly disappears.

The same re a c tio n takes place in

a mere histam ine so lu tio n , although much l a t e r .

This l a t t e r e ff e c t

ap p aren tly i s caused by the au to oxidatio n of histam ine.

The attem pts

to base a q u a n tita tiv e a n a ly sis of diamine oxidase upon t h is color re a c tio n app aren tly f a ile d .

The form ation of the peroxide can also

be demonstrated by the eth y l alcohol - c a ta la se method of K e ilin and

-2 0 -

H art ree ( Z e lle r : 17). We have alread y mentioned th a t the co n cen tratio n of the sub­ s t r a t e i s of marked in flu en ce upon the r a te a t which ammonia i s formed and we concluded th a t a su b stra te optimum must e x is t.

This

optimum according to the re p o rts ( Z e lle r. Schar and S ta eh lin : 27) J

app aren tly i s most pronounced in the case of histam ine or agmatine, l e s s pronounced in the case of spermine or spermidine, and hardly de­ te c ta b le in the case of p u tre sc in e or cadaverine.

A p o ssib le explan­

a tio n f o r t h i s s tr ik in g d iffe re n c e in the behavior of su b s tra te s of the seme enzyme could be th a t i n histam ine and agmatine the a f f i n i ty of one of the two b a sic groups i s by f a r g r e a te r than the a f f i n i ty of the o th er amino group; f o r p u trescin e and cadaverine on the other I hand, one may assume th a t both b asic groups have approxim ately equal a f f i n i t i e s to the enzyme. From what has been s a id in preceeding paragraphs we may as­ sume th a t a double binding takes place between the diamine oxidase and i t s s u b s tr a te s .

S im ilar suggestions have been made in regard to

o th e r enzymes (Haldane: 43).

In the case of histam ine or agmatine,

while the two groups consequently w ill compete f o r the corresponding group of the enzyme, a preference as to the binding of the iminazole o r guanidine group w ill occur.

The r e s u lt i s , then, th a t two sub­

s t r a t e molecules combine w ith one enzyme molecule or: ing and consequently enzymatic breakdown are prevented.

c o rre c t bind­ Thus, i t can

be explained th a t as long as not a l l p r o s th e tic groups p resen t are

occupied, an in crease in the su b s tra te c o n cen tratio n w ill cause an in cre ase in the number of s u b s tra te molecules broken down p er time u n it.

I f , however, the enzyme p resen t i s s a tu ra te d w ith s u b s tra te ,

a f u r th e r in c re a se of the su b s tra te w ill le a d to an in cre ase in the number of u n ip o la r bindings competing w ith b ip o la r bindings s t i l l i n ex isten ce and thus causing a decrease in the ra te of rea ctio n . But not only an in cre ase in s u b s tra te con cen tration may le a d to a u to in h ib itio n .

A decrease in the number of the fre e groups

of the enzyme may have the same consequence.

By blocking such

groups with potassium cyanide, choline, sem icarbazide, e t c . , a f o r­ merly optimal co n cen tratio n of su b stra te i s converted i n t o a superoptimal one and thus again a decrease in the ra te of re a c tio n be­ comes apparent ( Z e lle r : 44).

Under such co n d itio n s, a ls o , putre­

scine and cadaverine may e x h ib it an a u to in h ib itio n . By such c o n sid era tio n s we s h a ll be able to ex p lain various o th e r phenomenas a s, f o r in sta n c e , th a t a monamine may be bound by diamine oxidase w ithout being a c te d upon by the enzyme; b u t, thus p reventing a c o rre c t binding between the diamine oxidase and a dia­ mine, the monamine fu n ctio n in g as a com petitive in h ib ito r w ill pre­ vent the enzymatic breakdown of the diamine. We, th e re fo re , a rriv e a t the conclusion th a t a v a st number of p o ssib le in flu e n c e s upon the diamine - diamine oxidase re a c tio n e x is t.

E sp e c ia lly , the a u to in h ib itio n mentioned seems to be of im­

portance f o r the p h y sio lo g ic al re g u la tio n of the enzyme a c t iv i t y .

d) The Q u a n tita tiv e E stim ation of Diamine Oxidase R ecallin g the re a c tio n equation one re a d ily can see th a t v ario u s p o s s i b i l i t i e s e x is t f o r measuring the diamine oxidase pre­ se n t.

The determ inations of the amount of oxygen consumed, of the

ammonia lib e r a te d , and of the peroxide formed are based on chemical methods. The oxygen consumption may be measured by the a id of War­ burg' s apparatus# The amount of ammonia lib e r a te d can be determined according to van Slyke (McHenry and G-avin: 28) , or by the method of F olin (E dlbacher and Z e lle r : 45); the method of vacuum d i s t i l l a t i o n according to Farnas, and the d iffu sio n a n a ly sis according to Conway are other ways of determ ining the amount of ammonia ( Z e lle r : 17, 34).

The l a t ­

t e r methods by which the d i s t i l l e d ammonia i s measured by n e s s le riz a t io n seem to be of g re a t advantage because they perm it the determina­ t io n of ra th e r small amounts of ammonia and thus make i t p o ssib le to estim ate the ammonia even a few minutes a f t e r the enzymatic re a c tio n has s ta r te d . The peroxide which i s formed during the re a c tio n can be mea­ sured by making use of i t s a b i l i t y to oxidize various substances a s, f o r in sta n c e , in d ig o su lfo n a te .

The d eco lo ratio n of ind ig o su lfo n ate

can be follow ed up e it h e r by determ ining the time necessary f o r com­ p le te d eco lo ratio n or by determ ining p h o tom etrically the amount of

-2 3 -

incLigo l e f t unchanged a f t e r a given time ( Z e lle r ; 24, 25, 26, 36). The l a t t e r method proved to he of sp e c ia l value f o r the estim atio n of diamine oxidase in human serum. The s u b s tra te co n cen tratio n , furtherm ore, can a lso be determ ined purely chem ically by measuring the amino nitrogene accord­ in g to the method of van Slyke. Since histam ine i s a b io lo g ic a lly a c tiv e substance, i t s disappearance may be determined by pharm acological procedures.

This

method was the f i r s t one to be used to estim ate histam ine inactiva^* i

tio n .

In the pharm acological assay e ith e r the blood p ressu re re s­

ponse of the a n e sth e tiz e d c at or the c o n tra c tio n of the s t r i p of the Guinea p ig ileum as caused by histam ine i s recorded. In a l l these determ inations the pH must be optim al, between 6.8 and 7.6 ( B e st: 13).

Another p o in t of g r e a te s t importance i s the

co n cen tratio n of the s u b s tra te .

Histamine and agmatine, as mentioned

alre ad y , have a d i s t i n c t l y lim ite d co n cen tratio n optimum.

This op­

timum i s dependent upon the amount of enzyme p resen t and, th e re fo re , must be determined f o r each tis s u e or tis s u e e x tra c t to be te s te d f o r i t s enzyme conten t.

Only under these conditions i s a comparison

of enzyme a c t i v i t i e s p e rm issib le .

Cadaverine and p u tre sc in e are not

c h a ra c te ris e d by the ex isten ce of such a pronounced optimum and by u sin g these substances one u su a lly can omit the determ ination of the c o n ce n tra tio n optimum.

This i s the obvious reason th a t most chemical

e stim atio n s of diamine oxidase a c t iv i t y were done w ith cadaverine as

s u b s tr a te . In the follow ing paragraph we describe the b io lo g ic a l me­ thod as used in our experim ents.

We determined the diamine oxidase

a c t i v i t y in the blood of normal persons and of p a tie n ts , as well as i n the blood of experim ental anim als. F if te e n cc. of blood were taken from the vein and thoroughly mixed w ith 45 rngm. of sodium flu o rid e .

This a n tico a g u lan t does not

in te r f e r e w ith the enzyme a c t i v i t y ( Z e lle r : 25).

In order to avoid

a u to o x id atio n the incoagulable blood was used immediately.

Six

small t e s t tubes were prepared, in to each of which 1 cc. of flu o rid e blood was p ip e tte d .

To each of these t e s t tubes 0.2 cc. of histam ine

s o lu tio n were added, the co n cen tratio n of which in cre ased geometri­ c a lly .

The follow ing so lu tio n s of histam ine dihydrochloride in phy­

s io lo g ic a l s a l t so lu tio n had to be a v a ila b le :

1 cc. of the so lu tio n s

1 to 6 con tain in g 5, 10, 20, 40, 80, and 160

of histam ine dihydro-

c h lo rid e .

Immediately a f t e r having added the histam ine so lu tio n s to

the blood samples, the tubes were placed in to a water b ath of 37° C. and l e f t th ere f o r two hours.

Then the samples were tra n s fe rre d to

the ice box, where they remained u n t i l evaluated on the a n esth e tiz e d c a t.

In a d d itio n , we prepared 6 co n tro l tubes, each containing 1 cc.

of the same flu o rid e blood; these c o n tro l tubes were kept in the ice box and the histam ine so lu tio n s were added j u s t before making the assay. Meanwhile, the cat was se t up. follow ed by sodium b a rb itu r a te .

As a n e sth e sia we used e th e r

A fte r slow in je c tio n of 1.5 mgm.

a tro p in e s u lf a te the c a r o tid a r t e iy was connected w ith the blood p ressu re recordin g ap p aratu s.

We in je c te d 0 .2 cc. of the blood h is ­

tamine m ixture, to which 0 .3 cc. of p h y sio lo g ic al s a l t so lu tio n were added. the c a t.

These in je c tio n s were made in to the exposed femoral vein of Beginning w ith the lowest histam ine c o n cen tratio n , the in ­

je c tio n of the c o n tro l sample was always follow ed by the in je c tio n of the incubated sample.

The amount of histam ine destroyed was cal­

c u la te d as r e l a ti v e amount according to the follow ing formula:

S-i — Sp) i*. x - -ig _y _S1 In t h i s fomnula-

re p re se n ts the f a l l in blood pressure in

mm. Hg. due to the e ff e c t of the non-incubated sample, S-g to the ef­ f e c t of the incubated sample,

y ' stands f o r the amount of histam ine

dihydrochloride a c tu a lly in je c te d w ith the non-incubated control sam­ p le . In t h i s manner S x— values were obtained f o r each blood sample te s te d , which p erm itted the p lo ttin g of a curve in d ic a tin g the a c tiv ­ i t y of diamine oxidase found. As s ta te d b efo re, these x-values rep resen t only r e la tiv e values; the a c tu a l amounts of histam ine destroyed could have been c a l­ c u la te d only by means of an exponential curve which would have to be found se p a ra te ly fo r each c at due to the in d iv id u a l v a ria tio n s in h is ­ tamine s e n s i t iv i t y .

This could be om itted since we were in te re s te d

only in r e la tiv e changes in diamine oxidase a c t i v i t y w ith in the same

anim al, and in comparisons of diamine oxidase a c t iv i t y in normal and a l l e r g i c in d iv id u a ls . The follow ing curves rep resen t the average amount of d ia­ mine oxidase a c t i v i t y as found in twenty-two normal human and in t h ir t y - f o u r normal canine hlood samples.

We a lso in d ic a te in these

i l l u s t r a t i o n s the range of in d iv id u a l v a r ia tio n observed*

:?-

x - V' aA uce s

s

t f o v *VVi

Afei*-v» histam ine > cadaverine )> p u trescin e Most s u ita b le f o r such stu d ie s seems to be the examination of the ra te of decomposition of spermidine homologues ( Z e lle r. Sch&r and S ta e h lin : 27).

As s a id b efo re, two b a sic groups of the su b s tra te s

combine with the enzyme.

Triamines of the type of spermidine, th ere­

fo re , can form complexes with the enzyme in two d if f e r e n t ways.

The

attachm ent takes place e it h e r a t the longer or s h o rte r chain and thus, e it h e r the amino group of the longer or the one of the s h o rte r chain becomes oxidized.

Which of the two chains i s decomposed can be seen

from the re a c tio n v e lo c ity , since the l a t t e r i s a fu n ctio n of the le n g th of the methylene chain, in cre asin g from ethylene diamine to

cadaverine. The a f f i n i t y , however, i s not so le ly determined by the p o si­ tio n but a ls o by the c o n s titu tio n of the two b a sic groups, n e c e ssita ­ tin g t h e i r sep arate c o n sid era tio n . From i n h ib itio n experiments with monamines, guanidines, e t c ., one can see th a t the a f f i n i t y of c e r ta in guanidines i s by f a r g re a te r than the one of the monamines ( Z e lle r : 24, 44, 46).

The e x is tin g d if ­

ference between the a f f i n i t i e s of the two b a sic groups of histam ine and agmatine have been mentioned b efore. A knowledge of the a f f i n i t i e s e x is tin g between the diamine oxidase and i t s s u b s tra te s i s by no means merely of th e o r e tic a l in ­ te re s t.

The g re a t m ajo rity of substances re a c tin g in some way with

diamine oxida.se are normal c o n s titu e n ts of the animal organism.

Such

substances may be a c tu a lly s u b s tra te s of the enzyme or they may func­ tio n as in h ib ito r s blocking the diamine oxidase by v irtu e of th e ir g re a t a f f i n i t y .

Among these substances, thiamine and guanidine are

outstand ing examples.

N om ally and i n t r a vitam a com petition f o r

the diamine oxidase must a r is e between a l l these substances, where­ fo re not only t h e i r a c tu a l c o n cen tratio n , but a lso t h e i r a f f i n i t y i s of deciding in flu e n c e . Spermine, as an example, possesses a g re a t a f f i n i t y to the diamine oxidase, while i t s re a c tio n v e lo c ity i s r e la tiv e ly small. Thus, the enzyme i s prevented from a c tin g upon diamines w ith le s s a f­ fin ity .

T his, no doubt, n e c e ssa rily must le a d to an accumulation of

-3 4 -

diamines of l a t t e r order, thus u p s e ttin g normal balances which con­ s t i t u t e the b a s is f o r normal metabolism.

f ) On the I n h ib ito r s of Diamine Oxidase As i s known f o r a long time, ammonium ions ( Best and McHenry: 13) have only l i t t l e in h ib itin g e f f e c t.

Urethane, iodine a c e ta te ,

flu o r id e s , carbon monoxide, and malonates in co ncentration s commonly used a re w ithout in flu en ce upon the diamine - diamine oxidase reactio n ( Z e lle r : 17, 25; Edlbacher and Z e lle r : 45).

Calcium ions seem

to be s l i g h tl y in h ib itin g (McHenry and Gavin: 15). Various heavy m etal in h ib ito r s a s, f o r in sta n c e , pyrophos­ phate (McHenry and G-avin: 28) as well as th io u re a and hydrogen su l*

fid® ( Z e lle r : 44) seem to decrease the oxygen consumption.

These

l a t t e r i n h ib ito r s , however, e x e rt t h e i r influ ence during the second stage of o x id atio n ; we alread y mentioned t h is second stage of oxida­ tio n as not being concerned w ith the a c tio n of diamine oxidase proper. This can be concluded from r e s u lts of experiments in which these sub­ stances were te s te d as to t h e i r a b i l i t y to in h ib it the re a c tio n , and i n which the oxygen consumption did not exceed one atom, as i s u su a lly seen in the absence of such 11in h ib ito r s " . Probably a l l the d e riv a tiv e s of ammonia a c t as in h ib ito r s i f p resen t in s u f f ic ie n tly high co n cen tratio n s.

This in h ib itio n i s sim­

ply a com petitive one. Monamines, lik e methylamine or amylamine, are only very weak i n h ib ito r s .

The in h ib itio n caused by the presence of choline or ephe-

drine ( Z e lle r, S tern and Weak: 40; Z e lle r : 24) i s more marked and

seems of g r e a te s t p h y sio lo g ic a l s ig n ific a n c e .

Since the a f f i n i t y of

choline to diamine oxidase i s weak, the enzymatic breakdown of cada­ v e rin e appears to be le s s in flu e n ce d by t h is monamine than the break­ down of p u tre s c in e .

In t h i s connection we r e c a ll the in te r e s tin g

f a c t th a t histam ine re p re se n ts a very powerful in h ib ito r of choline e s te r a s e . As to ephedrine, i t seems im portant to r e a liz e th a t diamine oxidase and monamine oxidase in many re sp e c ts are sim ila r enzymes. The a c t iv i t y of both these enzymes can be expressed by the same equa­ tio n .

An im portant d iffe re n c e , however, l i e s in the f a c t th a t dia­

mine oxidase can be in h ib ite d by cyanides and carbonyl reagents which does not hold tru e f o r the monamine oxidase ( Z e lle r, S tern and Wenk: 40; Blaschko. R ich ter and Schlossmann: 47, 48).

This behavior of

monamine oxidase seemingly in d ic a te s the absence of a carbonyl group p a r tic ip a tin g in the re a c tio n of t h is enzyme.

Such conclusion i s

supported by the f a c t th a t monamine oxidase re a c ts veiy well with secondary amines a ls o a s, f o r in sta n c e , w ith a d re n alin .

I t can be

shown th a t ephedrine a c ts as com petitive in h ib ito r upon the monamine oxidase (Blaschko. R ich ter and Schlossmann: 47). Other experim ental data ( Z e lle r. S te m and Wenk: 40) in d i­ cate th a t 1-ephedrine very markedly in h ib i t s the breakdown of cada­ v erin e by diamine oxidase.

From these observations we may assume

t h a t the binding between monamine and monamine oxidase i s the same as the one between diamine oxidase and the replaceable amino group

-3 7 -

of a diamine.

Since the in h ib itio n of diamine oxidase by ephedrine

i s due to com petition, i t seems c le a r th a t the degree of such in h ib ­ i t i o n depends la r g e ly upon the a f f i n i t y between su b s tra te and diamine oxidase.

Thus, we can e a s ily i n te r p r e t those experim ental data

( Z e lle r . S tern and Wenk: 40) which demonstrate th a t the enzymatic breakdown of cadaverine i s more i n te r f e r r e d with by the presence of ephedrine than i s the breakdown of histam ine. The in h ib ito ry e f f e c t of guanidine d e riv a tiv e s upon the dia­ mine oxidase a c t i v i t y seems to be of g re a t p h y sio lo g ical i n te r e s t. * While some re p o rts ( Blaschko: 49) in d ic a te th a t guanidine prevents the enzymatic breakdown of diamines, o th er experiments ( Z e lle r: 25) demonstrate th a t such in h ib ito ry e ff e c t depends la rg e ly upon the m etby lation of the guanidine and th a t such in h ib itio n in cre ases with the degree of methylation*

Also, in th is in stan ce the in h ib itio n

seems to be due to com petition since experiments demonstrate th a t the degree of in h ib itio n i s g re a tly in flu en ced by the a f f i n i t y of the enzyme s u b s tra te . We must assume th a t the two re a c tin g groups of the enzyme d i f f e r from each o th er, since only one amino group of the su b stra te can be s u b s titu te d w ithout i n t e r f e rrin g w ith the enzyme re a c tio n . The guanidines seem to compete f o r th a t group of the enzyme which may re a c t with a s u b s titu te d amino group.

The in cre ased in h ib ito ry

e f f e c t with in cre ased m ethylation p o ssib ly i s a consequence of in ­ creased b a s ic ity ,

liven 0.001 molar methyl guanidine causes a marked

-3 8 -

ih h ib itio n of deam inization of histam ine ( Z e lle r : 25)* Such experim ental fin d in g s are p o ssib ly in some connection with, the o b serv atio n of sim ultaneous occurrence of histam ine and methyl guanidine in the u rin e of parathyroidectom ized dogs (Koch; 50)* One can imagine th a t due to a disturbance of the metabolism methy 1guanidine i s formed which, by i n te r f e r r i n g with the diamine oxidase a c t i v i t y , causes the occurrence of histam ine in the urine*

I t seems

to be of i n t e r e s t , furtherm ore, th a t a l l those guanidine d e riv a tiv e s (a rc a in e , s y n th a lin , e tc .) in flu e n cin g the diamine oxidase a c tiv ity play some ro le in the carbohydrate metabolism.

In a d d itio n , we re­

c a l l the re p o rts according to which methyl guanidine i s a powerful s tim u la to r of g a s t r o - i n t e s ti n a l glands (Krimberg; 51; Krimberg and Komarow: 52) and the observ ation th a t the c irc u la to ry co llapse found in guanidine in to x ic a tio n i s mainly due to the lo s s of plasma and p ro te in s from the blood (Minot and K e lle r; 55, 56; Minot; 53, 54). Arcaine and sy n th alin e are a ls o known to have a d ila tin g e ffe c t on c a p i ll a r i e s (Linneweh; 57) and to cause a f a l l in blood pressure (Junkmann: 58). Im inazole i t s e l f a ls o has a marked in h ib ito ry influence upon the diamine oxidase a c t iv i t y ( Z e lle r : 46), which in flu en ce i s g re a tly decreased i f a carbonyl group i s p resen t in a side chain. Curare has been claimed to cause a marked re le a s e of h i s t a ­ mine from the m usculature (Alam. Anrep. Barsoum, T a la a t, and Wieninger 59).

This a lc a lo id , however, in h ib i t s the diamine oxidase only in ex-

-3 9 -

ceedingly high c o n cen tratio n s ( Z e lle r. S tern and Wenk: 40).

Thus,

we do not b e lie v e th a t such in h ib ito ry e f f e c t of curare c o n s titu te s ■ I the und erlying mechanism f o r the rele ase of histam ine mentioned be­ fo re . ] We th in k i t probable th a t a lso o th e r a lc a lo id s have an in —

i

! h i b it i n g e f f e c t lik e ephedrine, but such fin d in g s would be without |

sp e c ia l p h y sio lo g ic a l i n t e r e s t .

;

Methylene blue (McHenry and Gavin: 15), pyocyanine and to luy lene blue ( Z e lle r : 25) seem to be very powerful in h ib ito r s of

i

diamine oxidase.

We may e x p la in the e f f e c t of these b a sic dyes with

t h e i r a b i l i t y to take up hydrogen, since o th er dyes used as redoxin d ic a to r s , no m atter whether they have a more p o s itiv e or more nega­ tiv e normal p o te n tia l, e x ert much le s s in flu en ce upon the enzyme ac­ tiv ity . The carbonyl reagents are very powerful in h ib ito r s ; even exceedingly low co n cen tratio n s are s u f f ic ie n t to demonstrate th e ir e f f e c t.

We f in d experiments rep o rted in the l i t e r a t u r e in which

sem icarbazide, thiosem icarbazide, hydroxylamine, dimethylcyclohexandione (dimedone), sodium h y d ro g en su lfite ( Z e lle r : 17, 25), potas­ sium cyanide (Best and McHenry: 13; Z e lle r : 44), phenylhydrazine and the k eto reag en ts: hydrazido carboxymetbyl pyridinium chloride and dime thy 1ami de a c e tic a c id bydrazide chi orme thy l a t e (Werle: 60) were used.

The f a c t th a t a l l these carbonyl reagents are of such marked

in h ib itin g in flu en ce seems to us to be an in d ic a tio n th a t the d ia-

mine oxidase possesses a carboxyl group necessary fo r the oxidation of the diamines.

Since monamine oxidase i s not in flu en ced by these

carbonyl re a g e n ts, even i f added in much h ig h er co n cen tratio n than i s necessary f o r in h ib itio n of the diamine oxidase, these reagents seem to be a convenient instrum ent f o r sep aratin g these two enzymes from each o th er. The h is t id i n e decarboxylase, however, i s markedly in h ib ite d by these reagents (Werle: 60).

The d ifferen c e in the concentrations

of those reagen ts necessary to in h ib it the histam ine forming enzyme and the histam ine in a c tiv a tin g enzyme i s too small to be safely used in se p ara tin g these two p r in c ip le s . The a c tio n of potassium cyanide upon the diamine oxidase a c t i v i t y ( Z e l le r : 44) can be dissolv ed in to several processes which are of i n t e r e s t f o r two reasons.

An understanding of the cyanide

a c tio n throws some l ig h t upon the p o ssib le stru c tu re of the diamine oxidase and, furtherm ore, new viewpoints are gained which are of value f o r enzyme chem istry in g en eral. The in h ib itin g e f f e c t of cyanide upon "histam inase11 has been known f o r a long time ( Best and McHenry: 13).

I t had already

been observed many y ears ago t h a t, under c e r ta in circum stances, such in h ib itio n was not n e c e ssa rily a complete one. I t i s g e n erally assumed th a t potassium cyanide possesses the a b i l i t y to de-ionize heavy m etals, thus causing an in a c tiv a tio n of heavy m etal enzymes.

Since the common heavy metal in h ib ito r s have

been found to be without e f f e c t upon the diamine oxidase, while the carbonyl rea g en ts, as s ta te d b efo re, rep re se n t very powerful inhib­ i t o r s of th is enzyme a c t i v i t y , we may suggest th a t the in h ib itio n of diamine oxidase by cyanide i s p o ssibly due to the a b i l i t y of the cyanide to re a c t w ith the carbonyl groups of the enzyme under f o r ­ mation of cyanhydrine.

Under such circum stances no complex forma­

tio n would take p lac e. Experimental r e s u l ts ( Z e lle r ; 44), show th a t cyanide has a much g re a te r a f f i n i t y to the enzyme than has cadaverine, and c le a r ly in d ic a te th a t such in h ib itio n of diamine oxidase by cyanide i s completely re v e rs ib le when the potassium cyanide i s withdrawn by adding pyruvic a c id or by absorbing i t by a l c a l i .

The f a c t th a t

cyanides may be absorbed by a l c a l i obviously has been overlooked by e a r l i e r workers ( G-ebauer-Euelnegg and A l t : 22) , who consequently a r­ riv e d a t m isleading conclusions.

F or s tu d ie s on the in flu en ce of

cyanide upon diamine oxidase one has to use a mixture of a lc a l i and cyanide of a cyanide vapor ten sio n equal to the one of the cyanide so lu tio n to be te s te d as to i t s a c tio n upon the enzyme system.

Al­

though u su a lly a l c a l i i s used in Warburg's manometric apparatus f o r absorbing carbon dioxide, we b e liev e th a t t h is i s not necessaiy when diamine oxidase i s stu d ied , since no carbon dioxide i s formed during the diamine - diamine oxidase re a c tio n . A withdrawal of cyanide, however, obviously can a lso be brought about by the diamine - diamine oxidase re a c tio n proper.

As

-4 2 —

s ta te d in a proceeding se c tio n , th is enzyme re a c tio n lead s to the form ation of an aldehyde which undoubtedly re a c ts w ith the cyanide. I f one chooses, th e re fo re , a concentration of cyanide a t which the J ■ enzyme re a c tio n i s not completely suppressed, the amount of cyanide |j

| hound to the aldehyde w ill in crease as long as fre e cyanide i s pre­ se n t.

The diamine * diamine oxidase re a c tio n recovers during th is

! p e rio d of f ix a tio n of in c re a s in g amounts of cyanide and f i n a l l y , when a l l fre e cyanide has disappeared, the re a c tio n v e lo c ity w ill \

re tu rn to i t s normal le v e l. ■I

Experimental d ata ( Z e lle r ; 44) in d ic a te th a t such a retu rn i

of the re a c tio n v e lo c ity to normal occurs ex actly when one molecule of oxygen i s consumed p er molecule of cyanide and thus, one molecule of su b s tra te i s changed in to the corresponding aldehyde* The cyanide, on the o th er hand, causes a withdrawal of the aldehyde from the re a c tio n e q u illib riu m , thus in c re a sin g the reac­ tio n v e lo c ity which i s seen i f s u ita b le q u a n tita tiv e r e la tio n s e x is t. I t seems to us th a t such r e s u l ts d e fin ite ly suggest a r e v e r s i b i li t y of the diamine oxidase a c t i v i t y and the a b i l i t y of the enzyme to synthesize diamines, even though under normal conditions the e q u illibrium seems to be markedly disp laced towards the side of the break­ down. F u rth e r experiments ( Z e lle r : 44) in d ic a te th a t potassium cyanide may have an in d ir e c t in h ib itin g influence upon the diamine — diamine oxidase re a c tio n .

As seen from experiments w ith sodium su l-

—43—

f id e and th io u re a , these l a t t e r substances i n h ib i t the second stage of o x id atio n , whereas they are of no influ en ce upon the f i r s t stage of oxidation* s id e ra tio n s :

We a rriv e a t such conclusion from the follow ing con­ during the diamine - diamine oxidase re a c tio n the

r a te of breakdown of the enzyme su b stra te complex determines the v e lo c ity of the t o t a l reaction*

At the beginning, t h is breakdown

re p re se n ts a monomoleeular reaction*

Thus, the oxygen consumption

takes place according to the follow ing formula (McHenry and G-avin: 28): C= ~

t

log • — a-x

in which uaH re p re se n ts the i n i t i a l amount of s u b s tra te , "x11 the amount of s u b s tra te decomposed, and 111" the time during which th is breakdown took place*

This constancy, however, e x is ts only during

the f i r s t m inutes; afterw ards the oxygen consumption in cre ases s te a d ily , which obviously i s due to the f a c t th a t the second stage of o xidation becomes more and more apparent.

In the presence of

sodium s u lfid e or th io u re a , however, "C1* remains p r a c tic a lly con­ s ta n t ( Z e lle r : 44) o r, in o th er words, the second phase of oxidation cannot take p lac e.

T herefore, these two substances do not in h ib it

the diamine - diamine oxidase re a c tio n proper.

Cyanide seems to have

a s im ila r e f f e c t b u t, in c o n tra st to the mode of i t s a c tio n during the f i r s t phase, the cyanide apparently e x e rts i t s e ff e c t during the second phase by forming a complex.

Thus, the mode of i t s actio n s

during the two stag es of o x idation vaiy widely,

Experiments ( Z e lle r :

4 4 ), in which the c o n cen tratio n s of the su b s tra te and the cyanide are such th a t not a l l cyanide i s fix e d by the re a c tio n product, demon­ s t r a t e th a t the re a c tio n comes to an end as soon as one atom of oxy­ gen i s consumed.

I f the s u b s tra te co n cen tratio n , however, i s in ­

creased so th a t a l l cyanide i s elim inated, then the oxid atio n con­ tin u e s .

These observations in d ic a te to us th a t the cyanide in d ir e c tly

may i n h ib i t the diamine - diamine oxidase re a c tio n proper by in h ib it­ in g the o x id atio n of the re a c tio n product which, th e re fo re , f a i l s to be removed from the re a c tio n equillibrium * In o th e r experim ents, in which the su b stra te co ncentration i s in cre ased w ithout changing the co n cen tratio n of the cyanide, the re a c tio n v e lo c ity decreases ( Z e lle r : 44).

This seems to be paradoxic,

however, bu t may be explained by the assumption th a t, due to the blockage of enzyme groups by the cyanide, the su b stra te concentration becomes superoptim al, thus causing a u to ih h ib itio n which we mentioned alre a d y . Assuming th a t cyanides combine w ith groups of the enzyme, we can understand th a t cyanides possess an a f f i n i t y of t h e i r own,, thus competing w ith the s u b s tra te .

This com petition seems to be the

reason fo r the observation ( Z e lle r : 44) th a t the in h ib itin g e ffe c t of the cyanide depends upon the a f f i n i t y of the su b stra te in use. The l a s t in h ib ito r to be discussed i s thiamine:

-4 5 -

c - m 2 HgO -

C

C -

ii

ti

H -

CH2 - IT

G

it

i>

CH

C - CH0 • CHo0H

\ /

3

S

This form ula re p re se n ts thiamine as a d e riv a tiv e of trim ethylene dia­ mine*

As such a diamine i t possesses a s tr ik in g a f f i n i t y to diamine

oxidase*

Experiments ( Z e lle r, Sch&r and S ta e h lin ; 27), however, dem­

o n s tra te th a t no enzymatic breakdown of thiamine takes place when in ­ cubated w ith diamine oxidase preparations*

Competition experiments

rev eal th a t the deam inization of a l l s u b s tra te s of diamine oxidase i s in h ib ite d in the presence of thiam ine; the deam inization of histam ine i s completely prevented i f thiamine and histam ine are of equal concentra tio n * Due to i t s g re a t a f f i n i t y to diamine oxidase, thiamine i s very lik e ly to a c t as an in h ib ito r a lso by producing superoptimal su b s tra te concentrations* As has been assumed f o r some time, the kidneys of r a ts do not contain any ,,h istam inase,, ( Rose, Karadv and Browne; 61; Rose and Browne: 63; McHenry and Gavin: 28). amin

I f r a t s , however, are fe d a v i t ­

d e fic ie n t d ie t, the kidneys become able to deaminize histam ine

and cadaverine ( Z e lle r , Sch&r and S ta e h lin : 27)*

Since data of such

experiments in d ic a te th a t kidneys of such thiamine d e fic ie n t r a ts do not contain any vitam in

any longer, we may assume th a t under normal

-4 6 -

circum stances the thiamine p resen t combines with a l l diamine oxidase contained in the kidney* I

I f such should be tru e we would have to

c o rre c t the assumption th a t the kidney of the r a t i s fre e from " h is tam inase". I t seems p o ss ib le , furtherm ore, to ex plain the remarkable

i!

ij

observ atio n th a t most of the histam ine in trav en o u sly in je c te d in to

:!

li

r a t s i s taken up by the kidneys*

We merely have to consider th a t

com petitive in h ib itio n i s governed to a high degree by the a c tu a l co n cen tratio n of the competing substrates* The obvious in fluence of thiamine upon diamine oxidase a c t i ­ v ity was a lso te s te d in our laboratory*

I s i t p o ssib le to d etect a

i

decrease in diamine oxidase a c t iv i t y of dog's blood a f t e r in fu sin g | i ;

a thiam ine s o lu tio n in to the animal? We b r i e f l y describe the methods and m a te ria ls used in these experim ents:

the dogs — 20-30 pounds in weight — were a n e sth e tiz e d

by means of nembutal.

The femoral veins were exposed and the a r t e r i a l

blood p ressu re was recorded.

We in fu sed a s o lu tio n in to the femoral

v e in which p er hour contained 10 mg». of thiamine hydrochloride in 120 cc. of p h y sio lo g ic al s a lin e .

This in fu sio n was continued f o r 2\- hours.

Blood samples were taken from the femoral v ein before the in fu sio n s t a r te d and 40, 80, 120, and 150 minutes a f t e r the beginning of the in fu sio n .

The l a s t sample was f i n a l ly taken t h i r t y minutes a f t e r the

in fu sio n had ended.

The blood samples thus obtained were examined as

to t h e i r content in diamine oxidase a c t iv i t y in a manner which was

describ ed in a previous se c tio n .

In th is fash io n we stu d ie d six a n i­

mals which a l l showed e s s e n tia lly equal changes in the enzyme a c t iv i t y . The blood p re ssu re in fo u r dogs f e l l s lig h tly during the in fu sio n and re tu rn e d to normal approxim ately t h i r t y minutes a f t e r the in fu sio n was in te r r u p te d .

The follow ing curves rep resen t the averages of the x -

values as o btained from the enzyme assay s.

Four o ther dogs, infused

I under equal experim ental c o n d itio n s, however, only with pure physio!l M ;! lo g ic a l s a l t so lu tio n , served as c o n tro ls. In these c o n tro l animals il

no changes in blood diamine oxidase a c t iv i t y were found.

V "

nh (Sl ' L

ul

e 5.

c~ ©

°1

Cn

6~-

r *

Ho

ih

in

rr

l 6n ..

r ' i c j i O *vu .

A O

X



o f- 'Vf .

X

-

v - (P u l u .

e

- . O

/V*^ t* W

6\.l-

.

P $

"

r o

3 0 'vwi'vi. < v ^ * t e r

>**j e c t / c J ' H

T

o

er

^j

i

j

t* O'vt.

-6 9 -

We a re , a t the p resen t time, in no p o s itio n to discuss or even to attem pt to exp lain the mechanism of th is h ep arin e f f e c t.

As

f a r as the th ree animals are concerned in which the in je c tio n did not cause any change in the diamine oxidase a c t iv i t y of the "blood, i t i s our im pression th a t the dose of h ep arin i s of deciding impor­ tance and th a t the amount of heparin necessary to produce the e ff e c t mentioned cannot he determined merely on the b a sis of body weight. We were encouraged by the fin d in g s in the m ajority of these experiments and attem pted to suppress the e f f e c t of try p sin upon the diamine oxidase by simultaneous a d m in istratio n of heparin*

Although

u sin g these agents in varying r a t i o s , these attem pts, so f a r a t l e a s t , were un su ccessfu l.

-

70 -

IV

THE OCCURRENCE AND PREPARATION OP DIAMINE OXIDASE Kidney Human

L iver

Intestins.1 Mucosa

Adrenal

Blood

Lung

4

4

4+

44

4+4

+4

Beef

++4-

+4

+++

•w

Sheep

44+

+4

Horse

44+

4+

Hog

++++

4 4

Rat

-

Guinea p ig

++

Rabbit

+

4

Dog

+++

4

Cat

+++

4

Jag uar

+++

?

Kangaroo

+

Chi cken

+

Pigeon

4+

+444

Muscle

Spleen

T

+4+ 4

4

4

-

-

-

-

+4+

4

4

4

4

4

4 4+ 4+

4

4

-

++

4+

4

4

Palcon

++

4+

Duck

-

44

Tawny owl

+

44

G-allus banciva

?

4

Magpie

+

44

S ta r lin g

4

+4

-

4

44

-7 1 -

The preceeding ta b le i s compiled from re p o rts by various a u th o rs.

The follow ing i n te r e s tin g p o in ts are to be added:

;

The human p la c e n ta i s ric h in diamine oxidase (Danforth:

184; Z e lle r. Schar and S ta e h lin : 27).

This observation augments the

'number of in d ic a tio n s th a t during pregnancy im portant changes in the ; diamine metabolism take p lace.

The idea th a t diamine oxidase a c t iv i t y

i s in some connection with reproduction i s a ls o supported by the ob­ se rv a tio n th a t sperraa f l u i d of human and v ario u s o th er mammals con­ ta in s a s tr ik in g ly high enzymatic a c t iv i t y ( Z e lle r : 35; Z e lle r and J o e l : 86) .

I t seems to be more than merely in c id e n ta l th a t sperma

a ls o contains a s u b s tra te and an in h ib ito r of diamine oxidase as w ell. ;We ra th e r b e lie v e th a t p h y sio lo g ic al sig n ific a n c e must be a scrib ed to ; the presence of t h i s complete enzym e-substrate system.

Whether i t i s

concerned w ith the biology of sperma or w ith the process of impregna­ tio n . Since enzyme and su b s tra te are p re se n t w ithin the sperma f l u i d , we can very re a d ily understand the e a r l i e r observation th a t 11r e s p ir a tio n ” i s continued even in the absence of spermatozoa (Windi s to s s e r : 87).

A study of c e ll r e s p ira tio n i s p o ssib le , th e re fo re ,

; only a f t e r a se p a ra tio n of c e l l s from f l u id .

The enzyrne system seems

.unimportant as f a r as the m o tility of the spermatozoa i s concerned, since m o tility i s a lso observed in a medium containing cyanide (Belonoschkin: 88). T e s tic le s , spermatozoa, p ro sta te gland and seminal v e sic le s

-7 2 -

ii

t;

j co n tain only l i t t l e diamine oxidase a c t i v i t y ( Z e lle r and J o e l : 86). ij ji Human pancreas i s another organ rich, in diamine oxidase l| j! ( Z e lle r . Birkh&user. M islin and Wenk; 69). I t i s an in te r e s tin g ji

f f a c t th a t pancreas i s c h a ra c te riz e d by a very high content in sp erji niine ( Z e lle r ; 85) which, as already discussed, i s a su b s tra te of

P

jj diamine oxidase. B rain and r e tin a ( Z e lle r. Birkh&user. M islin and Wenk: 69) ij ■ j seem to co n tain only l i t t l e diamine oxidase. The co ncentration in I; j the b ra in appears to be h ig h e st in the pallidum (Birkh&user: 89). li In cow* s m ilk ( Z e lle r. Birkh&user. M islin and Wenk: 69) , .

|| h e a r t, stomach, skin, u rin a ry bladder and u rin e of the dog (Best and ji

!' McHenry: 13) , and a lso in y e a st ( Z e lle r. Birkh&user. M islin and Wenk; '!

li 69), only tra c e s of diamine oxidase a c t iv i t y are found. ij

Throughout

j; the mammals diamine oxidase i s detected most freq u e n tly in the k id !: ney, i n t e s t i n a l mucosa and l i v e r .

Usually the kidney, e sp e c ia lly

!: the cortex, i s r ic h e r in t h is enzyme than i s the l i v e r .

Rodents

jj

j d i f f e r from o th er mammals in t h e i r diamine metabolism in several re s . p e c ts . As f a r s-s b ird s are concerned, the l i v e r u su a lly contains r more diamine oxidase than does the kidney. 1

The diamine

oxidase a c t iv i t y of various organs changes dur­

in g the ontogenesis of human, c a t tl e and s ta r lin g ( Z e lle r. Birkhftuser. M islin and Wenk; 69). 1

A marked in crease of diamine oxidase a c t iv i t y is found in

jj

U

human serum during pregnancy ( Z e lle r ; 34, 25, 26, 24, 37; Werle and

fj

Sffkemann: 30) ; a ls o , dog's "blood con tain s, "by f a r , more diamine oxi)i

jj dase during pregnancy than i t does normally, i|

As

f a r as the p re p a ra tio n of s u ita b le enzyme concentrates i s

i concerned, a l l the evidence in d ic a te s th a t h o g 's kidney i s the ric h ;! e s t and most s u ita b le source. | ta in e d from t h i s organ.

Very a c tiv e p rep a ra tio n s can be ob-

I t i s b e st to t r e a t the ground organ with

; ic e c o lle d acetone ( Z e lle r. S tern and Wehk: 40), thus obtaining a diy !, powder which can be used f o r months w ithout lo sin g in a c t iv i t y . By );i! | e x tra c tin g th is powder with p h y sio lo g ical s a l t so lu tio n one obtains the f i n a l enzyme so lu tio n , Shis l a t t e r s o lu tio n can be concentrated I; ]• (McHenry and Gravin: 15) by evaporation. The alterants made to p u rify

jj

si fj

the enzyme p re p a ra tio n s by p r e c ip ita tio n s , adsorptions and e lu tio n s \ jl jj were not extensiv e enough (McHenry and Gavin: 15; Hdlbacher and !j Z e lle r ; 45), N evertheless, today i t i s p o ssib le to work with enzyme |j jl jj p re p a ra tio n s (McHenry and G-avin; 28) which are ten times more conceni if* 1 t r a t e d than was the o rig in a l "liistam inase11.

-7 4 -

Y m m im

oxidase m d phegkaitciy

Technical reasons, u n fo rtu n a te ly , made i t im possible f o r us to follow up the diamine oxidase in dog’ s blood during pregnancy. We had the o p p ortu nity, however, to assay blood of e ig h t pregnant dogs.

By averaging our x -v alu es, we obtained the follow ing curve.

For the sa&e of comparison, we also show a curve obtained by averag­ in g the x-values of eig h t normal female dogs:

X

I

rr

'



ih-

"L

e. S.

-7 6 -

! I

From these curves i t i s c le a rly seen th a t the “blood of the pregnant anim als c o n tain s, hy f a r , more diamine oxidase than the

i' hlood of normal female anim als.

These fin d in g s agree w ith sim ila r

j} claim s rep o rte d in the l i t e r a t u r e .

I t seems to “be well worthwhile

to discuss them a l l more in d e ta il “because, “by doing so, we might I')

jj he ahle to throw new l i g h t upon quite a few problems concerning norj;

(j mal and p a th o lo g ic a l pregnancy. Sim ultaneously, we s h a ll demonstrate I* jj ag ain th a t the p h y sio lo g ic al sig n ific an c e of diamine oxidase i s , by j| f a r , g re a te r than i s commonly thought. ! Histamine very fre q u e n tly i s considered as the agent re s |] ,! p o n sib le f o r the onset of normal lab o r and, a ls o , as an e tio lo g ic L ( f a c to r in pregnancy toxemia* fj

j

Experim ental data in d ic a te th a t the d is tr ib u tio n of hista*-

;!l! mine in female anim als d if f e r s somewhat from the one in male animals. Thus, i t i s claimed th a t blood of female r a t s contains more histam ine > than blood of male r a ts (Rose: 90). these fin d in g s .

We were not able to confirm

In t h is re s p e c t, i t i s im portant, furtherm ore, th a t

the ovaries of r a t s contain ten times more histam ine than the t e s t i ­ c le s (Rose: 90).

In a d d itio n , i t i s shown th a t the tis s u e of the

: u te ru s i s enabled to absorb histam ine and to sto re i t ( Oehme: 2).

It

i s assumed (Oehme: 2) th a t the u te rin e tis s u e re le a se s th is sto red histam ine when surrounded by a f l u i d containing no histam ine.

Exper­

im ental data in d ic a te th a t not the a c tu a l amount of histam ine i s caus­ ing u te rin e c o n tra c tio n s, but ra th e r the abso rp tio n of histam ine from

-7 7 -

|| the surrounding medium by the u te ru s , or i t s re le a se in to the su ri J rounding flu id * These experiments show t h a t , as f a r as the human. | u te ru s and the u t e r i of many animals are concerned, stim u la tio n i s i| || caused r a th e r by the histam ine e n te rin g the tis s u e . In some anim als, | however, the histam ine re le a s e d from the u te rin e tis s u e seems to ex— jj j Jj e r t i t s pharm acological a c tio n upon the u te rin e m usculature. Such P p l a t t e r behavior i s claimed, f o r in sta n c e , f o r the u te ru s of Guinea j'i

j P ig s. i

■ Ii jj

Having enumerated th ese few o b servatio ns, l e t us think: of the suggested p o s s ib i l i ty th a t histam ine may play a decisive ro le in

|j

ji causing u te rin e c o n tra c tio n s. We have to th in k of two main p o s s ib ili* | itie s : u te rin e c o n tra c tio n s could be caused by the histam ine p resen t j! w ith in the bloodstream or by th a t histam ine re le a se d from the tis s u e |l

of the u te ru s . The f i r s t of these suggested p o s s i b i l i t i e s seems unI) |j l ik e l y because th ere i s no evidence th a t the histam ine content of

jj

!j

jj

the blood i s in cre ased during pregnancy.

As to the second sugges-

li t tio n , we have to assume the presence of r e l a ti v e l y larg e amounts of ii s| histam ine in the u te rin e tis s u e , which assumption appears to be supjj p o rted by experim ental observ atio ns (Harding and P o r t: 91; Bartholomew ji and Kracke; 92; Bartholomew and P a rk er: 93). ^

The next question which we obviously have to ask ourselves i s

ji

whether t h is histam ine p re se n t in the u te r in e tis s u e i s a c tu a lly a c tiv e

ji

during u te rin e c o n tra c tio n s.

Experimental experience apparently answers

i t h i s question m a p o s itiv e sense:

thus, one fin d s more histam ine in

the blood of the u te rin e v ein (Marcou and Atanasiu-Vergu: 94) a f t e r j; the c o n tra c tio n of a human u te ru s than b e fo re .

Somehow, comparable

J

!j ob servation s are rep o rte d f o r the histam ine content of blood from j| pulmonary vein s taken before and sh o rtly a f t e r the production of ana^j| p h y la c tic shock in Guinea p ig s (B artosch: 95); the histam ine content ji

j! of blood from canine h e p a tic veins shows an analogue behavior (Man-

jj

j waring: 96).

In a d d itio n , th ere i s some in d ic a tio n th a t the hi s ta ­

ll mine content of venous blood from muscles i s in creased a f t e r contrac|j tio n of these muscles (Anreo and Barsoum: 97) ; we a lso mention, in I

| t h i s connection, the observation th a t the histam ine content of blood j from the coronary veins i s g re a tly in flu en ced by the amount of work ji ; the h e a rt i s doing. S im ila rly , in conditions in which the oxygen .Si ji supply to the h e a r t i s in s u f f ic ie n t (Marcou: 98), blood from coronij

■ ary veins seems to contain a h ig her histam ine con tent.

A ll these ob-

i se rv a tio n s appear to support our b e lie f th a t we have to th in k , as f a r jj as u te rin e c o n tra c tio n s are concerned* of the e ff e c t of histam ine re ;j le a se d ra th e r than of histam ine formed ad hoc. We assume th a t during ii V ■ the in te r v a ls between the c o n tra c tio n p e rio d s, a loading and sto rin g i: 5 of histam ine w ith in the u te rin e m usculature takes p lace, j

I t seems safe to assume th a t th is histam ine, when lib e r a te d , a lso e x e rts i t s pharm acological a c tio n s upon the lo c a l c irc u la to ry

: system, thus f a c i l i t a t i n g the muscular a c t i v i t y by in c re a sin g blood ■ i and oxygen suoply. j

H istam ine, furtherm ore, seems to play an im portant role dur-

ing the m enstrual cycle*

As i s known, while the prolonged a c tio n of

histam ine causes changes in the p erm eab ility of the v a sc u la r bed, the sh o rt histam ine a c tio n in flu e n ce s ra th e r the width of the small ' v e s s e ls .

In creased p e rm e ab ility leads to the form ation of exudates

r ic h in protein*

A ll t h i s seems to h in t a t a p a r tic ip a tio n of h is ­

tamine in the mechanisms of changes occurring during the m enstrual .■ cycle. Experiments show th a t the u te ru s of immature Guinea pigs be, comes enlarged due to hyperemia follow ing the a d m in istra tio n of h is li

!l tamine (Bobinson and Zondek; 99)*

These ob serv atio ns, however, are

| not confirmed by o th er in v e s tig a to r s (fra n k : 100; Lewin: 101). i; |i from a l l th is we f e e l j u s t i f i e d in agreeing w ith those who * i! i ! assume th a t some connection e x is ts between histam ine metabolism and f; li

sexual fu n c tio n s.

This j u s t i f i c a t i o n i s in creased by those observa-

i;

tio n s which have been made on the behavior of diamine oxidase during i; j pregnancy* -i

I

The "histam inase" a c t i v i t y of blood i s markedly in creased

j!

;■ during pregnancy (Marcou and Atanasiu-Vergu: 94; Werle and Effkemann; 31, 32).

Subsequent in v e s tig a tio n s demonstrated th a t th is r,histam in-

ase" i s id e n tic a l w ith diamine oxidase ( Z e lle r and Birkhftuser: 37; i own experim ents) .

While the re p o rts in the lite ra tx ire are concerned

w ith in v e s tig a tio n s on human su b je c ts, we showed, as mentioned and ! i l l u s t r a t e d b e fo re , th a t corresponding changes seem to a lso occur in !; |i dogs. The p h y sio lo g ic al purpose of the in crease in the diamine o x i-

-8 0 -

dase content of blood, might be to p ro te c t the u te ru s from histam ine re le a s e d or m anufactured a t some o th er place of the organism and, ii

j a ls o , to prevent an a c tio n of the histam ine re le a s e d during u te rin e c o n tra c tio n s upon the general m aternal c ir c u la tio n . We may a lso men5 I1 tio n here th a t no histam ine appears in the u rin e of pregnant women I (Werle and Sffkemann: 31, 32) and th a t the histam ine content of the ! blood i s even s a id to decrease during pregnancy (Marcou and Atana si u Vergu: 102). In a d d itio n , th ere seem to be o th er f a c to rs a c tiv e which p ro te c t the u te ru s from u n d e sired histam ine a c tio n s .

The s e n s itiv ity

of the u te r in e m usculature to histam ine i s shown to decrease during pregnancy, mainly towards the end of pregnancy.

Another f a c to r appar-

i' e n tly of im portance, in t h is re sp e c t, i s the in creased e le c tr o ly te metabolism during pregnancy, which i s known from qu ite a few ex p eri; ments to in c re a se the re s is te n c e of animals to histam ine (hffkemann and Werle; 29, 30). As f a r as the diamine oxidase content of the u te rin e tis s u e i s concerned, the few a v a ila b le data show th a t a t b e st only tra c e s of the enzyme are p re se n t in t h i s organ (Werle and Effkemann: 31, 32). F in a lly , the p la c e n ta has to be discussed:

th ere are convinc­

ing experim ental d ata in the l i t e r a t u r e dem onstrating the presence of histam ine in t h i s organ (Harding and F o r t: 91; Bartholomew and Krackes 92; Hofbauer: 103; Bartholomew and P ark er: 93; Werle and Sf fkem«.nns 31, 32).

F u rth e r stu d ie s on t h i s problem rev ealed th a t the p lac en ta i s

-3 1 -

a ls o c h a ra c te riz e d "by a s tr ik in g ly high content in diamine oxidase. I t i s g e n e ra lly assumed ( P a n fo rth : 84; Werle and Effkemann: 31, 32; Marcou; 98) th a t the enzyme p re se n t in the p la c e n ta c o n s titu te s an­ o th e r f a c to r p ro te c tin g the u te ru s from f r e e histam ine.

The g rea t

number of mechanisms in p ro te c tin g the u te ru s from histam ine appears understandable i f we consider th a t the m aternal histam ine metabolism i s g re a tly in c re a se d (K aueller-A dler: 109, 110, 111; Tschoup and IschoiOT?; 105; B ickel: 104; Bloch and Pinttsch: 108; H o ltz : 106; Holtz and H eise: 107; and many o th e rs ) and th a t the blood coming from the f e tu s con tain s more histam ine than does the m aternal blood, but only very l i t t l e

diamine oxidase a c t i v i t y (Werle .and Effkemann; 31, 32;

Z e lle r and Birkh&user: 37; Z e lle r . Sch&r and S ta e h lin ; 27). Thus the m aternal histam ine metabolism d if f e r s very d is t in c t ly from the one of the fe tu s .

A pparently, the d e to x ific a tio n of the f e t a l

blood takes place i n the p la c e n ta .

This i s c le a rly in d ic a te d by q u a n tita ­

tiv e determ inations of the histam ine contained in venous and a r t e r i a l blood of the u m b ilica l cord. , The In creased form ation of histam ine in the f e t a l organism i s p o ssib ly in some connection with a r e la tiv e lack of oxygen su p p lied to the f e tu s , thus lead in g to conditions not o p ti­ mal f o r diamine oxidase a c t i v i t y .

That t h is l a t t e r p o in t i s of impor­

tance i s shown by experiments demonstrating a marked decrease of h is ­ tamine in c a t f s blood follow ing a r t i f i c i a l h y p e rv e n tila tio n of the a n i­ mals; t h is decrease la s te d fo r two hours (E ic h le r and Sneda: 112).

It

i s p o ss ib le , furtherm ore, to decrease the diamine oxidase a c t iv i t y of

-

32 -

jj dog* s blood by su b je ctin g the animals to anoxia ( own experim ents) . j| The follow ing ta b le i l l u s t r a t e s the p re v a ilin g r e la tio n s h ip s d isI | cussed above: OHJaH '" (F e ta l Blood ( (M aternal

HTSTSHniE—

' -

m M H zr'o x x m & d

Increased

Traces

ITormal

Increased

P la c e n ta

Present

Very high

Uterus

Increased

Traces

~

This ta b le c le a rly demonstrates th a t the fe tu s i t s e l f also appears to be w ell safeguarded a g a in st an overflow of histam ine from

I | the m aternal organism, jj

From what was s a id i n a preceeding chapter the e x is tin g d is-

j tr ib u t i o n of diamine oxidase obviously does not c o n tro l the metabolism of histam ine e x c lu siv e ly . ! j! ij |j ii ij ii |

In ev alu atin g the p h y sio lo g ic al sig n ific an c e

of diamine oxidase during pregnancy one has to keep in mind the ra u ltitude of s u b s tra te s of t h is enzyme and the g re a t v a rie ty of substances and fa c to rs in flu e n c in g the enzyme re a c tio n .

From such co n sid eratio n s

one could imagine th a t during pregnancy an in crease occurs p rim arily

I! in the metabolism of diamines and polyamines in g en eral, and th a t the iIjI jj in crease in diamine oxidase c o n s titu te s the r e s u lt of a p h y sio lo g ical I! jj a d ap tatio n , ;; A d istu rb e d balance between uroduction and breakdown of dia^i! ; mines can be expected to cause severe changes of normal m etabolic pro:} jj c esses, U n fo rtu n ate ly , in t h i s respect histam ine i s the only su b stra te

-3 3 —

of diamine oxidase to which some a tte n tio n was p a id so f a r .

Impor­

ta n t connections of diamine oxidase w ith the metabolism of pigments and carbohydrates; endocrine and nervous fu n c tio n s, e t c . , appear to have been overlooked e n tir e ly , although s tu d ie s on these problems very l ik e l y would le a d to new concepts on many com plications of preg­ nancy and puerperium.

A f u r t h e r d iscu ssio n on these problems seems

to be out of l i n e ; we f e l t j u s t i f i e d , however, in in d ic a tin g the nec­ essary d ire c tio n of fu tu re research . We b e lie v e th a t one more p o in t ought to be mentioned; namely, the p o s s i b i l i t y to diagnose an e x is tin g pregnancy by d e te m in in g the diamine oxidase a c t i v i t y of blood*

Although a l l proper chemical me­

thods can be used ( Z e lle r and Birkh&user; 37), the determ ination of the hydrogen peroxide, a re a c tio n product of the diamine - diamine oxidase re a c tio n , seems to be most p r a c tic a l ( Z e lle r : 36; Labhardt: 113, 114),

This method, as was mentioned b efo re, i s based on the de-

c o lo r iz a tio n of indigo su lfo n a te by hydrogen peroxide.

We b eliev e

th a t the experiences made w ith th is chemical pregnancy t e s t so f a r are y e t too meagre to perm it a r e lia b le e v a lu a tio n . ever, good reasons to consider th is t e s t as s p e c ific .

There a re , how­ The r e la tiv e ly

few data a v a ila b le a t p re se n t (Labhardt: 113, 114; Werle and fff fkemann 31, 32; Z e lle r : 36) in d ic a te th a t by means of th is method pregnancy may be diagnosed beginning with the s ix th week.

The t e s t , as f a r as

can be seen from these re p o rts , seems to be of value in the e a rly diagnosis of any kind of a b o rtio n .

This i s due to the f a c t th a t the

-3 4 -

disappearance of diamine oxida.se a c t iv i t y from blood of pregnant women ap p aren tly depends upon the fu n c tio n a l s ta te of the p la c e n ta r a th e r than upon the anatom ical connection between placenta, and u te ru s . 1'Toteworthy a ls o are the data on diamine oxidase a c tiv ity in blood of women s u ffe rin g from h a b itu a l a b o rtio n s (3ffkemann and Werle: 29, 30).

Almost no adaptive in crease in diamine oxidase ac­

t i v i t y takes p lace i n these p a tie n ts from the very beginning.

There

i s no mention made in the l i t e r a t u r e as to the p o s s i b i l i t y of pre­ v enting a b o rtio n s on the ba-sis of these fin d in g s .

-

85 -

VI A WOKKIm I; ||

a) S e n s itiz a tio n and Antigen-Antibody Reaction

i j

HYPOTHESIS Off ALLSRG-Y

R e su lts

of extensive stu d ie s on sk in t e s t s performed on

c a v a l l e r i s t s , b a k ers, e t c , , in d ic a te th a t every person acqu ires a

! h y p e rs e n s itiv ity to the a lle rg e n s of h is surroundings i f he was ,j

!’ a c tiv e in the same p ro fe ssio n f o r a s u f f ic ie n tly long p e rio d of > 1 j! tim e; however, only 20% of these s e n s itiz e d in d iv id u a ls develop a l— jj

l e r g i c m a n ife sta tio n s ( Salen and Ju e h lin -D a n n fe lt; 115),

These oh-

,!

l! jl

se rv a tio n s were made in so many su b je cts th a t we f e e l r ig h t in con-

!

Ii

eluding th a t s e n s itiz a tio n re p re se n ts a normal p h y sio lo g ic al process

!

occurring i n every individ ual* As f a r as those su b je c ts are concerned in whom m an ifests-

f.

j;

tio n s of a l l e r g i c disease develop, we b e lie v e in the presence of an

I

in h e re n t d is p o s itio n to respond to c e r ta in i n s u l ts in an abnormal fashion*

I

I t does not seem to be merely in c id e n ta l th a t the same p e r-

centage (20%) i s a lso given f o r the incidence of serum sickness

l ( Schmidt: 116)* In g en eral, we b eliev e these observations do not 3 i' f in d the a p o re c ia tio n they obviously deserve. They le a d to the sup-

j;

p o s itio n th a t i n t a c t mucous membranes and p o ssib ly normal skin, as i: |i w e ll, are permeable under ordinary conditions fo r a lle rg e n s . The q u estio n whether the a lle rg e n s resorbed in th is p h y sio lo g ic al manner a re f u l l a n tig en s or only p a r t i a l a n tig en s i s not under discussion* Im portant only i s the f a c t th a t these substances, being e ith e r of a

p r o te in n atu re or n o t, do not lo se t h e i r a n tig e n ic ity during th e i r passage in to the blood or lymph stream; because otherw ise no form ation of a n tib o d ie s could take p la c e . Thus, we b e liev e th a t three fundamental th eses in the common concept of a lle r g y should d e f in ite ly be c o rre cte d : 1) S e n s itiz a tio n of in d iv id u a ls to a lle rg e n s of t h e i r surroundings does not rep re se n t a p a th o lo g ic a l, but r a th e r a normal p h y sio lo g ic al response* 2) P e rm ea b ility of mucous membranes f o r un digested pro­ te in s should no lo n g er be considered a c h a ra c te ris ­ t i c of an a l l e r g i c in d iv id u a l, but r a th e r a physio­ lo g ic a l fa c u lty of mucous membranes and, p o ssib ly , of sk in in a l l normal in d iv id u a ls . 3) Thus, the a b so rp tio n of d ie ta ry p ro te in s by the in­ t e s ti n e s — in c o n tra s t to the o ld er concept — does not take p lace ex clu siv ely follow ing the breakdown of these p ro te in s in to u n sp e c ific amino a c id s , but a ls o before such breakdown occurs. I t i s a p e c u lia r f a c t th a t the l a s t two theses were c o rre cte d by immunologists q u ite some time ago; however, no a tte n tio n was paid to these fin d in g s , dem onstrating the p erm eability of i n t a c t normal mu­ cous membranes f o r und igested fo re ig n p ro te in s in human su b jects and anim als (R atn er: 117; Walzer: 118).

In t h is connection, we may also

mention stu d ie s showing a p o ssib le percutaneous a b so rp tio n of in s u lin

-3 7 -

(He rmann and Kassowitz: 119). Assuming, th e re fo re , th a t such p h y sio lo g ic al ab so rp tio n of a lle rg e n s not deprived of t h e i r species s p e c if ic ity a c tu a lly takes p la c e , we n e c e s s a rily must conclude th a t an tig en -an tib o d y rea c tio n s occur c o n s t a n t ^ in eveiy individu al*

Thus, the an tigen-antibody

r e a c tio n lik ew ise appears to be a normal p h y sio lo g ical process, con­ st©,ntly tak in g p lace in the a l l e r g i c and in the normal in d iv id u a l as well*

Prom these c o n sid e ra tio n s the follow ing question r e s u lts :

Why does the an tig en -an tib o d y re a c tio n cause symptoms only in the a l ­ l e r g i c and not i n the normal in d iv id u al?

I t i s obvious th a t the pro­

bable answer l i e s beyond concepts of c e l lu l a r pathology*

-8 8 -

b) Histamine and Diamine Oxidase To g ain in s ig h t in to t h is problem, we th in k i t b e s t to ima­ gine th a t the a l l e r g i c in d iv id u a l i s le s s r e s i s t a n t to a n tig e n -a n ti­ body re a c tio n s than i s the normal person.

Or, in o th er words:

the

a l l e r g i c in d iv id u a l i s , by f a r , more s e n s itiv e to the pathogenous products of the a n tig en -an tib o d y re a c tio n than i s the n o n -a lle rg ic in dividu al* We do not deem i t necessary to d iscuss in d e ta il the ques­ tio n whether or not histam ine i s a c tu a lly the substance responsible f o r the symptoms of a l l e r g i c shock.

In s p ite of s lig h t d ifferen c es

between the an ap h y lactic shock and the histam ine shock, we believe th a t histam ine re p re se n ts a substance f a c i l i t a t i n g stu d ie s on the com plicating mechanism of shock, a t lea,st in one d ire c tio n .

Those

s li g h t d iffe re n c e s could be caused by substances so f a r unknown and produced sim ultaneously w ith histamine* S trik in g ly l i t t l e a tte n tio n was p a id so f a r to the behavior of diamine oxidase in a l l e r g i c individuals*

In the l i t e r a t u r e we

f in d only vague d iscu ssio n s and remarks h in tin g a t the p o s s ib i l i ty th a t the diamine oxidase a c t i v i t y in a l l e r g i c in d iv id u a ls may d i f f e r from the one in normal persons (Horste r : 120; Tschoup and Tschopp: 105; Albus: 121, 122; Albus and Mttrbel: 123), We determined the diamine oxidase a c t iv i t y in blood of twentyfiv e a d u lt p a tie n ts who had a ty p ic a l h is to r y of a l l e r g i c bronchial

asthma f o r a t l e a s t e ig h t and a h a lf y e a rs.

A ll these p a tie n ts were

under s p e c if ic treatm ent and fre e from acute exacerbations when the blood samples were taken.

In twenty-one in sta n c e s the diamine oxi­

dase a c t i v i t y of the blood was d e fin ite ly le s s than normal.

The f o l ­

lowing curves re p re se n t the h ig h e s t enzyme a c t iv i t y found among these p a tie n ts as compared w ith the average a c t i v i t y of our normal su b je c ts. The d iffe re n c e i s obvious.

X



V-

A / o