Studies on Thiamine Antagonists

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FORDHAM UNIVERSITY GRADUATE SCHOOL

May 15

This dissertation prepared under my direction by

A lb e r t J* E u se b i

entitled

S T 1 ID IE S 0 N ..T H M M

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

has been accepted in partial fulfilment of the requirements for the

Degree of

p o e t o r o f Ph i l o sophy

Dr* L eop old H* C ereced o (Faculty A dviser)

19

STUDIES OH THIAMINE ANTAGONISTS

ByA lb ert J* Eusebi B.S.

Fordham U n iv e rsity

19^2

M.S.

Fordham U n iv e rsity

19^7

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF CHEMISTRY AT FORDHAM UNIVERSITY

NEW YORK

ProQuest Number: 10992950

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992950 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

iii

Acknowledgment s. I wish to ex p ress my g r a titu d e to Bey, Eugene G isel, S. J . and to Dr. Leopold 1 . Cerecedo f o r generous m a te ria l a id . G ra titu d e i s a ls o extended to th e Department of Organic Chemistry and to my co-w orkers in th e B iochem istry Department fo r t h e i r many fa v o rs , I s in c e re ly a p p re c ia te th e a s s is ta n c e o f my s is te r - in - la w , Mrs. McShane, who d id th e h u lk of th e ty p in g .

F in a lly , I am fo re v e r

in d eb ted to my w ife whose co o p eratio n and u n d erstan d in g made t h i s work p o s s ib le . This work was c a r r ie d out under th e d ir e c tio n of Dr. Leopold B. Cerecedo.

L

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iv r

i Table o f C ontents

Page

Acknowledgments

iii

L is t of Tables

y il

L is t o f F ig u res

x

L is t o f A b b rev iatio n s

xi

In tro d u c tio n

1

P a rt I

S y n th esis o f Compounds A llie d to Thiamine and Oxythiamine

Chapter I

3

S tu d ies on th e Deamination o f Cocarboxylase in a B uffered and in an Aqueous Medium

II

The S y n th esis of Qxythiamine-Diphosphate and Oxythiamine-Monophosphate

III

3

9

P h y sica l and Chemical P r o p e rtie s of Q xythiam ine-Diphosphate

19

IV A Hew S y n th esis o f the T hiazole Alcohol o f Thiamine.

P re p a ra tio n o f a Hew P y rim id in e-

T hiazole D e riv a tiv e

2?

P a rt I I B io lo g ic a l S tu d ie s With Thiamine A ntagonists Chapter V

F a ilu re of X an th o p terin and F o lic Acid to Replace Thiamine in R ats and

VI

31

Mice

31

The E ffe c t of Oxythiamine, OxythiamineMonophosphate, Qxythiam ine-D iphosphate, and N eopyrithiam ine in Mice

VII

36

S p e c if ic ity o f S tru c tu re f o r A ntithiam ine A c tiv ity

kfy

C ontin u ation o f Table of Contents Summary of B io lo g ic a l Work w ith Thiamine A ntagonists P a rt I I I

M icro b io lo g ical S tu d ie s With Thiamine A ntagonists

Chapter V III

E ffe c t of Oxythiamine, Oxythiamine D iphosphate, H eopyrithiam ine and U ra c il-T h ia z o le D eriv ativ e on the Growth o f S taph, aureus

IX

E ffe c t o f Thiamine A ntagonists on th e Growth o f L. fermentum

P a r t IV

The A ction o f Thiamine A ntagonists on C ertain Enzyme Systems R equiring Thiamine or Cocarboxylase

Chapter X Urea S yn th esis in Rat and Mouse L iver S lic e s , The E ffe c t of Thiamine and i t s I n h ib itio n by Oxythiamine XI

S tu d ies on th e E ffe c t o f Oxythiamine and H eopyrithiam ine on th e Glam and Carp Thiaminase

X II

A Study o f th e Carboxylase Enzyme of Yeast

P a rt V P ho sp h o ry latio n S tu d ie s With Thiamine and th e Role o f Oxythiamine and H eopyrithiam ine Chapter X III

^nzym atic P h o sp h o ry latio n o f Oxythiamine by B akerfs and Brewer1s Yeast

XIV

A ction o f Oxythiamine and H eopyrithiam ine on th e Enzymatic S y n th esis o f Cocarboxylase

Vi r C o n tin u atio n o f Table of C ontents

Page

G eneral Summary

125

B ib lio g rap h y

128

1_

v ii l_

n

L is t of Tables Table

Page

I-D eam ination o f Cocarboxylase in A cetate B u ffer by l i t r o u s Acid Gases a t pH

4 .1

5

II-R a te o f Cocarboxylase Deamination in A cetate B uffer by U itro u s Acid Gases

7

III-C © carboxylase A c tiv ity of th e Condensation Product o f T h iazo le Pyrophosphate and 2-Methyl-4-Amino-5“ Bromo. Methyl Pyrim idine

23

IV-Conrposition o f th e Thiamine D e fic ie n t B asal D iet ( d ie t C-28-A)

32

V - lff e c t o f A d im in istra tio n o f F o lic Acid in Mice on a Thiamine D e fic ie n t D iet

33

V l-S ffe c t o f F o lic Acid and X anthopterin in Thiamine D e fic ie n t R ats

34

V ll-C om parative A ntithiam ine E ffe c t o f Oxythiamine, Q xythiam ine-D iphosphate, and N eopyrithi amine in Mice

40

V lll-C om parative E f f e c t o f Oxythiamine and N eopyrithiam ine in dba and G3H Mice

42

IX-Comparative E ffe c t o f Thiamine A ntagonists a t a Level o f 1/25 X -E ffect o f H eopyrithiam ine in X I-E ffe ct o f Bromoxythiamine in

44 Mice a t Low Levels

4?

Swiss and Rockland Mice

X II-E ffe c t o f Oxythiamine on th e Growth o f Staph, aureus

62

X III-E ffe e t o f Q xythiam ine-D iphosphate, H eopyrithiam ine and a U ra c il-T h ia z o le D e riv a tiv e on th e Growth of S tap h .au reu s L

63 _j

Continuation of L ist of Tables Table X lV -Sffect of Oxythiamine on th e Growth of L aetob a c illu s fermentum XV-Effect o f Qxythiam ine-D iphosphate on th e U t i l i ­ z a tio n o f Thiamine and Goearboxylase by Lacto­ b a c illu s fermentum XYI-Xffect o f Neopyri thiam ine on the U tiliz a tio n of Thiamine and Cocarboxylase by L a c to b a c illu s fermentum XYII-Urea S y n th esis in Thiamine D e fic ie n t Rat L iver S lic e s and I t s I n h ib itio n by Oxythiamine X V III-Urea S y n th esis in Thiamine D e fic ie n t and Normal Rat L iv er S lic e s XlX-Urea S y n th e sis in Thiamine D e fic ie n t and Normal Mouse L iv er S lic e s XX-Effect o f Oxythiamine and N eopyrithiam ine on th e Thiamine D estroying F a c to r o f Clam XXI-JSffeet o f Oxythiamine on th e Thiaminase of Carp X X I-I-Inhibition o f th e Carboxylase Enzyme System by Oxyt hiam i ne-D ipho sphat e X X III-E ffeet o f Thiamine and N eopyrithiam ine on th e P u r if ie d Carboxylase Enzyme o f Brewer*s Yeast XXIY-Failure o f Qxythiam ine-D iphosphate to D isplace Goearboxylase from th e Apocarboxylase

ix r

i C o n tin u atio n o f L is t o f f a b le s

Table X XV-lffeet o f Oxythiam ine-Biphosphate on th e P u r if ie d Carboxylase o f Brewer*s Yeast

108

XXVI-Oxythiamine I n h ib itio n of Cocarboxylase S y n th esis by Pro-pionibacterium pentosaceum

119

X X V II-In h ib itio n of Cocarboxylase S y n th esis in Brew er’s Yeast by Oxythiamine

122

X X V III-Failure o f N eopyrithiam ine to I n h ib it S ynthesis o f Cocarboxylase by Brew er’s Yeast

L

123

L ist o f Figures Table

1- •U ltra-V io let A bsorption S p ectra o f Oxythiamine and Qxythiam ine-Diphosphate 2- •Comparative S tru c tu re s o f Oxythiamine, Bromoxythiamine and a U rac il-T h ia z o le D e riv a tiv e

xi r

~i L is t of A bbreviations For convenience, p a r t ic u la r l y in a number of th e T ables, c e r ta in

a b b re v ia tio n s have been adopted in t h i s work. CoB^ f o r Cocarboxylase Cys. fo r C ysteine Met. f o r M ethionine f o r H eopyrithiam ine OB^ fo r Oxythiamine OBiDiPOh f o r Oxyt h i am:ine-B ipho sphat e B^ f o r Thiamine B^I f o r Thiamine Iodide T.S. f o r T issue S lic e .

L

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-I

In tro d u c tio n During re c e n t y ears numerous in v e s tig a tio n s have been c a r r ie d out

which show th a t compounds e x is t in n a tu re or can be sy n th esized which a re c lo s e ly r e la te d s tr u c t u r a l ly to th e v itam in s, and which have th e c a p a c ity o f producing in a number of s p e c ie s the sig n s of a s p e c if ic av ita m in o sis o f th e n a tu r a l substance a l l i e d to them.

The o b serv atio n o f Q uastel and

Woolridge ( l ) on th e r e v e r s ib le in h ib itio n by m alonate of th e enzym atic o x id a tio n o f su c c in a te p robably marked th e b i r t h o f the concept o f co m p etitiv e in h ib itio n between s tr u c tu r a lly r e la te d substances* However, i t was not u n t i l th e work of Woods, (2 ) because o f i t s r e la tio n s h ip to p r a c t i c a l th e ra p y , th a t a s u f f ic ie n t im petus was given to th e study of b io lo g ic a l antagonism s.

Woods showed th a t the growth

in h ib itin g or b a c t e r i o s t a t i c a c tio n o f su lfa n ila m id e in c e r ta in b a c te r ia was re v e rse d by sm all amounts o f p~aminobenzoic a c id , a s tr u c tu r a l analogue o f th e sulfonamide* Follow ing th e se o b se rv a tio n s, a number o f v itam in s and c e r ta in o th e r m e ta b o lite s have had t h e i r chem ical s tr u c tu r e s changed to a g r e a te r or l e s s e r degree, and many o f th e se d e r iv a tiv e s have been found to behave co m p e titiv e ly w ith th e r e la te d m etabolite*

Thus, s p e c if ic

a n tim e ta b o lite s have brought about b a c t e r i o s t a s i s , th e appearance of s p e c if ic d e fic ie n c y sig n s in anim als, th e m a n ife sta tio n of c e r ta in new p harm acological e f f e c t s , and th e in h ib itio n o f d e f in ite enzymic re actio n s* The very e x a c tin g requirem ent in s tr u c tu r e f o r a c t i v i t y on th e p a r t of th e m e ta b o lite , o r i t s i n h ib itio n by the s t r u c t u r a l analogue has serv ed to throw new and prom ising l i g h t on th e mechanism o f a c tio n of c e r ta in enzyme systems*

This re p re s e n ts one asp ect of th e use o f

a n tim e ta b o lite s th a t has not been w idely explored in th e case o f th e 5-

^1

2

a n tith ia m in e s .

Some re s e a rc h alo n g th e se l in e s has been done h u t most

o f th e work has concerned i t s e l f w ith in vivo s tu d ie s . The work to he d isc u ssed d e a ls w ith the p re p a ra tio n o f new s p e c if ic a n tith ia m in e s , w ith th e a c t i v i t y o f th e se a n ta g o n is ts in mice and in m icroorganism s, and w ith t h e i r a c tio n on c e r ta in enzyme systems re ­ q u irin g thiam ine o r cocarboxylase f o r a c t i v i t y .

I t was only through

th e a p p lic a tio n of a number o f d e r iv a tiv e s such as oxythiam ine, oxythiam ine-m onophosphate, oxythiam ine-diphosphate, and hromoxythiamine, th a t c e r ta in con clu sio n s could he drawn as to t h e i r a c tio n in v iv o , and u ltim a te ly , as to the n a tu re o f th e mechanism of oxythiam ine in h ib itio n .

L

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“i PART I THE SYITHESIS OP COMPOUNDS ALLIED TO THIAMINE AND OXYTHIAMINE Chapter I

S tu d ies on th e Deamination o f Cocarboxylase in a B uffered and in an Aqueous Medium

Prom a knowledge of th e manner in which thiam ine i s a c tiv e or f u n c tio n a l in viv o and in v i t r o , namely in th e form o f i t s diphosphate, i t was very p o s s ib le th a t in th e in h ib itio n o f thiam ine by oxythiam ine, th e diphosphate form o f oxythiam ine was in v o lv ed .

This concept re ­

ceiv ed support from th e work o f Soodak (3 ) w ith chloroxythiam ine.

This

compound when a d m in iste re d to mice a t high le v e ls was found not to be to x ic .

The replacem ent o f th e hydroxyl group of th e th ia z o le alco h o l

m oiety o f oxythiam ine ren d ered th e compound in a c tiv e .

This in d ic a te d

t h a t a p h o sp h o ry latio n mechanism might be involved. I t was w ith t h i s knowledge in mind th a t atte m p ts were made to sy n th e siz e oxythiam ine-diphosphate and oxythiamine-m onophosphate. The a v a i l a b i l i t y and a p p lic a tio n o f such oxythiam ine e s te r s , r e la te d c lo s e ly to th e thiam ine e s te r s , should add co n sid e ra b ly to our under­ sta n d in g o f th e mode o f oxythiam ine in h ib itio n . This s e c tio n d e a ls w ith th e attem p ts to sy n th e siz e oxythiam inediphosphate by th e deam ination o f cocarboxylase in a b u ffe re d and in an aqueous medium. The problem of s y n th e s iz in g oxythiam ine-diphosphate was approached from two d i f f e r e n t a n g le s.

The most d ir e c t approach and one which

o ffe re d prom ise, involved a deam ination of cocarboxylase to oxythiam inedip h o sp h ate.

T his re a c tio n which has proven very s u c c e ss fu l fo r the

p re p a ra tio n o f oxythiam ine in e x c e lle n t y ie ld from thiam ine has never Lbeen s u c c e s s fu lly a p p lie d to cocarboxylase.

The deam ination of

,

4

cocarboxylase "by n itro u s a c id gases has been stu d ie d by only two p re­ v io u s w orkers.

W eil-Malherbe (4 ) found th a t in an aqueous medium,

cocarboxylase was about 28% deam inated a f t e r 6 hours.

Soodak, (3 ) employ­

in g q u a n titie s of cocarboxylase as high as 250 mg. was ab le to deam inate com pletely th e coenzyme but o b tain ed a t th e same time v ary in g degrees o f d ep h o sp h o ry latio n .

In h is work, Soodak used an aqueous medium as was

used f o r th e p re p a ra tio n o f oxythiam ine from thiam ine*

No attem pt

was made to c o n tro l th e a c i d ity by means o f a b u f fe r and th ere b y p re­ vent d ep h o sp h o ry latio n .

He d id not fo llo w up t h i s method because of

th e a tte n d a n t e x te n siv e h y d ro ly sis o f th e phosphate which accompanied th e deam ination. The tech n iq u e of m ain tain in g a co n sta n t a c id ity by means o f a s u ita b le b u f fe r , w hile deam inating a compound w ith very l a b i l e phosphate groups, was s u c c e s s fu lly employed by K le in z e lle r (5)*

This

worker found th a t in th e case o f a d e n o sin e -trip h o sp h a te , a compound w ith v ery l a b i l e phosphate groups, complete deam ination could be e f­ f e c te d w ithout d ep h o sp h o ry latio n when th e r e a c tio n was c a r r ie d out in a b u ffe re d medium o f pH 4 .0 .

In t h i s manner K le in z e lle r o b tain ed

in o s in e -trip h o s p h a te in f a i r y ie ld . Experim ental In t h i s work, th e deam ination of cocarboxylase was follow ed by a ra p id thiochrom e p ro ced u re.

The e x te n t of dephosphorylation was

follow ed by th e d e te rm in a tio n of phosphoric a c id according to th e method o f F isk e and Subbarow ( 6 ).

P re lim in a ry experim ents were

c a r r ie d out in an aqueous medium w ith sm all q u a n titie s of cocarboxy­ la s e .

Two to fo u r mg. b atch es of cocarboxylase were d iss o lv e d in

5 ml. o f w ater, and tr e a te d w ith th e n itro u s a c id gases f o r 2 to 6

_1

5 r

i h o u rs.

Under th e se c o n d itio n s 50 to 100$ dephosphorylation took p la c e ,

depending upon th e tim e o f th e r e a c tio n .

Complete deam ination d id not

occur u n t i l th e tim e th a t i n i t i a l dephosphorylation was observed. When t h i s r e a c tio n was c a r r ie d out in an a c e ta te b u f fe r o f pH 4 .1 , of

i t was found th a t h y d ro ly s is of th e l a b i l e phosphate groupAcocarboxy­ la s e d id not o ccu r, and a t th e same tim e complete deam ination to oxy— th iam in e-d ip h o sp h ate could be accom plished.

P re lim in a ry experim ents

were se t up employing 2 to 20 mg. o f cocarboxylase, and th e r e s u lts of th e se experim ents are shown in Table I . Table I D eam ination o f Cocarboxylase in A cetate B u ffer by N itro u s Acid Gases a t pH 4 .1 Experiment Number

Q u antity o f Cocarboxylase mg.

Deamination Time Hours

$ Deamination

$ Dephosphoryl a t ion

1

2

5

100

0

2

4

6

100

0

3

10

24

100

0

4

10

24

100

0

5

10

24

100

0

6

20

24

100

0

In th e s ix t h experim ent i n which 20 mg. o f cocarboxylase was employed, an attem p t was made to i s o la te th e oxythiam ine-diphosphate. The problem of i s o l a t i o n from th e a c e ta te b u f fe r proved to be a very d i f f i c u l t one.

A number o f d if f e r e n t p r e c ip ita n ts were t r i e d in an

attem p t to p r e c i p i ta te th e oxythiam ine-diphosphate d i r e c t l y from the b u ffe re d medium. L

The fo llo w in g were used:

tu n g s tic , s il ic o tu n g s tic

6

r n and phosphotungst ic a c id s , a c r id in e , a ls o barium , s i l v e r , le a d , mercury, go ld and uranium .

The f i r s t group of p r e c i p ita n ts proved o f no a v a il,

f o r in no case did a p r e c i p i ta te form. w ith th e heavy m etal p r e c i p i ta n ts .

Somewhat b e tte r success was had

Imm ediately follow ing th e complete

deam ination o f th e cocarb o x ylase, a d ir e c t p r e c ip ita ti o n o f th e oxy­ th iam in e-d ip h o sp h ate could be o b tain ed by b rin g in g th e s o lu tio n to pH 5*6 s&d p r e c i p i ta ti n g w ith le a d and u ran y l a c e ta te .

Blank s o lu tio n s

s e t up sim u ltan eo u sly and c o n ta in in g e v ery th in g but cocarboxylase, d id not give a p r e c i p i ta te under id e n tic a l c o n d itio n s w ith a n e u tr a l le a d a c e ta te s o lu tio n .

I t was decided, th e r e fo r e , to work w ith a n e u tr a l

le a d a c e ta te s o lu tio n as th e method o f ch o ice. With t h i s i n mind, a la rg e s c a le experim ent was s e t up w ith 200 mg. o f cocarboxylase and a s e r ie s o f an aly ses were run a t v ary in g in te rv a ls # Such p e rio d ic d e term in a tio n s have value in dem onstrating th e r a t e of deam ination o f cocarboxylase under th e c o n d itio n s of our experim ent. The complete b u ffe re d s o lu tio n c o n s is te d of 200 mg. of cocarboxylase, 273 Eg* o f sodium a c e ta te , 1 ml. o f g la c i a l a c e tic a c id , and 6 ml. of 60$ sodium n i t r i t e s o lu tio n . o f 10 ml.

This was brought to a t o t a l f in a l volume

The pH of t h i s s o lu tio n was 4 .0 ,

The r e s u l t s of th e an aly ses

a t d if f e r e n t tim e in te r v a ls are shown in Table I I .

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7

Table I I Rate o f Deamination of Cocarboxylase in A cetate B u ffer by N itro u s Acid Gases o f D eam ination in hours

^Deamination

f> Dephosphoryl a t ion

2

28.6

0

5

*1.5

0

21

68.7

0

Zk

86.7

0

25 .5

86.9

0

28

100

0

A phosphate a n a ly s is a t th e end of th e 28 hour p e rio d showed th a t no in o rg a n ic phosphate was p r e s e n t.

The pH of th i s s o lu tio n was

brought to 5*3 w ith N NaOH, and t r e a te d w ith a n e u tr a l 10^ le a d a c e ta te s o lu tio n u n t i l p r e c i p i ta ti o n was com plete.

The p r e c i p i t a te was washed

tw ice w ith sm all q u a n titie s o f w ater and th en suspended in 3 ° ffll. o f w ater.

The le a d was removed by p a ssin g laydr o gen s u lf id e through th e

suspension u n t i l no f u r th e r le a d s u lf id e would form.

The p r e c ip ita te

o f le a d s u lf id e was c e n trifu g e d down and washed th re e tim es in th e c e n trifu g e w ith w a te r.

The combined s o lu tio n s were taken to dryness

in vacuo a t room tem p eratu re, and th e re sid u e taken up in 5 b1* o f 0.1N HOI. A sm all amount o f in s o lu b le m a te ria l was c e n trifu g e d down, and th e y e llo w ish s o lu tio n tr e a t e d w ith a sm all amount of ch arco al fo r d e c o lo n iz a tio n .

The c o lo r le s s f i l t r a t e was then tr e a te d w ith 80 ml. o f

a b s o lu te a lc o h o l, and allow ed to stan d in th e ic e chamber f o r 2* hours. A sm all amount o f c o lo r le s s p r e c i p i ta te had formed which was c e n trifu g e d down, washed w ith e th e r , and d rie d in vacuo.

The f i n a l

8 r pro d u ct amounted to only 3 Big* and was very hygroscopic*

A phosphorus

i

a n a ly s is showed th a t th e compound contained phosphorus.

This m a te ria l

decomposed over a range o f 200-2^0°C. On th e b a s is o f t h i s experim ent, i t i s v ery probable th a t cocarboxy­ la s e could be s u c c e s s fu lly deam inated to oxythiam ine-diphosphate, a l ­ though, in t h i s work a pure p re p a ra tio n could not be is o la te d . I t i s , of co u rse, p o s s ib le th a t in such a r e a c tio n th e cocarboxylase molecule was s p l i t in to i t s p y rim id ine and th ia z o le fragm ents.

This p o s s i b i l i t y

appears u n lik e ly on th e b a s is of th e analogous r e a c tio n w ith thiam ine, in which oxythiam ine can be o b tain ed in e x c e lle n t y ie ld . The lim ite d a v a i l a b i l i t y of cocarboxylase and i t s d i f f i c u l t and expensive p re p a ra tio n , re p re s e n te d serio u s, o b je c tio n s to t h i s method o f p re p a ra tio n .

L

9 r

i

Chapter I I

The S y n th esis o f Qxythiam ine-Diphosphate and OxythiamineMonophosphat e

The second approach which was open fo r th e s y n th e s is o f oxythiam ined iphosphate in v o lv ed th e p h o sp h o ry latio n o f oxythiam ine to th e d i­ phosphate, in a manner, s im ila r to th a t used in th e s y n th e s is o f cocarb o x y lase.

This procedure has been s u c c e s s fu lly employed hy

W eijlard and Tauber (7 ) and by W eijlard ( 8 ,9 ) f o r th e s y n th e s is of cocarboxylase in about 10$ y ie ld . The i n i t i a l experim ents were C arried out by a procedure s im ila r to t h a t used by W eijlard and Tauber ( 7 ).

In t h i s s y n th e s is , phosphory­

l a t i o n i s brought about by th e use of a m ixture o f sodium pyrophosphate and pyrophosphoric a c id , and th e product is o la te d from th e re a c tio n m ixture by p r e c i p i ta ti o n w ith phosphotungstic a c id .

The phospho-

tu n g s ta te was th en decomposed w ith la rg e volumes of aceto n e, and th e cocarboxylase i s o l a t e d by f r a c tio n a l p r e c ip i ta tio n w ith aceto n e. Employing th e procedure o f W eijlard and Tauber f o r th e p re p a ra tio n of oxythiam ine-diphosphate gave l i t t l e su ccess.

Only on th re e occasions

could a f in a l product be is o la te d and an a n a ly s is on each of th e se showed a tendency f o r th e phosphorus and n itro g e n to run low and fo r th e carbon and hydrogen to be h ig h .

Repeated d i f f i c u l t y was u s u a lly

encountered in th e decom position o f th e p hosphotungstate w ith aceto n e. The ex p erien ce from t h i s work s tro n g ly in d ic a te d th a t th e procedure employed f o r th e s y n th e s is o f cocarboxylase could not be a p p lie d to th e sy n th e sis o f o x y th iam ine-diphosphate.

In p a r t ic u l a r , th e

tech n iq u e fo r th e f r a c t i o n a l p r e c ip ita tio n o f th e cocarboxylase ap­ p a r e n tly could not be a p p lie d to th e p u r if ic a tio n o f oxythiam ineL

_!

10

r

i diphosphate* The problem re so lv e d i t s e l f , th e r e fo r e , to one of determ ining "byt r i a l th e b e st r a t i o so lv en t to p r e c ip ita n t to use f o r i s o la t in g a pure p ro d u ct.

This phase o f th e work was c a r rie d out employing a method

s im ila r to th e new sy n th e s is of cocarboxylase re p o rte d by K arrer and co-w orkers (1 0 ).

K arrer has found th a t cocarboxyla.se can be syn­

th e s iz e d and is o la te d as a phosphate s a l t in b e t t e r th an 50 ^ y ield* His e a r l i e r work on t h i s s y n th e s is was confirm ed and extended in a more re c e n t p u b lic a tio n ( l l ) .

In t h i s more re c e n t work, th e e a r l i e r

procedure was m odified by K arrer so th a t a p u re r cocarboxylase prep­ a r a tio n was o b ta in e d .

The p h o sp h o ry latio n o f th e thiam ine was c a r rie d

out a 100-105°C r a th e r than a t 135“ 1^0°C. which gave a p u re r product t h a t was as a c tiv e as cocarboxylase sy n th e siz e d by th e method of W eijlard and Tauber.

T h iam in e-trip h o sp h o ric a c id was an i n i t i a l

s te p in t h i s s y n th e s is , which in th e presence of phosphoric or hydro­ c h lo r ic a c id r a p id ly underwent h y d ro ly s is to cocarboxylase.

I t was

expected th a t oxythiam ine might be p h o sphorylated in the same manner and th a t th e f r a c t i o n a l p r e c ip i ta ti o n f o r the diphosphate might be worked o u t.

L

11 r

i Experim ental The p re p a ra tio n o f th e diphosphate was brought about in th e fo llo w in g manner! Five grams of 85$ phosphoric a c id was h eated in a sm all beaker to 290-310°C. o r u n t i l c r y s t a ls appeared on i t s w a lls .

The a c id was

allow ed to cool and th en p la c e d in an o i l b ath p re v io u sly h eated to 135-1^0°C.

A fte r tem p eratu re e q u ilib r a tio n , ^ g. of oxythiam ine was

added a l l a t once and kept a t t h i s tem perature fo r 15 m inutes w ith co n sta n t s t i r r i n g .

The fu sed mass was allow ed to cool somewhat and

th en washed tw ice w ith 5° ml* p o rtio n s of acetone b efo re i t s o li d i f i e d . The s o lid mass was d iss o lv e d in 50 ml. of w ater and k drops of 85$ phosphoric a c id added.

The dark brown s o lu tio n was th en f i l t e r e d and

150 ml. o f acetone added.

A fte r stan d in g o v ernight in th e ic e box, a

d ark brown o i l had se p a ra te d .

The o i l was tak en up in 50 ml. of

0.1M phosphoric a c id and d e c o lo riz e d w ith a sm all amount o f l o r i t A and f i l t e r e d .

To th e f i l t r a t e 150 ml. of acetone was added, and a

clo u d in e ss formed im m ediately. in th e ic e c h e s t,

This was allow ed to stan d overnight

The yellow s e m i- c r y s ta llin e m a te r ia l was taken up

in 15 ml. o f phosphoric a c id and d e c o lo riz e d a second tim e w ith N orit A and p r e c ip ita te d w ith 80 ml. o f aceto n e.

T his f r a c tio n a tio n was

re p e a te d a second tim e and th e c o lo r le s s product th e n c r y s ta l liz e d by tre atm e n t w ith 50 ml. o f an equal m ixture of a b so lu te a lc o h o l and aceto n e.

On sta n d in g in th e fre e z in g chamber w ith p e rio d ic s c ra tc h in g ,

t h i s m a te ria l became c r y s t a l l i n e .

I t was f i l t e r e d , washed w ith a b so lu te

a lc o h o l, aceto n e, and e th e r , and d r ie d in vacuum.

In o rd e r to follow

th e e x te n t of p u r if i c a t i o n , phosphorus an aly ses were run a t each stag e Lpf

th e i s o l a t i o n .

The c r y s t a ll in e product is o la te d a t t h i s s ta g e was

j

12 r

i c o n sid e ra b ly low in i t s phosphorus c o n te n t. C a lc u la te d f o r ^3.2 ^ 20^1 Free P, 5.93? Pound:

H ydrolyzable P, 11.87;

Free P, 2 .0 ;

T otal P, 17.80$

Hydrolyzable P, 9*2;

T o tal P ,l6 .1 $

The f r a c tio n a tio n was c a r r ie d f u r th e r by s o lu tio n in 10 ml. of 0 . 1M phosphoric a c id and tre atm e n t w ith $0 ml. o f aceto n e.

A fte r

sta n d in g in th e ic e ch est o v e rn ig h t, the s tic k y o i l was c r y s ta lli z e d w ith a b so lu te a lc o h o l and aceto n e, washed and d rie d as b e fo re .

A

phosphorus a n a ly s is a t t h i s p o in t gave th e fo llo w in g figure® ! Free P, 4 .0 ; H ydrolyzable P, 1 0 .0 ;

T o tal P, 1 6,5%

I t was apparent th a t only a p a r t i a l p u r if ic a ti o n of oxythiam inediphosphate was accom plished under th e se c o n d itio n s.

To o b ta in a

p u re r p ro d u ct, w ithout re g a rd to th e f in a l y ie ld , i t was decided to use a la r g e r volume o f so lv en t and to determ ine th e minimum amount of acetone needed to b rin g about an i n i t i a l c lo u d in e ss.

The e n tir e b atch

from th e p rev io u s sta g e was d iss o lv e d in 75 ml. of phosphoric a c id and acetone added g ra d u a lly . 150 ml. o f acetone was added.

An i n i t i a l dense clo u d in ess formed when This was allow ed to stand in the ic e

chamber f o r th re e days, and th e acetone su p ern atan t d isc a rd e d .

The

s tic k y g la s s y loo k in g m a te ria l was c r y s t a l l i z e d w ith $0 ml. of a b so lu te a lc o h o l w ith p e rio d ic s c ra tc h in g and stan d in g in th e fre e z in g chamber.

This product s ta r t e d to brown a t 180°C and decomposed a t

240~250°C.

The y ie ld amounted to only 500 mg. or approxim ately 10%,

A s a ti s f a c t o r y a n a ly s is fo r phosphorus on t h i s compound was substan­ t i a t e d by an a n a ly s is fo r carbon, hydrogen, and n itro g e n . Anal. C alcd. f o r iJST, 8 .0 3 ; Free P, 5 .9 3 ; Byd. P, 11 .8 7 ; T otal P, 17.80

3*82;

13 r

-i Found:

C, 2 7 ,2 ; H, 4 .0 4 ; H. 8*22; Free P, 5*56»

3yd. P, 10,68;

T o tal P, 1 7 .8 1 .

This s y n th e s is was re p e a te d on a second occasion ta k in g advantage of p a s t e x p erien c e,

^he procedure was m odified somewhat in an attem pt

to o b ta in b e t t e r y ie ld s and a p u re r p ro d u ct. Four grams o f oxythiam ine was fused w ith pyrophosphoric a c id as d e sc rib e d p re v io u sly ,

The p h o sp h o ry latio n was c a r rie d out a t 145°C

f o r 20 m inutes w ith c o n sta n t s t i r r i n g , allow ed to cool and washed tw ice w ith 50 ml* p o rtio n s o f a c eto n e.

The brown s o lid mass was d isso lv e d in

50 ml, o f w ater, 4 drops o f co n cen trated phosphoric a c id added, follow ­

ed by 150 ml. of aceto n e.

A d ark brown th ic k o i l se p a ra te d overnight

and was tak en up in 50 ml. o f w ater, d e c o lo riz e d w ith N o rit A, and p r e c ip ita te d w ith 150 ml, o f aceto n e. o v e rn ig h t.

This was kept in th e ic e -c h e s t

The l i g h t yellow s o lu tio n was d e c o lo riz e d a second time

and p r e c ip ita te d w ith 125 ml. of aceto n e.

The m ilky acetone super­

n a ta n t was d ecanted, and th e s tic k y p r e c i p i ta te d isso lv e d in 20 ml. of 0.1M phosphoric a c id and p r e c ip ita te d w ith 100 ml. o f aceto n e, and p lac ed in th e f re e z in g chamber over the weekend.

The p r e c ip ita te d

m a te ria l was tak en up in 50 ml. of 0.1M phosphoric a c id and p re­ c i p i t a t e d w ith 150 ml. of a c e to n e .

The tu r b id su p ern atan t was de­

can ted , and th e re s id u e d iss o lv e d in 10 ml. o f ’phosphoric a c id and p r e c ip ita te d w ith a m ixture o f 15 ml. of a b so lu te a lc o h o l and 35 ml. o f aceto n e.

The s e m i- c r y s ta llin e m a te ria l was tr e a te d w ith 50 ml. of

an-, equal m ixture o f a b so lu te a lco h o l and acetone to a id c r y s t a l l i z a ­ tio n .

The c r y s t a l l i n e m a te ria l was f i l t e r e d , washed as b e fo re , and

d rie d f o r s e v e ra l days in a good vacuum over s u lf u r ic a c id . L

This j

14 r product s ta r t e d to "brown a t 180°C. and decomposed a t 230-240°C.

A

phosphorus a n a ly s is on t h i s .m aterial gave a t o t a l P value o f 17* 0$. Another f r a c tio n a tio n was th e re fo re c a r rie d out by s o lu tio n in 75 ml. of phosphoric a c id ,and p r e c ip ita ti o n w ith 150 ml. of aceto n e. cloudy su p ern ata n t was decanted

and th e s e m i-c ry s ta llin e

up in 25 ml. o f phosphoric a c id and p r e c ip ita te d

product taken

w ith 50 ml. of aceto n e.

The f i n a l product was c r y s t a l liz e d w ith a b so lu te a lc o h o l. y ie ld was 1 .9 g. or 30 % o f th e o ry .

The

The f i n a l

The oxythiam ine-diphosphate s ta r t e d

to ta n a t 180°C and decomposed a t 245-255°0.

An a n a ly s is o f t h i s pro­

duct e s ta b lis h e d i t as e s s e n t i a l l y pure oxythiam ine-diphosphate. Anal. Calcd. f o r Hyd. P, 1 1 .87; Hyd. P, 11.64

8 . 03 ; Free P, 5*93;

T o tal P, 17.80; Found N, 7 .7 7 ;

Free P, 5 .6 0 ;

T o tal P, 17.55*

The a p p l i c a b i l i t y o f t h i s method was shown on a t h i r d occasion. The procedure was changed s l i g h t l y b u t gave about th e same y ie ld and a complete a n a ly s is t e s t i f i e d to th e p u r ity of th e compound.

The f in a l

procedure which i s recommended f o r th e p re p a ra tio n of t h i s new a n t i ­ thiam ine i s as fo llo w s; The p h o sp h o ry latio n i s c a r r ie d out a t 135~T40°C f o r 15 m inutes, cooled and washed as d e sc rib ed p re v io u sly .

The s o lid mass i s d isso lv e d

in 50 ml. o f w ater p lu s 4 drops o f co n c e n tra te d phosphoric a c id and p r e c ip ita te d w ith 150 ml. o f aceto n e.

A fter stan d in g o v e rn ig h t, th e

d a rk brown o i l i s d isso lv e d in 15 ml. of w ater p lu s 4 drops of con­ c e n tra te d phosphoric a c id , and d eco lo riz e d w ith U orit A, f i l t e r e d , washed w ith w a te r, and brought to a t o t a l volume of 75 ml.

This i s

th en tr e a te d w ith 150 ml. o f acetone to give an i n i t i a l dense c lo u d i­ n ess.

This i s th en p la c e d in th e ic e chest f o r s ix hours, a f t e r which

15 r

i th e tu r b id su p ern atan t i s d ecanted and d isc a rd e d .

The y ello w ish o i l

i s taken up in 15 ml. o f phosphoric a c id and d e c o lo riz e d a second time w ith a sm all amount o f H orit A, f i l t e r e d , washed and "brought to a t o t a l volume o f 25 ml.

P r e c ip ita tio n i s c a r rie d out w ith 125 ml. of aceto n e.

This i s p lac ed i n the ic e -c h e s t o v e rn ig h t.

The c o lo r le s s o i l i s d is ­

so lv ed in 20 ml. o f phosphoric a c id and p r e c ip ita te d w ith 100 ml. of aceto n e.

A f u r th e r f r a c tio n a tio n i s then c a r rie d out hy s o lu tio n in

25 ml. o f a c id and p r e c i p i ta ti o n w ith $0 ml. of aceto n e.

The g la ssy

loo k in g m a te ria l i s f i n a l l y tak en up in 10 ml. of phosphoric a c id and p r e c i p i ta te d w ith an equal m ixture of a b so lu te a lco h o l and acetone. C r y s ta lliz a tio n i s accom plished by t r e a t in g th e s tic k y m a te ria l w ith 50 ml. o f a b s o lu te a lc o h o l and le a v in g in the fre e z in g chamber.

A fte r two days, ^with o c c a sio n a l sc ra tc h in g , th e diphosphate becomes c r y s t a l l i n e and can be f i l t e r e d .

This i s washed and d rie d as b efo re

and th e y ie ld amounted to 1 .7 g* o r 27.5$ o f th e o ry .

The oxythiam ine-

diphosphate s t a r t e d to brown a t 185-190°C and decomposed a t 245~255°0* Anal. Pound:

C, 27*83; H, *f.06; 3J, 7*76; Free P, 5*65;

Hyd. P, 11.62; T o tal P, 17*70. F u rth e r a ttem p ts have sin c e been c a r r ie d out but in no case out o f elev en runs could a b e t t e r than 30$ y ie ld be o b tain ed .

On th e

b a s is o f th e re c e n t paper by K arrer (1 1 ), i t i s v ery probable th a t an improved s y n th e s is w ith h ig h e r y ie ld s could be o b tain ed by c a rry in g out th e p h o sp h o ry latio n a t lower te m p eratu res. B)

S y n th esis o f Gxythiamine-Monophosphate.

To com plete th e analogy between th e oxythiam ine and thiam ine s e r ie s o f compounds, th e p re p a ra tio n of oxythiamine-monophosphate was L

J

16 r u n d ertak en .

As in th e case o f th e diphosphate, two approaches were

open f o r th e s y n th e s is of t h i s d e r iv a tiv e .

I t has "been w ell e s ta b lis h e d

by o th e r workers and a ls o in t h i s work th a t cocarboxylase on treatm ent w ith n itro u s a c id g ases in aqueous medium undergoes dephosphory­ l a t i o n q u ite r a p id ly .

The second phosphate group o f cocarboxylase i s

extrem ely more d i f f i c u l t to remove.

This f a c t a ffo rd e d one p o s sib le

approach to th e s y n th e s is o f oxythiam ine-monophosphate.

A second

pathway was based on th e s im i l a r i t y of behavior o f cocarboxylase and oxythiam ine-diphosphate tow ards H HC1 a t 100°C.

This procedure has

been employed by numerous w orkers fo r th e p re p a ra tio n of th iam in emonophosphate, and was adopted in th i s work f o r th e p re p a ra tio n of oxythiam ine-m onophosphate. Oxythiamine-monophosphate was sy n th esized by both methods.

The

deam ination and dep h o sp h o rylation o f co.carboxylase was c a r rie d out in an aqueous medium much in th e same manner as in th e p re p a ra tio n of oxythiam ine from th iam in e.

To o b ta in th e monophosphate from cocarbo­

x y la se , th e l a t t e r compound had to be sy n th e siz e d .

The method used

was th a t o f E a rre r in which he o b tain ed th e phosphate s a l t in about 551° y ie ld .

Follow ing E a rre r* s procedure, a $0f> y ie ld o f cocarboxy­

la s e phosphate s a l t was o b ta in e d .

This product was not analyzed fo r

i t was o f no s ig n ific a n c e w hether th e product contained a sm all amount o f thiam ine-m onophosphate sin c e i t was to be used in th e p re p a ra tio n o f oxythiam ine-m onophosphate. Four grams o f cocarboxylase phosphate s a l t was d iss o lv e d in 50 ml. of w ater and n itro u s a c id gases were p assed in fo r k h o u rs.

This was

allow ed to stan d o v ern ig h t and th e p ro cess m s re p e a te d on a second .and Lt h i r d day.

i

A n e g a tiv e thiochrom e t e s t was obtain ed on th e t h i r d day*

The excess acid was removed "by p assing the so lu tio n through a column of the r e sin IR-4B which was kept cold .

This technique fo r the re­

moval o f excess acid in the preparation of oxythiamine from thiamine was employed w ith succes hy Mr. G-. Bonvicino of the Organic Chemistry Department.

Following the r e s in treatm ent, the elu ate was a c id ifie d

w ith F HC1 and concentrated to an o i l in a vacuum at room temperature. The o i l wa.s taken up in alcoh ol and p recip ita ted w ith an excess of eth er and l e f t in the ic e box overnight.

An o i l separated, and t h is

was d isso lv ed in 150 ml. of ab solu te a lco h o l, and the so lu tio n sa t­ urated w ith dry HG1 gas and l e f t in the ic e chamber.

Ho p r e c ip ita te

formed at t h is point so the a lc o h o lic so lu tio n was p recip ita ted with an excess of ether and a fte r 5 hours in the ic e box, the s lig h t ly turbid supernatant was decanted, and the p r e c ip ita te d iss o lv e d .in 10 ml. o f 0.1H HC1, d ecolorized w ith Norit A, washed and d ilu ted to 20 ml. w ith the washings.

This was then p recip ita ted w ith 175

acetone and l e f t

in the ice-b o x .

The p r e c ip ita te was taken up in

ab solu te alcoh ol

and HC1 gas passed in to saturation at ic e bath

temperature.

This was then p recip ita te d with ether to g iv e a dense

clo u d in ess.

A fter standing overnight, the p r e c ip ita te which s e tt le d

liras treated w ith 20 ml. o f ab solu te alcohol to aid c r y s ta lliz a t io n . The y ie ld obtained was 1 .1 g.

The oxythiamine-monophosphate was very

hydroscopic and melted at 185-190°C. Anal. Calcd. fo r C^gH^gO^H^ClgS. Free P, 0;

Hyd.

H, 4.49; Free P,

P, 0;

T o tal P, 7 .4 1 .

C, 3 4 .4 5 , H, 4*315 Found C, 35.05;

0; Hyd. P, 0: Total P, 7.18

The a n a ly sis indicated that the product is o la te d probably con­ t a in e d a very small amount o f free oxythiamine.

However, i t was su f-

18 r

f i c i e n t l y pur& f o r use in th e animal and enzyme s tu d ie s to he re p o rte d

n

la te r. The p re p a ra tio n o f oxythiamine-monophosphate was a ls o accom plished hy h y d ro ly sis o f oxythiam ine-diphosphate w ith d ilu te a c id . y s is was c a r rie d out w ith IT HC1 a t 100°C#

The hydrol­

Five hundred mg. of oxy—

th iam in e-d ip h o sp h ate was h e ated in a h o ilin g w ater h ath w ith IT HC1 f o r one hour.

Follow ing th e h y d ro ly s is , th e s o lu tio n was co n cen trated to an

o i l a t room tem p eratu re.

The o i l was then Jtaken up in a b so lu te alc o h o l,

and HC1 gas p assed in to th e s o lu tio n u n t i l l com pletely s a tu ra te d , and th en th e monophosphate p r e c ip ita te d w ith e th e r .

The p r e c ip ita te was

tak en up in w ater and p assed through a column of IR-4B.

The e lu a te

was a c id if ie d w ith HC1 to Congo red , and co n ce n tra te d to an o i l a t room tem perature in a vacuum.

The o i l was tak en up in 0.1N HCl and

p r e c ip ita te d w ith acetone to th e f i r s t appearance o f a clo u d in e ss and allow ed to stan d in th e ic e - c h e s t o v ern ig h t.

A s e m i- c r y s ta llin e

m a te ria l s e t t l e d down which was tr e a te d f u r th e r w ith a b s o lu te a lo c h o l. A fte r such tre a tm e n t, the product became c r y s t a l l i n e . and d rie d .

This product a ls o m elted a t 185-190°C.

I t was f i l t e r e d

A y ie ld of 150 mg.

was o b tain ed . Anal. Calcd. fo r

°* 3 ^ 5 ; H, 4 .3 1 ;

Free P, 0; Hyd. P, 0; T otal P, 7.41. H, 4 .4 7 ; Free P, 0:

Hyd. P, 0;

Found: C, 3 3 .6 ;

T otal P, 6 . 90

I t seems th a t a p u re r p re p a ra tio n was o b tain ed by way of deam ination o f th e co carb o x y lase.

I t was t h i s p re p a ra tio n of oxythiamine-mono­

phosphate th a t was employed in a l l of th e anim al and enzyme work to fo llo w . L

-J

19

Chapter III#

P h y sica l and Chemical P roperties of QxythiamineLiphosphate

The s im ila r ity in the behavior of cocarboxylase and oxythiaminediphosphate towards h yd rolysis w ith d ilu te a cid at 1G0°C has been d is ­ cussed and ap plied in the preparation o f oxythiamine-monophosphate# We were fu rth er in te r e ste d in studying the a ctio n of s u l f i t e on oxy­ thiam ine-diphosphate and a ls o in i t s u lt r a - v io le t absorption spectrum as eoicpared to that o f oxythiamine, thiam ine, and coc£arboxylase# The s p lit t in g a ctio n o f s u l f i t e on thiamine was ea rly observed by Williams and coworkers, (1 2 ) and u ltim a tely proved to be an e f f e c t iv e a id in e s ta b lish in g the structure o f thiamine#

The products o f t h is

rea ctio n were id e n tifie d as 2-m ethyl-4-am inopyrim idine-5-m ethylsulfonic a cid and k -m ethyl-5-beta-hydroxyethyl th iazole#

Later, W eijlard and

Tauber (7 ) stu d ied t h is rea ctio n w ith cocarboxylase and esta b lish ed th a t the same type o f displacement took place w ith the diphosphate# In th e ir work they id e n tif ie d the pyrimidine su lfo n ic a cid and th ia z o le pyrophosphate#

In the recent work o f Soodak and Cerecedo (13)» t h is

rea ctio n was stu d ied w ith oxythiamine#

A m odification o f the method

o f Williams e t a l . was used, and the reaction w ith oxythiamine was found to proceed in e s s e n t ia lly the same manner as w ith thiamine# One exception was noted in th e ir work, namely, that in the case o f oxythiamine no p r e c ip ita tio n o f the pyrimidine fragment occurs, sin ce the oxysulfon ic acid i s more solu b le than the am inosulfonic a cid . When the rea ctio n was complete, the pH was adjusted to the a lk a lin e sid e and the th ia z o le extracted w ith chloroform#

The aqueous so lu tio n

containing the oxysulfon ic acid was treated according to the method ipf Cline and Williams ( lh ) in the is o la tio n o f the oxysu lfon ic acid

obtained by s u lf it in g the corresponding 5**ethoxy-methyl pyrimidine* In the case o f oxythiamine-diphosphate the is o la t io n o f the two fragments fo llo w in g the s p lit t in g a ctio n of s u l f i t e offered some d if ­ fic u lty *

The d if f ic u lt y was f i r s t encountered in an i n i t i a l run in

which the exact procedure used by Soodak and Gerecedo (1 3 ) fo r oxy­ thiamine was employed.

The rea ctio n was carried out at pH 5*0 t o r k

days at room termperature.

As in the case w ith oxythiamine, no p r e c ip i

ta t ion occurs w ith the diphosphate.

To obtain a separation o f the two

fragments, the so lu tio n was made s lig h t ly a lk a lin e and extracted w ith chloroform sev era l tim es.

The chloroform ex tract when taken to dryness

gave no resid u e, in d ica tin g th at the th ia z o le . pyrophosphate, in con­ tr a s t to the fr e e a lco h o l, was not solu b le in the organic so lv en t.

The

o x ysu lfon ic acid in the aqueous phase was Iso la te d by the method of Gline and Williams ( lh ) and rep recip ita ted from 5 parts water and 8 p a rts o f a lco h o l. Calcd. fo r GgBgO^PgS.

C, 35*30;

H, 3*92;

Found G, 35*^5;

H, 3*80 From t h is r e s u lt i t was apparent that oxythiamine-diphosphate was s p l i t by s u l f i t e in the same manner as oxythiamine.

This was in ­

d icated by the is o la t io n o f the oxysulfonic acid and i t s a n a ly sis, although in t h is run the thiazole-pyrophosphate could not be is o la te d . Therefore, a new approach had to be worked out fo r separating the two fr a c tio n s .

Prelim inary experiments w ith gold ch lorid e and s ilv e r

n itr a te had shown th at the p yrim idine-oxysulfonic acid was not pre­ c ip ita te d by th ese m etals in an acid medium.

This approach was there­

fo r e chosen as a means o f separating the two fragments. jwhich gave s a tis fa c to r y r e s u lt s was as fo llo w s:

The procedure

21

r k%2 mg. o f oxythiamine-diphosphate was d isso lv ed in 8*0 ml. o f water and the so lu tio n adjusted to a pH o f 5*® w ith $T NaOH.

Eight

hundred mg. o f s o lid sodium "bisu lfite was added, and the mixture allowed to stand at room temperature fo r h days.

In th is method o f is o la t io n ,

the thiazole-pyrophosphate was Iso la te d f i r s t "by making the so lu tio n s lig h t ly a lk a lin e to litm us w ith ammonium hydroxide, treatment with 10 drops o f hydrogen peroxide and warming s lig h t ly to ox id ize s u lf i t e ion s to s u lf a t e .

The so lu tio n was then made acid to Congo red with

10$ HNOj added dropwise.

To t h is , a s lig h t excess of concentrated

barium n itr a te so lu tio n was added dropwise, follow ed hy a s lig h t ex cess o f 10$ s ilv e r n itr a te solution* and washed*

The p r e c ip ita te was f ilt e r e d

The f i l t r a t e was made neutral to .Congo red w ith d ilu te

ammonium hydroxide and concentrated to 10 ml* .in a vacuum at room temperature.

To t h is so lu tio n 60$ s ilv e r n itr a te was added dropwise

9

u n t il no fu rth er p r e c ip ita tio n occurred. l e f t in the ice-b o x overnight.

The grayish p r e c ip ita te was

The s ilv e r p r e c ip ita te was f ilt e r e d ,

washed w ith a lc o h o l, and eth er and d ried.

The y ie ld o f s ilv e r s a lt o f

the thiazole-pyrophosphate was 13P mg. The f i l t r a t e from the s ilv e r - t h ia z o l e-pyrophosphate, containing the oxysulfonic; acid , was trea ted w ith concentrated HC1 u n t il no fu rth er p r e c ip ita tio n toofc p la ce, and then f il t e r e d .

The f i l t r a t e was

then trea ted w ith HC1 gas to satu ration and l e f t in the Ice-b ox.

In

4 hours a white p r e c ip ita te had formed and t h is was f ilt e r e d o f f .

The

f i l t r a t e was concentrated to dryness in a vacuum and the residue d is­ solved in concentrated HC1 and further trea ted w ith HC1 g a s.

A small

amount o f undissolved residue was f ilt e r e d o f f , and the f i l t r a t e (taken to dryness.

A small amount o f residue remained at t h is point

22

r and tM s was ta k en up in 5 ml* o f w ater and tr e a t e d w ith 8. ml. o f

a b s o lu te alcohol*

This was l e f t in th e ic e - c h e s t and a p r e c i p i ta te

s e t t l e d o v e rn ig h t.

The o x y su lfo n ic a c id was f i l t e r e d , washed and

d r ie d .

i

The y i e l d was 23 mg.

Anal. C alcd. f o r Found:

C6Bq0i^ 2S.

G, *3^.85?

C, 35*3®?

H, 3.92?

H. 3 .? ^ .

To dem onstrate t h a t th e th iazo le-p y ro p h o sp h ate f r a c tio n had been o b ta in e d , t h i s was condensed w ith 2-m ethyl-4-am ino-5-brom om ethylp y rim id in e to g iv e co carb o x ylase.

The a c t i v i t y and y ie ld o f th e syn­

th e s iz e d cocarboxylase was determ ined w ith th e carboxylase system m anom etrically. The e n t i r e y ie ld o f th e s ilv e r-th ia z o le -p y ro p h o s p h a te , 130 mg., was suspended in w ater and decomposed w ith hydrogen s u lf id e , u n t i l no f u r th e r p r e c i p i t a t e would form a f t e r c e n trifu g in g and p a s sin g in more hydrogen s u lf id e .

The s i l v e r s u lf id e was f i l t e r e d o f f and washed w ith

sm all p o rtio n s o f w ater.

The f i l t r a t e p lu s washings were c o n cen trated

in vacuo to dry n ess a t room tem p eratu re. phosphate was o b ta in e d amounting to 39 mg.

A y ie ld o f th ia z o le -p y ro ­ This m a te ria l d id not

c r y s t a l l i z e and had th e appearance o f a s ilv e r y g la s s .

The s y n th e s is

o f th e cocarboxylase was c a r r ie d out by condensation o f 39 mg. o f th e f r e e th iaz o le -p y ro p h o sp h ate w ith hO mg. o f 2-methyl*-h-amino-5-bromom eth ylpyrim idine.

A m ixture o f th e two fragm ents was tr e a t e d w ith 0*5

ml. o f li q u i d p etro la tu m and h e a te d in an o i l b a th a t 100°C f o r f iv e m inutes w ith continuous s t i r r i n g . washed out w ith e th e r .

The m ixture was cooled and th e o i l

The re s id u e was d iss o lv e d in 3 m l. o f w ater

and t r e a t e d w ith 3® ml. d f a ceto n e.

A clo u d in e ss formed im m ediately.

(This was l e f t i n .t h e ic e c h e st o v e rn ig h t.

The m a te ria l which s e t t l e d

^

23

was then trea ted with an equal mixture o f absolute a lco h o l and acetone to aid c r y s ta lliz a t io n .

On standing in the freezin g chamber with

occasional scratching the product became c r y s ta llin e , washed w ith eth er and d ried .

It was f ilt e r e d ,

The f in a l y ie ld o f condensed product

was 18 mg. To a sc er ta in the cocarboxylase content o f t h is product, i t s a c t iv it y in the Warburg was determined on pyruvate decarboxylation and compared w ith a su ita b le c o n tr o l.

The main chamber of each Warburg

v e s s e l contained 100 mg, o f the cocarboxylase fr e e apoenzyme, prepared by washing brewer*s yeast th ree tim es in 50 volumes o f 0,1N W^HPO^, at 30°C.

To each chamber 0 ,5 ml, o f 0,05 M sodium pyruvate was added,

follow ed by 1 ,0 mg, each o f manganese and magnesium.

Two con trols w ith

pure cocarboxylase, at a l e v e l o f k gamma, were employed.

The product

syn th esized by the condensation mentioned above was employed at a le v e l o f 20 gamma.

The data o f t h is experiment are summarized in Table I I I , Table III

Cocarboxylase A c tiv ity o f the Condensation Product o f the Iso la ted Thiazole-Pyrophosphate and 2-Methyl-4-*Amino~5“Sromo Met hylpyr imidine , V essel Ho,

Contents

u l C©2 in 15 min.

1

Control

191

2

Control

182

3

Condensation Product (20 V )

65

Condensation Product (20 V )

63

Condensation Product(20 V )

58

Average

Gocarboxylase Content {f> Y ield )

187

k 5

62

6 ,5

24

On the "basis o f th ese r e s u lts 20V o f the condensation product showed approximately l / 3 the a c t iv it y o f 4Y o f cocarboxylase.

This

would in d ica te the presence o f 1*3V o f cocarboxylase or about a 6*5$ y ie ld . B) The U ltra -V io let Absorption Spectra of. Oxythiamine and Oxythiamine-Diphosphate. The u lt r a - v io le t absorption o f oxythiamine-diphosphate was d eter­ mined to complete the p ictu re w ith the two s e r ie s o f compounds, namely, thiamine and cocarboxylase on the one hand and oxythiamine and i t s diphosphate on the oth er.

In a recent report o f Soodak and Cerecedo

(1 3 ), the maxima in the u lt r a - v io le t absorption curve o f oxythiamine were reported.

In a phosphate b u ffer o f pH 7*2, oxythiamine was found

to have two maxima at 223 and 266 mji.

The u ltr a - v io le t curve for

oxythiamine was checked in 0.02M phosphate b u ffer o f pH 6 .8 and com­ pared w ith t h a t , o f oxythiam ine-diphosphate.

Oxythiamine was found to

g iv e two maxima at 222 and 267 mp. and a minimum was found in the curve at 241 mji.

Oxythiamine-diphosphate was found to g iv e two maxima at

222 and 265 ^

and a minimum at 242 mp..

The u lt r a - v io le t absorption curves fo r th ese compounds are shown in Figure 1.

I t w i l l be n o ticed that the absorption curves fo r

oxythiamine and i t s diphosphate run very c lo se and t h is , of course, i s a lso found to be the case w ith thiamine and i t s diphosphate.

The

curve obtained fo r oxythiamine checks w ell w ith that reported by Soodak and Cerecedo.

With both s e r ie s of compounds, phosphorylation

has l i t t l e e f f e c t on the absorption curve as would be expected. However, the importance of the amino group, as fa r as the behavior l

J

in the u lt r a - v io le t region i s concerned, i s seen in the sharp s h if t in the i n i t i a l maximum from 235 fo * thiamine to 222 mp. for oxythiamine, when the amino group i s replaced by a hydroxyl*

This s h if t in the

curve i s a lso found to be the case w ith the minimum from 252 for thiamine to 24 1 .fo r oxythiamine.

A study o f these curves and those

fo r thiamine and cocarboxylase shows that d if f e r e n t ia l absorption spectrophotometry can be n ic e ly applied to the determ ination o f thiamine or oxythiamine in a mixture o f the two compounds *

The same

a p p lie s, o f course, to a p o ss ib le assay for cocarboxylase and oxy­ thiam ine-diphosphate in a m ixture.

DNf ANVdWOO D M lN n u a S 3 “IMVHD

t ra

mOTtnjF=

u

t_(-i-r -U^.4

Chapter IV.

A)

A New Synthesis o f the Thiazole Alcohol o f Thiamine*

B)

Preparation o f a New Pyrim idine-Thiazole D erivative.

A p r a c tic a l and convenient syn th esis o f the th ia z o le alcoh ol of thiamine i s o f obvious value commercially.

A new pathway to the prep­

aration o f 4 -meth y l-5 -b etahydroxyetbyl th ia z o le was worked out w ith the use o f the new reagent LiAlH^.

The h isto r y of t h is reagent in

organic chemistry i s a very recent one and one of rapid development, 'ihe su c cessfu l e x p lo ita tio n o f t h is reagent in the reduction of a v a r ie ty o f organic compounds was carried out by Nystrom and Brown (15, 1 6).

Since t h is time a host of workers have made use of the

unique p ro p erties o f t h is reagent for th e ir p a rtic u la r needs.

In

t h is work, LiAlHjj, was found to be an e f f e c t iv e agent in the reduction o f ethyl-4-m ethy 1 -th ia z o le -5 -a c e ta te to the corresponding a lco h o l. A p r a c tic a l preparation o f the th ia z o le e s te r has been reported by Cerecedo and Tolpin (l? )*

These workers synthesized eth yl-^ -m ethyl-

th ia z o le -5 -a c e ta te by condensation o f eth yl-alp h a brom o-levulinate. w ith thioformamide.

Their attem pts to reduce t h is compound to the

a lco h o l, h-methyl-5-betahydroxy eth y l th ia z o le were u n su ccessfu l. The sy n th esis o f t h is a lcoh ol was carried out as fo llo w s in t h is work: To a so lu tio n o f 2*5 g . (0.066 m oles) o f LIAIB^ in 100 ml. o f anhydrous eth er was added a so lu tio n o f 18.5 £• (0 .1 0 m oles) o f ethyl-*J-methyl th ia z o le -5 -a c e ta te in 150 ml., o f anhydrous ether at a rate to produce m ild r e flu x o f the eth er.

The rea ctio n was a l­

lowed to continue fo r 15 minutes a fte r the l a s t a d d ition .

The

mixture was then cooled and water added dropwise to decompose the ex ce ss LiAlHty*

The p r e c ip ita te was then trea ted w ith an excess o f

a lk a li and the mixture extracted w ith three 100 ml# portion s o f ether# The combined eth er ex tra cts were dried with sodium s u lfa te and the ether removed in a vacuum#

The lig h t brown residue was d i s t i l l e d under

diminished pressure at 91-95°C at 2 mm. was ?#0 g# or 49$ o f theory#

The y ie ld o f th ia z o le alcohol

A p icra te and p icrolon ate o f the alcoh ol

were prepared fo r analysis# The p icr a te o f 4-methyl-5-betahydroxy eth y l th ia z o le was prepared in 95$ alcoh ol and r e c r y s ta lliz e d tw ice from the same solvent#

It

melted at l62°C# (uncorr#)♦ Anal. Calcd# for C^H^O qN^S. S,

8 .6 0 . Found:

C, 38.71;

G, 39*05; H, 3*06;

H, 3*22?

H, 15.05;

I , 14.95; S, 8 .3 0 .

The p icrolon ate o f the a lco h o l was prepared in the same manner.

It

melted at 183-185°0* (u n c o rr.)* Anal. Galcd. fo r G^H^OgH^S. S,

7*86; Found:

G, **7.17?

G, 47.10; H, 4.18;

H, 4 .1 8 ;

N, 16.94;

N, 17*19;

S, 7 .8 4 .

In an attempt to reduce 4-methyl th iazole-5-*acetic a cid

by a

sim ila r technique in eth er, only a few mg. of a p ic r a te could be ob­ ta in ed which melted a t l60°G.

The very s lig h t s o lu b ilit y o f the

fr e e acid in eth er lim ite d the method and i s fu rther evidence for the s o lu b ilit y lim ita tio n s w ith th is reagent.

Since 1947 t h is reagent

has found in creasin g use w ith more polar so lv en ts such as dioxane and tetrahydrofurane.

This should su b sta n tia lly increase the further use­

fu ln e s s o f LiAlHh ^or tk °se compounds not solu ble in eth er. At th is poin t i t may be worth w hile to summarize the reaction s which have been d iscu ssed .

The reaction s which have been carried out

in t h is work are shown in the follow in g scheme:

29

Thiazole E ster

Oxythiamine

Thiazole Alcohol 2-Methy 1 -4 -hydroxy Pyr imi a ln e-5 -S u lfo n ic Acid

Thiazole-Pyrophosphate + 2-Met hy1-4-Amino5-bromo-methylPyrimidine*

HONO Oxyt h i&mine-D ipho s - the th ird group received no supplementation but remained on the thiamine d e fic ie n t d ie t and served as c o n tro ls.

The con trols died a fte r having lo s t 73 grams, on the

average, on the 22n^ day o f the experiment*

The pigeons receiv in g

xanthopterin and f o l i c acid m anifested a sharp r is e in weight f o l ­ lowing the i n i t i a l d ep letio n p eriod .

From t h is work the authors con­

cluded, f i r s t , that xanthopterin and f o l i c acid could cure the symptoms o f thiamine d e fic ie n c y in pigeons and secondly, that th ese p terin s had th erefo re , in the pigeon, an a ctio n resembling that o f vitam in B^. A s e r ie s o f experiments were se t up in t h is work employing both f o l i c acid and xanthopterin in mice and rats*

The b asal thiamine

d e fic ie n t d ie t used was the C-28-A d ie t , the conposition o f which i s [Shown in Table IV.

Table IV Composition o f the Thiamine D eficien t B asal D iet (G-28-A) Amount per H ilo g . Casein

25©

Sucrose

53©

Crisco

100

Lard

50

S a lts*

50

Ruff ex

20 Supplements added per Hilo*

mg.

PyridoXine

10

R ib oflavin

10

Calcium Pantothenate

100

Alpha-to cophero1

40 mg.

Beta-Carotene

20

Choline

1*5 grams

Vitamin D (D risd o l)

5000 USP u n its

* Osborne and Mendel s a lt m ixture.

The quantity o f manganese

was doubled. Experimental The f i r s t group o f experiments were carried out w ith mice em­ ployin g f o l i c a cid which was r e a d ily a v a ila b le .

In th ese experiments,

the f o l i c acid was given o r a lly by in clu sio n in the d ie t , and a lso by in je c tio n .

Three str a in s o f mice were used in both types o f experiment,

in clu d ing Sw iss, Rockland and Fordham s tr a in s .

Ho str a in d ifferen ces

were noted in t h is work and fu rth er, no d ifferen ce was found when the jfo lic acid was included in the? b asal d ie t at a le v e l o f 20 mg. per

33 r i K ilo, or whether i t was in je c te d subcutaneously at a le v e l o f 50 gamma p e r day,

T h erefo re, th e anim als a re t r e a te d as one group.

The r e s u l t s

found when f o l i c acid was adm inistered to mice ©n the b asal thiamine d e fic ie n t d ie t are shown in Table V, Table T E ffe c t o f F o lic Acid AdMni s t r a t i on in Mice on a Thiamine D eficien t D iet Group

Somber o f Animals

Survival Time in Days

Controls

10

23-30

F o lic Acid

16

17-31

The su rv iv a l time o f those animals receiv in g f o l i c acid was comparable to th at o f the c o n tr o ls.

Further, the weights o f the

animals in the two groups showed th at both groups lo s t weight s te a d ily w ith no tendency on the part o f those receiv in g f o l i c acid to gain once they were thiamine d e f ic ie n t .

This fa ilu r e on the part o f f o l i c acid

to replace thiamine can not be attrib u ted to the low food intake, sin ce the animals rec eiv in g 5$ gamma of f o l i c acid by in je c tio n be­ haved as did the co n tro ls and those receiv in g f o l i c acid in the d ie t . It was decided-to fu rth er check t h is point w ith r a ts , and in t h is case f o l i c acid and xanthopterin were used.

Prelim inary to

s e tt in g up t h is experiment, xanthopterin had to be synth esized. The sy n th esis o f th is p ter in was f in a lly accomplished by the method o f Totter (2 0 ),

The xanthopterin was obtained in pure form as

esta b lish e d by i t s u lt r a - v io le t absorption spectrum which agreed w ith th at reported in the lite r a tu r e by T otter, up w ith s ix 22 day old Sherman str a in r a t s .

The experiment was se t These animals were put

gn the same C-28-A d e fic ie n t d ie t and divided in to three groups,

j

34 *“ At the time th at th e ir weight curves had shown a decided thiamine de­

i

f ic ie n c y , a group o f 2 animals served as co n tro ls, 2 received 5® gamma o f f o l i c acid by in je c tio n and 2 animals received $0 gamma of xantho­ p te r in by in je c tio n .

It may he sta ted b r ie f ly that the r e s u lts o f the

experiment agreed w ith those o f the previous one w ith m ice,

The data

obtained w ith r a ts are given in Table T I . Table ?I The E ffect o f F o lic Acid and Xanthopterin in Thiamine D eficien t Eats Group

Ho* of Animals

Survival Time in Days

Controls

2

34 and 35

F o lic Acid

2

30 and 34

Xanthopterin

2

30 and 37

The behavior o f the young Sherman ra ts e n tir e ly confirmed the observations w ith three s tr a in s o f mice.

It appears th erefo re, that

in mice and r a ts maintained on our sy n th etic d ie t, f o l i c acid and xanthopterin can not replace thiamine fo r growth or for maintenance. It i s s ig n ific a n t th at in a more recent p u b lica tio n , Busnel and h is group (21) observed that t h is in te r r e la tio n sh ip between thiamine and th e p ter in s depends to a great extent on the in t e s t in a l f lo r a . In experiments in which they adm inistered su lfa.d ru gs together with the p te rin s there liras a very pronounced decrease in the thiamine­ lik e a c t iv it y of th ese compounds.

This fin d in g i s o f obvious s ig n i­

fic a n ce in in ter p r etin g the data obtained in th is work.

The b asal

thiamine d e fic ie n t d ie t employed by Busnel and the C-28-A d ie t used in t h is work and in a l l o f the work with thiamine an tagonists to be reported, are b a s ic a lly d iffe r e n t.

However, even w ith t h is point

4n mind i t i s s t i l l d i f f i c u l t to conceive o f the in te r r e la tio n sh ip

j

35 r between th ese e n tir e ly d iffe r e n t groups of vitam ins, sin ce t h is same e f f e c t was n o ticed w ith r ib o fla v in d e fic ie n t animals.

I f these p terin

d e r iv a tiv e s could cure the ty p ic a l symptoms o f thism ine d e fic ie n c y in pigeons and r a t s , at most the cure i s a temporary one.

I t appears

very u n lik ely that the p te r in s act as precursors fo r thiamine sy n th esis. The experiments on mice did not show t h is e f f e c t .

Since the

o r ig in a l workers did not use mice in th e ir work, i t was p o ssib le that we had here a sp e cie s d iffe r e n c e , p a r tic u la r ly sin ce the e f fe c t was shown to depend on in t e s t in a l b a cteria .

However, the same r e s u lts

obtained w ith r a ts elim inated t h is p o s s ib ility *

L

Chapter VI.

The I f f e e t o f Oxythiamine, Gxyt hiamine-mono-phosphate, Oxythiamine-diphosphate, and Neopyrithiamine in Mice*

Uhlifce the non-com petitive in h ib itio n w idely studied w ith the s u lf a drugs, thiamine a n tim etah olites have found l i t t l e use as chemo­ th erap eu tic drugs.

This i s to he expected sin ce, as was shown hy

Wyss (22) w ith pyrithiam ine, blood concentrations which are non to x ic to experimental animals are not b a c te r io s ta tic .

N everth eless, the

b io lo g ic a l a p p lica tio n o f thiamine an tagon ists, p a r tic u la r ly o f oxy­ thiam ine, has afforded valuable inform ation w ith regard to the n u tri­ tio n o f sev era l s p e c ie s.

We have a lso in th ese s p e c if ic thiamine

a n ta g o n ists, a means o f ra p id ly d ep letin g animals o f th e ir vitam in sto r e s w ith the production of a s p e c if ic avitam in osis.

This i s part­

ic u la r ly exem plified in thiamine d efic ie n c y in the case o f the mouse. In t h is s p e c ie s, the ty p ic a l thiamine d efic ie n c y syndrome that i s obtained in other sp e c ie s, i s seldom produced when the mice are main­ tain ed on a thiamine d e fic ie n t d ie t .

The p o ly n eu ritic syndrome can

rap id ly be produced, however, by the use o f a s p e c ific antithiam ine such as neopyrithiam ine.

Thus, we have a rapid means o f studying the

p h y sio lo g ic a l and p a th o lo g ica l e f f e c t s of thiamine d efic ie n c y . The a p p lica tio n o f two such s p e c if ic antithiam ines in the mouse has been reported by lu s e b i and Cerecedo (2 3 ).

In t h is work, a

comparative study was made in mice employing oxythiamine and neopyri­ thiam ine.

It was observed that when neopyrithiamine was adm inistered

to mice at a le v e l o f thiam ine/neopyrithiam ine of l / 50* symptoms c h a r a c te r istic o f thiamine d efic ie n c y were obtained.

The same syndrome

was not observed w ith oxythiamine at an equivalent molecular concen­ t r a t io n .

Neopyrithiamine was found to be considerably more to x ic

37 r i than oxythiamine, and t h is observation has been confirmed and extended w ith much lower le v e ls o f neopyrithiamine and w ith other mouse strains# (The r e s u lts o f t h is work c o n s titu te s the subject matter of th is se c tio n . According to Wilson and Harris (2 4 ), pyrithiam ine does not have the structure assigned to i t .

These workers have synthesized a new compound

which they c a l l neopyrithiam ine, and which shows chemical and p h ysical p ro p erties that would he expected from the pyridine analogue o f thiam ine. The compound referred to as pyrithiam ine and f i r s t synthesized by Tracy and E ld e r fie ld (25) was found to be a complex mixture.

The early

work employing pyrithiam ine in mice and in microorganisms i s open to c r itic is m at le a s t from a q u an titative point of view.

The neopyri­

thiamine employed in t h is work was obtained from Merck and Go., Rahway, N. J. Oxythiamine, which was f i r s t synthesized by Bergel and Todd (26) was found by th ese workers to be b io lo g ic a lly in a ctiv e as a vitam in when employed In a one mg. dose in a rat electrocardiograph assay. It was not -until the work o f Soodak and Cerecedo (27, 28) that oxythiamine was found to be a potent thiamine an tagon ist.

These workers

found th at oxythiamine was a s p e c if ic antithiam ine in mice maintained on one gamma o f thiamine per day.

This work was confirmed by lu se b i

and Cerecedo (2 9 ) who a lso found that 25 gamma o f oxythiamine could n u llif y the e f f e c t o f one gamma of thiam ine.

Since the fin d in g of

Soodak and Cerecedo, sev era l workers have found oxythiamine to be a potent thiamine in h ib ito r in other sp e cie s and under varying con­ d itio n s . In a recent p u b lica tio n by Jones and coworkers (3 0 ), oxythiamine iwas found to be extremely to x ic to mice when fed o r a lly by p ip e tte .

,

In th e ir work, th ese workers adopted Woolley’s (31) method fo r ex­ p ressin g an in h ib itio n index*

This was the quotient obtained by div­

id in g the concentration o f in h ib ito r by the concentration of m etabolite necessary to n eu tr a liz e the e f f e c t o f the in h ib ito r .

Jones et al*

found that at three d iffe r e n t le v e ls of thiamine, the in h ib itio n index obtained fo r oxythiamine was th ree.

Their work was prim arily d irected

towards a study o f the e f f e c t o f thiamine d e fic ie n c y on the onset of the symptoms o f the Lansing s tr a in of p o lio m y e litis v ir u s .

The onset

o f symptoms was found to be delayed in thiamine d e fic ie n c y , and by the use o f oxythiamine they had a rapid means for producing a con trolled and severe thiamine d e fic ie n c y . Frohman and Day (3 2 ), employing oxythiamine in r a ts, found th is an tivitam in to be h ig h ly to x ic to th is species*

Their work was p a r ti­

c u la r ly s ig n ific a n t sin ce i t pointed to the;manner in which oxythiamine may exert i t s e f f e c t in v iv o .

When oxythiamine was fed to r a ts by

th ese workers, there was a pronounced excretion o f thiamine in the urine o f these anim als.

This was interpreted by ffrohman and Day as

a displacement o f thiamine in the t is s u e s o f the animals by oxy­ thiam ine.

The to x ic e f f e c t of oxythiamine was a lso observed to bring

about a marked r is e o f pyruvate in the blood o f th ese r a ts .

This

in d ica ted that oxythiamine in some way in h ib ited or blocked pyruvate metabolism* Norris and coworkers (33 )t working w ith ch ick s, found oxythiamine to be extrem ely a c tiv e a lso in t h is sp e c ie s.

It i s to be expected

th at in any sp e cies that requ ires thiamine as an e s s e n t ia l growth fa c to r , oxythiamine w ill m anifest i t s antithiam ine e f f e c t .

It is

s ig n if ic a n t that in the work o f Norris with ch ick s, oxythiamine was

found to give in h ib itio n in d icfiss ranging from 2? to 39*

®fees'e workers

arrived at the in h ib itio n index by determining the r a tio o f the amount o f in h ib ito r to the $jnount o f vitam in antagonized by the inhibitor# This o f course, i s e s s e n t ia lly the method used by Soodak and Cerecedo, and which i s d iffe r e n t from the method adopted by Wooley and other workers# Experimental. The experim ental procedure used in a l l o f t h is work w ith thiamine a n tagon ists was the fo llo w in g: Weanling mice were placed on the thiamine d e fic ie n t, G-28-A d iet and kept on t h is d ie t u n til they sta rted to lo s e weight a fte r reaching th e ir maximum w eigh ts.

At the time when they were ea tin g one gram o f

food or l e s s per day, they were in jec ted subeutaneously w ith one gamma o f thiamine per day fo r about a week.

This prelim inary treatment

served to d ep lete our animals and standardize them before the in je c tio n s o f the an tivitam in s were begun.

Controls were always se t up and th ese

were maintained on one gamma o f thiamine per day. A comparative study was devised w ith the in ten tio n o f determining the t o x ic it i e s o f oxythiamine, oxythiam ine-diphosphate, and neopyrithiamine on the same molecular b a s is . C-57 black str a in s were used.

Mice o f the Swiss albino and

In the case o f the three compounds

the e f f e c t .on the food intake was apparent overnight.

There was a

s ig n ific a n t drop in the food intake o f th ese animals and a lo s s in weight#

The r e s u lts which are shown in Table VII in d icated that the

an tivitam in e f f e c t o f neopyrithiam ine i s considerably more pro­ nounced than that o f oxythiamine or i t s diphosphate#

40

Table VII Comparative Antithiamine E ffe c t o f Oxythiamine, Oxythiamine-Diphosphate and Neopyrithiamine in Mice, Substance Tested

No. o f Mice

____

Wt. at Start Incidence o f In j. of o f Poly* Ant i thiamine n eurit i s & _____________ i -

Survival Time in ^ays. ___________________

Oxythiamine

9

10.6*13.6

0

13*21

OxythiamineDiphosphate

5

9*6-13.6

0

11-17

Neopyrithiamine

17

1 0 .2 -1 6 .4 ___________ 100__________ 6*8

In tbese experiments the a n ti thiam ines, in each case, were administered at a molecular ra tio n o f thiamine /antith iam ine o f 1 /5 0 .

A to ta l of 8

co n tro ls were used in th ese experiments and in a ll cases th ey maintained th e ir weight and survived u n t il they were s a c r ific e d 1 -2 weeks a fte r the l a s t animal in the corresponding experim ental group had died. In comparing the behavior o f th ese animals towards the d iffe r e n t compounds used, i t was g en era lly found that oxythiamine-diphosphate had about the same t o x ic it y as oxythiamine.

With n eith er o f th ese

compounds does the ty p ic a l syndrome of severe thiganine d efic ie n c y show up in the mice.

The animals become hunched but are otherwise

ab le to move around.

On the other hand, the e f fe c t w ith neopyrithiamine

was quite pronounced.

This m anifested i t s e l f in the development of

p o ly n e u r itic symptoms and in a shorter survival p eriod.

The poly­

n e u r itic syndrome co n sisted o f tremors and convulsions, follow ed by s p a s t ic it y of the le g s and head.

The mice were apt to hold th e ir

heads on one sid e and they f in a lly developed complete p a r a ly sis of the hind le g s .

41 r We were fu rth er in te r e ste d in studying the e f fe c t ©f oxythiamine and neopyrithiamine in two cancer str a in s o f mice.

These mice were

obtained from the Jackson Memorial Laboratory, Bar Harbor, Maine.

The

dba and C3H s tr a in s represent h igh ly inbred cancer s tr a in s which are found to have a high incidence of spontaneous mammary tumors.

The

experiment was designed in t h is case fo r a study o f p o ssib le changes in the t is s u e s o f such mice when they were made c r i t i c a l l y d e fic ie n t in thiamine by means o f oxythiamine and neopyrithiam ine. ly , the animals were s a c r ific e d before they died#

Consequent­

These t is s u e s were

sent to Br. A.Y* P is c io tt a , Milwaukee Co. Ceneral H ospital, Milwaukee, W isconsin.

His report was not received in time to be included in

t h is work. In the case o f neopyrithiam ine, the appearance o f p a r a ly tic symptoms o ffered an e x c e lle n t means for judging the proper time to s a c r ific e the anim als.

In the case o f oxythiamine, we had no such

way to go by, although the w eights and appearance of the animals, togeth er w ith th e ir d a ily food intake, gave a good idea as to the extent o f the thiamine d e fic ie n c y .

The experimental technique was the

same as that described before and in t h is case a d ifferen ce in the behavior o f the dba and C3H animals was noted as regards th e ir sus­ c e p t ib ilit y to oxythiamine. two str a in s sep a ra tely .

fo r t h is reason i t i s best to trea t the

The oxythiamine and neopyrithiamine used in

t h is experiment were given at molecular le v e ls of thiam ine/antithiam ine o f 1/50 so that couparison could again be made with the Swiss and 0-57 str a in s o f mice.

L

42

Table VIII Comparative E ffec t o f Oxythiamine and Neopyrithiamine in dba and C3H Mice. S train

Substance Tested

No. o f Mice.

Wt. at Start o f In j.

Incidence o f Poly­ n e u r itis $

dba

Oxythiamine

4

1 2 .5 -1 5 .1

0

dba

Neopyrithiamine

5

1 1 .6 -1 5 .6

100

C3H

Oxythiamine

6

1 3 .2 -1 6 .0

0

C3H

Neopyrithiamine

6

1 2 .2 -1 6 .2

100

Period o f Survival or Time at Which the Mice Were S a crific ed . 13-14 3-6

4-6

On the whole both the dba and C3H animals behave as do the Swiss and C-57 mice towards a high dose o f neopyrithiam ine.

The thiamine

d e fic ie n c y syndrome observed in these cancer str a in s was e s s e n t ia lly sim ila r to th at seen w ith the other s tr a in s , appears in 4 to 6 days.

This c lin ic a l p ictu re

At t h is point the animals were s a c r ific e d ,

and the t e s t e s and thyroids removed from h is to lo g ic a l examination. The behavior o f the two s tr a in s towards oxythiamine however, seems to in d ica te a sp e c ie s d iffe r e n c e .

A ll o f the dba animals f i t into the

general pattern se t by the Swiss and C-5 7 anim als.

Of the 4 dba

animals re ceiv in g oxythiamine at a le v e l of l / 50> one animal died on the 13th day and the other three s a c r ific e d on the 14th day o f the experiment a fte r having dropped to approximately 10 grams or l e s s . At th is point the oxythiamine animals were ea tin g between 0 .7 and 1 .0 grams o f food per day.

This p ictu re was p r e tty much as was found w ith

the other s tr a in s . The r e s u lts were somewhat d iffe r e n t w ith oxythiamine and the G3H l

j

*3 r anim als.

i A ll s i x o f the C3H mice receiv in g a regime of thiam ine/oxy-

thiamine o f 1/5 0 appeared to he more s e n s itiv e to t h is antivitam in.

Of

the s ix animals receiv in g oxythiamine, three died before they were s a c r ific e d fo r h is to lo g ic a l stu d ie s.

These animals succumbed on the

4 th , 7th, and 8th day a fte r beginning the in fe c tio n s o f oxythiamine# The remaining three animals behaved in much the same manner and had to be s a c r ific e d on the 4th, 8th, and 8th day, at which time th e ir appearance, body w eight, and food intakes ind icated they would not survive another day.

For a period o f 24 hours preceding the s a c r ific e of these animals

they ate l e s s than 0 .4 g. o f food# It was obvious at t h is point that in a l l o f the experiments carried out, the le v e ls o f an tivitam ins used were q u ite high.

A comparative

study was th erefore carried out w ith th ese antithiam ines in which the animals received a le v e l o f thiamine /a n ti thiamine o f 1/25*

In th is

work oxythiamine, oxythiamlne-monophosphate, oxythiamine-diphosphate, and neopyrithiamine were employed with Eockland and Swiss mice.

The

r e s u lts w ith both str a in s were the same and are shown in Table IX#

L

44

Table IX Comparative E ffe c t o f Thiamine Antagonists at a Level o f l/25« Substance Tested

lo* o f Mice

Incidence of P olyn eu ritis*

$

Week by the End o f Which Death *%sued 1 2 3 4

Oxythiamine

5

©

0

2

2

1

OxythiamineMonophosphat e

8

0

0

1

6

1

100

5

Diphosphate Heopyrithiamine 5

The general p attern o f potency observed at the higher le v e ls of the an tivitam in s was confirmed at t h is lower lev el#

Qxythiamine-mono-

phosphate which was a lso employed in t h is experiment i s found to have about the same t o x ic it y as oxythiamine and oxythiamine-diphosphate# neopyrithiamine i s again found to be much more to x ic than the oxythiamine d erivatives*

The same r e s u lt s were obtained at h a lf the concentration

used p rev io u sly , in d ica tin g that a certa in prelim inary period i s required to d ep lete the animals before the ty p ic a l p o ly n eu ritic p ictu re appears#

This d ep letio n o f the animals and p a r a ly sis can be

carried out w ith neopyrithiamine at a le v e l o f 1/25 as rap id ly as at l/5 0 «

This in d icated that neopyrithiamine was considerably more to x ic

than at f i r s t expected and c e r ta in ly more so than was reported by Woolley (34) fo r pyrithiam ine in mice* The E ffec t o f Heonyrithiamine In Mice at Very Low Levels* The r e s u lts obtained in the previous experiments w ith neopyri­ thiamine in mice made i t necessary to determine the in h ib itio n index fo r th is an tivitam in .

I t was furth er planned to determine i f a

tpossible sy n e r g is tic e f fe c t could be observed when both neopyrithiam inej

45 and oxyt h i ami ne-d ipho sphat e were given together*

To determine whether

any ad d itiv e e f f e c t was being obtained when both compounds were in je c te d togeth er, the t o x ic it y o f neopyrithiamine at an equivalent concentration had a lso to be ascertain ed .

In t h is way i t was found that much lower

concentrations o f neopyrithiamine produced p a ra ly sis and death in a short period o f time and th at the in h ib itio n index fo r neopyrithiamine was about 1* In an i n i t i a l experiment the e f f e c t of neopyrithiamine at a le v e l o f 1/25 was checked in Swiss mice*

In ad d ition , a group o f animals in

t h is experiment were in je c te d w ith a combination of oxythiam ine-di­ phosphate and neopyrithiamine which was given at the same molecular l e v e l o f l/2 5 » each being given at a le v e l o f 1/12*5*

The data ob­

tain ed w ith a group o f ten animals are summarized below* The E ffect o f Neopyrithiamine and of a Combination o f Neopyrithiamine and Qxythiamine-Dipho sphat e in Mice at a Level o f 1/25* A ntivitam in

Level

No* o f Animals

Survival Time in Bays*

Neopyrithiamine

1/25

5

6-8

Neopyrithiamine p lus

1/12*5

5

6 -8

OxythiamineDiphosphate

1/12*5

A ll o f the animals o f both groups showed a complete p a r a ly tic syndrome in 5 to 6 days and died 24 to 48 hours later*

These r e s u lts

are e s s e n t ia lly the same as those obtained w ith neopyrithiamine at a l e v e l o f 1/50*

A lso, the previous r e s u lts in Bockland and Swiss mice,

employing neopyrithiam ine at a le v e l of l /25 were confirmed in t h is experiment w ith Swiss mice*

We can rea d ily see that the concentrations

46

o f an tivitam in s used were too high in order to determine whether there was any ad d itiv e e f fe c t when both antivitam ins were given togeth er. From t h is experiment i t became apparent that the index fo r neopyri­ thiamine had to be determined. In a fu rth er experiment ten Swiss mice were se t up w ith s t i l l lower concentrations of neopyrithiam ine.

In th is experiment the neo­

pyrithiam ine was given at a molecular le v e l o f 1/10 and the combination o f both compounds at 1 /5 .

Two animals served as con trols and 4 animals

were put in to each group. A ntivitam in

Level

Ho. o f Animals

Survival Time in Days.

Neopyrithiamine

1/10

4

7-3

Neopyrithiamine p lus

l/5

4

7-8

OxythiamineD iphosphate.

1/5

As in the f i r s t experiment, here a lso a l l o f the animals o f both groups showed complete p a r a ly sis in 5 to 6 days and were dead one to two days la t e r .

The index fo r neopyrithiamine was obviously

much lower than 10. A further experiment was s e t up which u ltim a te ly e sta b lish ed the extreme potency o f t h is an tivitam in in mice.

Both Swiss and Itockland

mice were employed in th is experiment w ith id e n tic a l behavior.

The

data which e sta b lish e d the index fo r neopyrithiamine are shown in Table X.

L

Table X The E ffect ©f Neopyrithiamine in Mice at Low Levels* Antivitam in

Level

Neopyrithiamine

1/5

7-9

Neopyrithiamine

1/3

11

Neopyrithiamine

1/1

No* o f Animals

Survival Time in Days.

12

13-16

Several observations are worth mentioning in t h is experiment. Those animals which received a le v e l of 1/5 ju st about maintained th e ir weight or showed a s lig h t drop fo r the f i r s t two days.

However, they

then began to drop f a s t in weight and a l l the animals showed the same p a r a ly tic symptoms as those on higher le v e l s .

These animals had a

su rv iv a l time o f only 7 to 9 days .which i s approximately the same as that obtained on the 1/50 l e v e l . When the dose was decreased to 1/3 and l / l , the animals a l l showed a s lig h t gain in weight of about one to two grams.

At the lower le v e l,

the animals show th is gain fo r a period of 8 to 10 days.

Following

t h is period o f growth, they begin to lo s e weight rap id ly and succumb in the next h to 8 days, a fte r having shown the ty p ic a l symptoms. The experiments reported here serve to e s ta b lis h the extreme t o x ic it y o f t h is an tivitam in in mice, and the r e s u lt s obtained in d icate that the in h ib itio n index for neopyrithiam ine i s about 1,

It i s

p o ssib le at t h is point to make comparisons between the comparative e ffe c tiv e n e s s o f oxythiamine and neopyrithiamine as ant i t hi amine s . We fin d th a t, u sin g our system of expressing an in h ib itio n index, neopyrithiamine i s approximately 50 tim es more to x ic than oxythiamine on a molecular b a s is .

In other words, the same e f fe c t was obtained

r i w ith oxythiamine at a le v e l o f 1 /50 as was found with neopyrithiamine at a le v e l of l / l . There i s every in d ic a tio n from th ese experiments that neopyrithiamine ex erts i t s e f f e c t "by accumulation or storage when given at these very low l e v e ls .

Ho immediate harmful e f f e c t s are observed at such concen­

tr a tio n s as are seen in the case o f the higher le v e ls , in which case an e f f e c t on food intake and body weight i s observed overnight.

This

may be one explanation for the extreme t o x ic it y of neopyrithiam ine. In view o f the fa c t that animals receiv in g low le v e ls o f neo­ pyrithiam ine showed no harmful e f f e c t s at f i r s t but a c tu a lly a s lig h t gain , i t was decided to t e s t the e f fe c t o f neopyrithiam ine in a short term experiment.

In t h is experiment mice were put on the d e fic ie n t

G-28-A d ie t from weaning and continued on t h is d ie t throughout the experiment without r e ce iv in g any thiam ine.

Under such cond itions

mice u su a lly succumb in 20 to 30 days without showing any p a rticu la r thiamine d efic ie n c y sig n s. were se t up in th is case.

Two con trols and 7 experim ental animals The con trols were maintained on the def­

ic ie n t d ie t and received no thiam ine, while the experim ental animals were maintained on the same d ie t but received one gamma o f neopyri­ thiamine per day by in je c tio n .

These two groups were found to behave

in e s s e n t ia lly the same manner, ,vin d iea tin g that in the absence of thiam ine, neopyrithiamine could not in \any way replace i t or show any vitam in a c t iv it y at low l e v e ls .

The animals receiv in g neopyri­

thiamine a lso showed p a r a ly sis which was not observed w ith the co n tro ls.

L

Ji

49

r Chapter VII

S p e c if ic it y o f Structure fo r Antithiamine A ctivity*

To throw some lig h t on the p o ssib le mechanism by which oxythiamine ex erts i t s e f f e c t as a thiamine antagon ist, stu d ies w ith mice were car­ r ie d out employing bromoxythiamine and 4-me t h y l-5 -b et ahydroxy ethyl-H (1*4* d im eth yl-u racil ( 5 ! )-methyl thiazolium ch lorid e.

For the sake

o f convenience t h is d e riv a tiv e w ill be referred to as the u r a c ilth ia z o le d er iv a tiv e .

A comparison of the stru ctu res o f oxythiamine,

bromoxythiamine and the u r a c il-th ia z o le d eriv a tiv e i s shown in Figure 2. The preparation and a n a ly sis o f the u r a c il-th ia z o le d eriv a tiv e has been reported in a previous s e c tio n .

Bromoxythiamine, used in th is work

was obtained from Dr. Soodak and was synthesized by the method of Soodak and Cerecedo (13)* Anal. Calcd. fo r Oj^H^B^SOBr^. Br, 49.00

Found:

C, 29.51;

C, 29*38;

H, 3*86;

H, 3*26;

Br, 49.05

The r e s u lts w ith bromoxythiamine in d icated an exact s p e c if ic it y w ith regard to the th ia z o le hydroxy group.

The u r a c il-th ia z o le d eri­

v a tiv e , m odified considerably when compared to oxythiamine, in dicated th at a certa in degree o f s p e c if ic it y o f structure was necessary on.the pyrimidine portion o f oxythiamine to obtain in h ib itio n . In the work of Soodak ( 3 ) , chloroxythiamine was found to produce no a n ta g o n istic a ctio n when 100 gamma o f the compound was given to mice r ec eiv in g one gamma o f thiam ine.

Thus, under the same conditions

where a 25/1 oxythiam ine/thiam ine regime produced a d e fin ite a n ti­ vitam in e f f e c t , 100/1 o f chloroxythiam ine/thiam ine produced no harm­ f u l or b e n e fic ia l e f f e c t whatsoever. l

Bromoxythiamine was employed in t h is work at s t i l l higher le v e ls

50

figure 2 Comparative Structures o f Oxythiamine, Bromoxythiamine and the O racilThiazole D erivative Oxythiamine

2= ^ = C -C H 2 ,6

r= c -0 H

HoC-C G------------3 II II H— : CH

GHr,---- ----------- N % * \ XG H Bromoxythiamine

[=G-QH HoC-C ^ II V

G---------II OH

------------------N

/

C-CHg-CB^Br



h-H ethyl^-^etahydrosy-ithyX -H r (XllM, D im ethyl-Uracil ( 5 1)

-MethyX

Thiazolium Chloride.

J

ch3

HoC-N— 0 = 0 0=G

C'-

HH— C-Cfo

G-CHjj-OHgOH

.— —CH--

♦ \G

S

H

L

-I

51 r and the work w ith chi or oxythiamine was confirmed, Swiss and Bockland

i

mice were employed and the bromoxythiamine was adm inistered at very high molecular le v e ls o f 1/ 50, 1/ 100, 1 / 200, and 1/ 500* Table XI E ffe c t o f Bromoxythiamine in Swiss and Eockland Mice Level of Bromoxy­ thiamine

Ho. o f Mice

Wt, at Start o f In f. Brom­ oxythiamine.

Wt. at Time of S a c r ific e _ _*•

Av. Pood Intake Per Day.

Controls

3

1 1 ,3 -1 6 .4

12. 0- 17.0

1 .6

1/50

2

1 0 .7 -1 2 ,7

12. 0- 15.0

1 .7

1/100

2

11. 8- 13 .2

14.5-15*3

1 .8

1/200

2

11. 5- 16.6

1 4 .0 -1 7 .3

1 .8

1/500

2

1 1 .2 -1 3 .9

1 2 .8 -1 3 .8

1 .6

The r e s u lts w ith hoth stra in s were id e n tic a l and the two stra in s are treated to g eth er.

The data o f ta b le XI demonstrate that at a l l the

le v e ls used o f the bromoxythiamine, there was no harmful e f fe c t pro­ duced in th ese anim als. The experimental period was 5 weeks from the point o f sta r tin g the in fe c tio n s of the bromoxythiamine.

At the end of t h is time no e f fe c t

o f t h is compound was obtained, the animals receiv in g the bromoxythiamine behaving much lik e the co n tro ls as regards body weights and food in tak es. A ll the animals were th erefore s a c r ific e d at the end o f the f iv e week experimental period. Thus, i t was found that at a concentration o f bromoxythiamine 20 tim es greater than th at required by oxythiamine fo r in h ib itio n , no e f f e c t o f the bromo compound was n o tice d .

On the b a sis o f these r e s u lts , and

th ose o f Soodak (3)» and in view o f the c lo se s im ila r ity o f oxythiamine and bromoxythiamine, there i s a strong in d ica tio n that oxythiamine may L -I

52 r tie involved in a phosphorylation mechanism*

I

A ce rta in degree of stru ctu ral s p e c if ic it y was a lso apparent in the pyrimidine rin g when the u r a c il-th ia z o le d eriv a tiv e was in jec ted in to mice at high le v e ls without to x ic e f f e c t s .

I f oxythiamine i s involved

in a phosphorylation mechanism, then the enzyme i s concerned w ith both h alves o f the molecule as regards the s p e c if ic it y of enzyme action on i t s surface.

This o f course, i s to be expected, and was in d icated in

the work o f Emerson (35) w ith the 2-butyl analogue of thiamine which was found to be an antithiam ine in r a ts . In t h is work w ith Rockland mice, the u r a c il-th ia z o le d eriv a tiv e was in jec ted at le v e ls o f 1 /2 5 , l/5 °» and l/lOO* period was 6 weeks.

The experimental

Three animals were used in a long term experiment

and behaved as did three con trols in the same group.

The e f f e c t o f

t h is d eriv a tiv e was checked further in a short term experiment in which three animals were maintained on a C-28-A d ie t .

In such an

experiment, three con trols o f t h is group survived for periods o f 10 to 16 days a fte r reaching the peak in th e ir growth curves. experim ental animals receiv in g in je c tio n s o f 50*

Three

and 200 gamma

o f the u r a c il-th ia z o le derovative survived fo r periods o f 10 to 14 days from the point o f maximum w eight.

The food intake of these

animals was e s s e n t ia lly the same as th at of con trols maintained on the C-28-A d ie t and r eceiv in g no supplementation. D iscussion Thiamine i s present in organisms to a great extent as the d i­ phosphate, and t h is i s the form in which i t a cts in the carboxylase system.

It is a lso the a c tiv e form in the oxid ation and dism utation

Lrea ctio n s in volvin g pyruvate.

Several explanations may be advanced

j

53 r

to account fo r the fa ilu r e of bromoxythiamine to in h ib it in vivo at very high le v e ls* 1) I f i t i s assumed that oxythiamine i s phosphorylated in the same manner as thiamine, then the d ifferen ce in the a c t iv it y o f t h is com­ pound and that of chloro and bromoxythiamine can be explained.

This

would in d ica te that the a c tiv e form o f the in h ib ito r i s oxythiaminediphosphate.

This compound would then act to in h ib it the v i t a l func­

tio n in g o f the cocarboxylase systems in a manner sim ilar to i t s e f fe c t observed in v itr o w ith the yeast system. 2 ) It i s common knowledge th at thiamine i s phosphorylated by y ea st, l i v e r , and other tis s u e s of the animal body.

It i s reasonable to

expect that oxythiamine may in h ib it the phosphorylation o f the vitam in, thereby preventing i t from becoming fu n c tio n a l. 3 ) The th ird p o s s i b i l it y i s that in h ib itio n by oxythiamine might be brought about in both ways* It was w ith th ese concepts in mind that the work in the follow in g s e c tio n s , p a r tic u la r ly that w ith certa in enzyme systems in v it r o , was planned in an attempt to answer as best we could the question as to the manner in which oxythiamine a cts as a thiamine in h ib ito r .

L

-J

5^ r Summary o f the B io lo g ic a l Work With Thiamine Antagonists The present work has confirmed e a r lie r work that oxythiamine i s a potent antagonist in mice.

Comparative stu d ies carried out w ith oxy­

thiam ine, oxythiamine-monophosphate, and oxythiamine-diphosphate in ­ d icated that the mono oxythiamine.

and diphosphate had about the same t o x ic it y as

Sim ilar stu d ies were made in which the potency o f neo­

pyrithiam ine was compared to that o f oxythiamine and i t s d e r iv a tiv e s. He©pyrithiamine was found to be considerably more to x ic to mice on a molecular b a sis than oxythiamine.

This observation was a ttrib u ted to

the fa cto r of storage on the part of neopyrithiam ine.

This concept was

su b stan tiated by experiments in which neopyrithiamine was employed at very low con cen tration s.

In such experiments a cumulative e ffe c t of

neopyrithiamine was observed and no such e f f e c t was seen in the case of oxythiamine.

The in h ib itio n index for neopyrithiamine in mice was

found to be about 1 * Experiments w ith mice were a lso carried out w ith bromoxythiamine which e sta b lish e d the n e c e s s ity o f the th ia z o le hydroxyl group for in h ib itio n .

L

The sig n ific a n c e o f these fin d in gs i s d iscu ssed .

55 r Part III M icrobiological Studies With. Thiamine A ntagonists. Chapter 7 III

The S ffe c t o f Oxythiamine, Oxythiamine-Diphosphate, Eeopyrithiam ine, and the U racil-T hiazole D erivative on the Urowth o f St a-phvlo coccus aureus.

It has been w ell esta b lish ed that thiamine i s an e s s e n tia l growth fa c to r for a number o f b a cteria , for p la n ts, and for higher animals. It i s a lso recognized as a component of a number o f enzyme systems, p a r tic u la r ly those concerned with pyruvate metabolism.

As regards the

n u tr itio n of b a cte r ia , the thiamine molecule can be regarded as made up o f two m o ie ties, the pyrimidine and thi& zole h alves. 9H3 , c = c - c h 2ch2oh

H For the sake of convenience we can a r b itr a r ily d ivide micro­ organisms in to four groups as regards th e ir a b ilit y to synth esize thiam ine. 1 ) Those which syn th esize thiamine from simpler co n a istitu e n ts o f the medium. 2) Those which can syn th esize thiamine i f supplied w ith one h a lf o f the m olecule.. 3 ) Those that require both halves of the m olecule, but do not require the in ta c t molecule for growth. 4 ) Those which cannot syn th esize thiamine even i f supplied with both m o ie ties. L

56 r

i

For the purpose o f t h is work, i t was planned to make a study of a

rep resen ta tiv e organism of the th ird and fourth c la s s ific a tio n s *

Staph*

aureus and L actob acillu s fermentum were therefore s e le c te d for study, and i t was obviously of greater sig n ific a n c e to make such a study employing several thiamine d e r iv a tiv e s.

The e f f e c t of thiamine an tagon ists on

the growth o f Staph, aureus was made, employing oxythiamine, oxythiamine diphosphate, neopyrithiam ine, and the u r a c il-th ia z o le d e riv a tiv e . For the growth o f Staph, aureus a mixture of the pyrimidine and th ia z o le fragments can replace the in ta c t m olecule.

With such organ­

isms as Staph, aureus and others of group th ree, i t i s u su a lly assumed th at the organism p o ssesses an enzyme capable o f u n itin g the two p arts. Some work along th ese lin e s , in d ica tin g that the organism a c tu a lly requires the in ta c t molecule and can combine the two m oieties i s that of Tatum and Bel l (3 6 ) and th at of Tatum and Beadle (37)*

These

workers employed a th iam in eless str a in of mutant of Neurospora and observed that althought the organism could s t i l l synth esize the two halves o f the thiamine m olecule, i t could not grow because i t was unable to jo in the two togeth er. In connection w ith the in h ib itio n stu d ies on Staph, aureus to be reported in th is se c tio n , the work o f Knight and Mcllwain (38) on the e f f e c t of stru ctu re on the a v a ila b ilit y of pyrim idines and of th ia z o le s as growth fa c to r s fo r Staph, aureus, i s of immediate importance.

These workers te s te d a number o f su b stitu ted pyrimidines

and th ia z o le s .

The growth obtained w ith a v a r ie ty of su b stitu ted

pyrim idines and th ia z o le s was compared with that obtained w ith the pyrimidine and th ia z o le fragments o f thiamine which give optimum .growth o f the organism. ■■

The growth obtained w ith these d eriv a tiv e s

s

57 r th erefo re, was referred to the maximum growth taken as a standard for referen ce.

n

The optimum growth obtained w ith 2-methyl-4-amino-5-amino

methyl pyrimidine and 4-methyl-5-betahydroxy eth yl th ia z o le i s represent­ ed as four p lu s.

The growth found w ith the su b stitu te d d eriv a tiv e s i s

represented a lso on t h is

basis*

The data of Knight and Mcllwain are summarized in the follow in g ta b le which shows the nature of the su b stitu te d pyrim idines and th ia z o le s and the extent o f grov/th obtained w ith these d e r iv a tiv e s.

L

58

E ffect o f Structure on the A v a ila b ility of Pyrimidines and of Thiazoles as Growth Factors for Staph. aureus Pyrim idines

Thiazoles

N = C -R 2

5=3 = o -b 2

i

Bj-C II

I

C-Bo

N \

II 3

3ST----- G-H^,

%

CHg.NHg

H ■+•■+"■+'+"

H

CH2.CH2OH

4* + + 4-

13%

ch2 oh

H ++••+■ +-

H

CH2*G%0Ac

+- + ■+■

CH3

hh2

CHg.HH« CSH

H + +■

H

ch2. choh. ch3 + +

ch3

©Me

CH2.HH2

H

H

CH2.GH2.CH20]I + ■*“

gs3

W-2

GH2.GOHH2

H —

H

-CB-CEg

4-

ch3

OH

ch2.hh2

H

H

CHOH. CEj



CEj

0H

ch2. oh

H —

ch3

CHg.CHgOH



GH3

m2

gh3

H —

OH

CH2.CH2©Ac



CEj

OH

nh2

ch3 —

H

ch2.gh3



ch3

OH

h

H H g-

H

H

H

H

*2

ch3

HHg

CEj

OH

Ej

Growth

R^ Growth

*1

-





%

— H



The pyrim idines were te s te d in the presence o f an excess o f the th ia z o le h a lf and v ic e -v e r sa .

A study o f t h is ta b le w il l show that

replacement o f the amino group on the pyrimidine rin g o f the thiamine m olecule by an OH group renders the molecule in a c tiv e fo r growth o f Staph, aureus.

I t w i l l be r e a liz e d on the b a sis o f the work to be

reported in tl^is s e c tio n th at t h is compound, which, o f course, i s

59 r oxythiamine, i s not only in a c tiv e as a growth fa cto r hut at proper con­ cen tra tio n s i s an a c tiv e and potent in h ib ito r o f Staph, aureus growth. This in h ib ib itio n by oxythiamine was found to be considerably more pronounced than that produced by neopyrithiam ine.

The r o le of the

amino group thus becomes An a l l Important question.

That the amino

group was e s s e n tia l fo r a c t iv it y was e sta b lish ed in the work of Knight and Mcllwain already mentioned and in that by S in c la ir (39) w ith Phvcomyces blak esleean us.

This organism i s in many resp ects very

sim ila r to Staph, aureus in i t s requirements. In th is connection, and as regards in h ib itio n of such organisms, i t would ob viou sly be o f great value to study the e f fe c t on Staph, aureus o f the hydroxy d eriv a tiv e o f neopyrithiam ine.

I f the amino

group o f neopyrithiamine could be removed by n itrous acid gases, as was found for thiamine in the preparation o f oxythiamine, th is compound should give some in sig h t in to th is problem.

This reaction

w ith neopyrithiamine i s at present being stu d ied in the hope of ob­ ta in in g oxy-neopyrithiam ine. In the e a r ly work o f Woolley and White (kO) with pyrithiam ine, they esta b lish ed a s a tis fa c to r y method o f expressing the e f fe c t of antithiam ines on microorganisms.

They employed an in h ib itio n index

to express t h is ca p a city , which was the r a tio o f ant i t hiamine/thiamine which caused a 50> in h ib itio n o f growth in comparison w ith the growth obtained without the an tagon ist.

It was observed by these

workers th a t, in general, the index found could be correlated w ith the requirements o f the organism.

In the case o f those organisms

which required in ta c t thiamine for growth, in h ib itio n in d ices in Lthe range o f 7 to 19 were obtained.

With organisms such as Staph.

aureus. which had l e s s strin gen t requirements in so far as they could u t i l i z e the two fragments o f the thiamine m olecule, pyrithiam ine was found to give an index o f 2000. Soodak (3 ) studied the action of oxythiamine on Staph. aureus. and observed that in t h is case the general m icrob iological pattern se t up by Woolley was not adhered t o .

An in h ib itio n index o f about

40 was obtained with oxythiamine fo r t h is organism*

This fin d in g was

s ig n ific a n t fo r i t represents a rev ersa l in the comparative t o x ic it ie s o f oxythiamine and neopyrithiamine* It was the purpose o f t h is work to confirm th is fin d in g and to elaborate upon t h is , employing the newly synthesized neopyrithiamine in a comparative study with oxythiamine and oxythiam ine-diphosphate. The method employed by Woolley (40) for expressing the in h ib itio n index was adopted in th is work. Experimental The basal medium employed for Staph, aureus was that used by Woolley (4 0 ), which was so made up so that 10 ml. o f the medium contained 0.01 gamma of thiamine per ml.

In a l l o f these experiments

11 experimental tubes were se t up including a blank as a check on s t e r i l i t y and a con trol which gave optimum growth.

The general

plan o f th ese stu d ie s was to maintain the amount o f thiamine con­ stan t at a le v e l o f 0.01 gamma per ml. o f medium and to a lt e r the concentrations o f the a n tivitam in s.

In order that a comparison could

be made betiireen the d iffe r e n t antithiam ines employed, the concen­ tr a tio n s were always used on the same molecular b a s is .

The concen­

tr a tio n s o f thiamine and antithiam ines employed in each case, cor­ responding to the experimental tube numbers are shown in the follow in g

scheme • Tube Ho.

Thiamine gamma/ml

1

0*01

2

Oxythiamine garama/ml.

Oxythiamine Diphosphate gamma/ml.

Heopyrithiamine gamma/ml.

U racilThiazole gamma/ml.

0 .0

0 .0

0 .0

0 .0

0.01

500

775

650

500

3

0.01

50

77.5

65

50

k

0.01

10

15.5

13

10

5

0.01

5

7.75

6.5

5

6

0.01

1

1.55

1 .3

l

7

0.01

0.50

©.78

0.65

0.50

8

0.01

0.10

0.15

0.13

0.10

9

0.01

0.05

0.078

0.065

0.05

10

1 .0

1 .0

1.55

1.3 0

1.0 0

11

Blank-------

Control

The experiments were carried out in Evelyn tubes which served the tw ofold purpose o f culture and colorim etric tubes*

In a l l cases two

in d iv id u a l runs were made on d iffe r e n t days w ith a fresh culture of Staph* aureus*

The b asal medium was p ip etted in to the s t e r i l e tubes

under s t e r i l e co n d itio n s, and t h is was follow ed by the ad dition o f the an tim eta b o lites in varying concen trations.

The f in a l volume

was adjusted to 10 ml* and the tubes then inoculated w ith a 2h hour growth o f Staph, aureus which had been washed three times with water and then suspended in s t e r ile water u n t il the suspension was very f a in t ly tu rb id .

One drop o f th is suspension was employed in

each tube except the blank.

The tubes were incubated at 37°C for

19 to 20 hours a fte r which time they were read in an Evelyn photo­ e l e c t r i c colorim eter w ith a 5^0 f i l t e r .

The tubes were then placed

back in the incubator and l e f t fo r a t o ta l of 40 hours, a fte r which time the tubes were read again. Table XII E ffect o f Oxythiamine on the Growth o f Staph, aureus. Oxythiamine gamma/ml.

Thiamine gamma/ml.

19 hour Galv. Heading Sun #1

20 Hour Galv. Heading Hun #2

40 Hour Galv. Heading

0 .0

0.01

64

63

52

500

0.01

96

95

53

50

0.01

95

96

55

10

0.01

95

94

53

5

0.01

94

96

51

1

0.01

93

90

52

0 .5 0

0.01

89

86

4l

0.1 0

0. 01

73

76

31

0.05

0.01

71

75

32

1 .0

1 .0

66

68

32

These r e s u lts show a marked e f f e c t of oxythiamine on the growth o f Staph, aureus in 19 to 20 hours incubation. fo r oxythiamine o f 45 i s obtained.

An in h ib itio n index

From the data i t i s a lso seen

that the marked in h ib itio n by oxythiamine i s e a s ily and com pletely overcome by the organism on prolonged incubation.

The r e s u lt s a lso

show that the in h ib itio n i s r e a d ily overcome by the ad d ition of fu rth er thiam ine.

A fu rth er in te r e s tin g point is apparent from the

data obtained w ith the lower le v e ls of oxythiamine, namely that on prolonged incubation a stim ulatory e f fe c t i s observed.

The organism

appears to have the cap acity o f s p lit t in g oxythiamine and u t iliz in g Lthe th ia z o le moiety for growth.

63 i

r

Sim ilar experiments were set up w ith the other an tagonists mentioned in the previous general scheme.

The concentrations employed

were those shown in the scheme and represent concentrations o f oxy­ thiam ine-diphosphate, neopyrithiam ine, and the u r a c il-th ia z o le d e riv a tiv e , ca lcu la ted on a molecular h a sis w ith regard to oxythiamine.

The

r e s u lts obtained w ith th ese three d e riv a tiv e s are shown in Table X III. fa b le XIII E ffe ct o f Oxythiamine-^iphosphate, Neopyrithiamine and the U racilThiazole D erivative on the Growth o f Staph, aureus. Tube No.

19 Hour Galv. lea d in g s. OBjDlPQjj,

NPB]_

UT.

40 Hour Galv. Headings QEjDiPO^

NPB^

1

6l

61

66

53

52

2

98

98

69

60

78

3

93

96

70

60

61

k

90

93

66

5k



5

90

79

63

48

51

6

90

72

62

50

55

7

86



64

55

5k

8

79

67

62

3k

51

9

72

65

60

k3

51

10

70

68

6k

36

60

As was found in vivo w ith mice, oxythiamine-diphosphate seems to e x h ib it the same order o f t o x ic it y as oxythiamine.

In the case o f

oxythiamine-diphosphate an index of about 30 was obtained, w hile neo­ pyrithiam ine was found to g ive an in h ib itio n index o f 1300.

It was

found that both in the case of oxythiamine and oxythiam ine-diphosphate, L

j

the in h ib itio n index fo r Stash* aureus was s ig n if ic a n tly lower than th at obtained w ith neopyrithiam ine.

It was a lso observed th a t, although,

the e f f e c t o f oxythiamine i s more pronounced than that o f neopyrithiam ine, i t s e f fe c t a fte r 40 hours incubation did not la s t as lon g.

The organism

can apparently overcome the in h ib itio n by oxythiamine more rea d ily than th at caused by neopyrithiam ine. The data a lso in d ica te that the u r a c il-th ia z o le d eriv a tiv e employed in t h is work was not in h ib ito ry even at very high concentrations. This agrees w ith the observations made with mice.

65 r

n Chapter IX.

E ffe ct o f Thiamine Antagonists on the Growth of L a ctob acillu s fermentum.

A second s e r ie s o f m icrob iological stu d ies was concerned with L a cto b a cillu s fermentum. an organism requiring the in ta ct thiamine mole­ cu le fo r normal growth.

A second important c h a r a c te r istic of th is

organism i s i t s growth behavior when thiamine or cocarboxylase i s sup­ p lie d as n u tr ie n t.

On a m olecular b a sis cocarboxylase causes a 25 to

30^ b e tte r growth than thiam ine.

In t h is work the e f fe c t o f oxythiamine,

oxythiamine-diphosphate and neopyrithiamine was studied in the presence o f both growth fa c to r s . In a very in te r e s tin g study by Sarett and Gheldelin ( 4 l) w ith t h is organism, i t was found that pyrithiam ine was more to x ic to the organism when cocarboxylase was supplied than w ith thiam ine.

They obtained an

in h ib itio n index o f 50 when thiamine was supplied as the growth fa c to r , said an index o f 10 w ith cocarboxylase.

On the b a sis o f t h is work,

S arett and C heldelin proposed the idea that fr e e cocarboxylase was not a normal interm ediate in the u t iliz a t io n o f thiamine in the case o f L. fermentum. but rather that thiamine before phosphorylation could combine w ith the apoenzyme to form an u n d issociated enzyme system.

They reasoned that i f thiamine was converted to cocarboxylase

before being attached to the p rotein , i t would be in h ib ited to an equal or greater degree than was observed fo r coparboxylase.

This

work o f Sarett and C haldelin, as i s that of Woolley* s i s subject to c r itic is m at le a s t q u a n tita tiv e ly on the b a sis of the p u rity of the pyrithiam ine employed.

L

66 r

~\ Experimental The "basal medium of Sarett and Cheldelin (42, 4$) was employed fo r preparing the inoculum and for the growth stu d ie s.

The organism was

kept in stah cu ltu res in the ice-h ox and d a ily inocula were prepared as needed.

A very d ilu te suspension o f the organism in s t e r i l e sa lin e

was used for in o cu la tin g the tubes.

The readings in a l l cases were

o made a fte r 18 hours incubation at 37 C and the growth was measured tu r b id im e tr ie a lly w ith a 5^0 f i l t e r . A set of 20 experimental tubes were used for each s e r ie s , employing varying amounts of thiamine and cocarboxylase and three le v e ls o f the a n tiv ita m in s.

The thiamine and cocarboxylase were p ip etted in to s t e r i l e

Iv ely n tubes and th is was follow ed by the an tivitam in s.

S te r ile water

was added in appropriate amounts to make a t o t a l volume o f 5* 0 ml. Five ml. o f the b asal medium was then added to each tube and the tubes steamed and in ocu lated w ith the exception o f the blanks.

The readings

in a l l cases are given in terms of the o p tic a l d en sity or 2 -lo g . galvanometer reading. The data obtained with oxythiamine employing both thiamine and cocarboxylase as growth fa c to r s are shown In Table XIV#

L

67 Table XIV The E ffe ct o f Oxythiamine on the Growth o f Staph, aureus Gamma Qxythiamine per tube

0.00

Thiamine-Gamma per Tube 0.01 0.02 0.01

0.0 4

0.00

0.036

0.161

0.252

0.328

0.409

0.10

0.027

0.168

0.269

0.328

0.398

0.30

0.009

0.174

0.276

0.310

0.420

1 /00

0.004

0.013

0.155

0.^84

0.444

Gamma Oxyt h i amine per Tube

Cocarboxylase-Gamma per Tube 0 .0 0

0.01

0.02

0. Q3

0.04

0.00

0.032

0.208

0.301

0,347

0.432

0.10

0.032

0.149

0.276

0.328

0.444

0.30

0. 004

0.027

0.066

0.268

0.398

1 .0 0

0.004

0,009

0.009

0.013

0.022

Employing thiamine as a growth fa c to r , the in h ib itio n with oxy­ thiamine i s q u ite pronounced and even more so when cocarboxylase i s used.

In the presence o f thiam ine, oxythiamine i s found to give an

index of 37 fo r h a lf maximal in h ib itio n .

I f we compare the growth

w ith thiamine and coearboxylase in the absfence o f any in h ib ito r , the organism i s found to ex h ib it approximately 25% b e tte r growth w ith cocarboxylase than with thiam ine, when compared on a molecular b a s is .

At the same time i t i s found that oxythiamine i s more to x ic

in the presence o f cocarboxylase, in which case an index o f 10 was found. Sim ilar stu d ies were made w ith oxythiamine-diphosphate and with neopyrithiam ine.

In th ese experiments, the concentrations of the

Lan tagon ists were calcu lated on a molecular b a sis to oxythiamine.

The

r

n fin d in g s are shown in Tables XV and XVI* Table XV E ffec t o f Oxythiandne-Biphosphate on the u t iliz a t io n of Thiamine and Cocarboxylase by L actob acillu s fermentum Gamma of OBxDiPO^ x>er tube

0.00

Thiamine-Gamma per Tube O.OOL 0.02 0.03

0.04

0.00

0.046

0.161

0.237

0.337

0.432

0.16

0.018

0.137

0.222

0.347

0.398

0.47

0.009

0.081

0.237

0.319

0.409

1.55

0.004

0.013

0.155

0.301

0.432

0.00

Cocarboxylase-Gaimna per Tube 0.04 0.01 0.02 0.03

0.00

0.036

0.215

0.319

0.367

0.444

0.16

0.027

0.081

0.237

0.347

0.409

0.47

0.013

0.018

0.056

0.222

0.367

0.009

..0 * 0 0 9 ....

0.009

0.018

Gamma o f OBjBiP©/* per Tube

.........

L

JI

S ab le XVI

E ffe c t o f Neopyrithiamine on the U tiliz a tio n o f Thiamine and Cocarboxy­ la s e by L actob acillu s fermentum Gamma o f NPB^ per Tube

0.00

Thiamine-Gamma per Tube 0.01 0.02 0.03

0.04

0.00

0.041

0.155

0.260

0.347

0.398

0.12

0.022

0.076

0.222

0.310

0.377

0.36

0.013

0.056

0.125

0.222

0.319

1.20

0.004

0.004

0. 004

O.O56

0 .U 9

Cocarboxylase-Gamma per Tube Gamma Neopyri­ >.00 0.01 0.02 0.03 thiamine per Tube C

0 .0 4

0.00

0.Q46 0.208

0.301

0.328

0.444

0.12

0.036 0.009

0.056

0.181

0.347

0.36

0. 018 0.009

0.046

0.086

0 .U 4

1 .2 0

0.009 0.004

0.004

0.009

0.013

A study of the data fo r oxythiamine-diphosphate and fo r neo­ pyrithiam ine shows that a s ig n ific a n t to x ic e f f e c t i s a lso obtained w ith oxythiam ine-diphosphate.

In t h is case, an index of 4? i s found

w ith thiam ine, and an index o f 15 when the organism i s supplied w ith c©carboxylase.

We a lso n o tic e th at usin g fr e sh cu ltu res o f L. fermentum

in the absence o f any in h ib ito r , the growth curves arb p r e tty w ell reproducible, that i s , 20 to 25$ b e tte r growth with cocarboxylase than w ith thiam ine. The experiments w ith animals had demonstrated that neopyrithiamine was considerably more to x ic than oxythiamine.

This p ictu re was com­

p le t e ly reversed in the stu d ie s w ith Staoh. aureus in which case oxy-

70

thiamine was found to he ah out JO tim es more to x ic than neopyrithiamine fo r an 18 hour growth period.

The present s e r ie s of experiments with

L. fermentum again e sta b lish ed neopyrithiamine as more to x ic than oxy­ thiam ine.

In t h is case in d ices w ith thiamine and cocarboxylase, em­

ploying neopyrithiam ine, o f 10 and 5* r e s p e c tiv e ly , were obtained.

It

would appear from the work w ith mice and from that with Staph, aureus and L. fermentum that neopyrithiamine i s more to x ic than oxythiamine fo r those organisms requiring in ta ct thiamine for growth. It was a lso found with the three in h ib ito r s employed that the in h ib itio n by th ese d e r iv a tiv e s was s p e c if ic , for in a l l cases the in h ib itio n was r ea d ily overcome by the organism in the presence of excess thiamine or cocarboxylase. D iscu ssion A ll o f the evidence seems to in d ica te th at in the case of the two w idely used thiamine a n ta g o n ists, oxythiamine and neopyrithiam ine, the mode of in h ib itio n i s e s s e n t ia lly d iffe r e n t.

In the case of oxythiamine

we are dealin g w ith a stru ctu ral analogue o f thiamine in which the amino group has been replaced by a hydroxyl group.

Neopyrithiamine represents

a stru ctu ra l a lte r a tio n in which the th ia z o le alcoh ol has been replaced by an equivalent pyridine a lco h o l.

The two compounds show a d iffe r e n t

s e le c t i v i t y o f a ctio n as was demonstrated w ith mice and L. fermentum on the one hand and w ith S. aureus on the other.

The r e s u lts o f the

experiments in vivo in d icated that the point of in h ib itio n w ith oxy­ thiamine was through the th ia z o le alcoh ol grouping. The work o f S arett and G heldelin ( h i ) in d icated the importance of the amino group in the in h ib itio n o f L. fermentum. fo r they obtained in h ib itio n a lso w ith higher concentrations o f ju st amino-pyrimidine

71 r d er iv a tiv e s but not w ith u racil#

i However, com petition through the

amino group i s not the only point fo r in h ib itio n and t h is i s borne out by the very to x ic e f f e c t s o f oxythiamine and oxythiamine diphosphate# Contrary to the b e l i e f of S arett and Cheldelin an amino pyrimidine i s not e s s e n tia l to obtain in h ib itio n w ith L» fermentum.

The th ia zo le

moiety in t h is resp ect i s o f obvious sig n ific a n c e sin ce hydroxy pyri­ midines alone did not in h ib it#

L

J

72 r

i

Part Hr The Action o f Thiamine A ntagonists on Certain Enzyme Systems Requiring Thiamine or Gocarboxylase Chapter X,

Urea Synthesis in Rat and Mouse Liver S lic e s . The E ffect of Thiamine and i t s In h ib itio n by Oxythiamine.

Among the many p h y sio lo g ic a l ro le s o f thiam ine, i t s fu n ction with resp ect to urea sy n th esis in rat liv e r s li c e s has recen tly been report­ ed by Leuthardt (4 4 ).

In the case of t is s u e from r a ts p reviou sly made

d e fic ie n t in thiam ine, urea sy n th esis i s markedly reduced.

When

thiamine was added to such t is s u e s , urea formation was restored to normal.

This observation i s in lin e w ith the much more comprehensive

stu d ies o f Barron (45) on the ro le o f thiamine in rat t is s u e s w ith resp ect to carbohydrate sy n th e sis.

I t was further observed by

Leuthardt that a d d itio n o f thiamine to liv e r s li c e s from normal animals showed no augmentation of urea sy n th e sis.

Several experi­

ments were carried out by t h is worker w ith thiamine-monophosphate and cocarboxylase, and i t was observed that th ese e s te r s had but a s lig h t e f fe c t on urea s y n th e sis,

n e v e r th e le ss, the ro le o f thiamine

in such a system must be a ttrib u ted d ir e c tly to the metabolism o f pyruvate a ctin g as su b str a te.

The observed d ifferen ce between

thiamine and cocarboxylase can be a ttrib u ted to a phenomenon o f d iffu s io n .

This concept i s borne out by the la te r work by Eahrlander

and Leuthardt (4 6 ), in which they studied th is mechanism employing rat liv e r homogenates.

When liv e r homogenates of thiamine d e fic ie n t

r a ts were employed, the observations were reversed.

With pyruvate

and ammonium ch lorid e as su b stra te, urea formation was g r e a tly gngmented w ith the ad d ition o f cocarboxylase whereas thiamine had l i t t l S j

a c t iv it y .

This observation was o f obvious s ig n ific a n c e and c le a r ly

Indicated the p h y sio lo g ic a l ro le played by the cocarboxylase.

The d if ­

feren ce in the behavior, o f the liv e r s li c e s and that of the homogenates was a lso s e l f ev id en t.

The d ifferen ce in the d iffu sio n ra tes of

thiamine and eocarboxylase i s elim inated when liv e r homogenates are employed.

The greater d iffu sio n rate o f thiamine in the case o f liv e r

s l i c e s , can be a ttrib u ted to the sm aller nature o f the molecule and to the d ifferen ce in the io n ic charges in thiamine and cocarboxylase. Ex-peri mental Prelim inary experiments were earried out w ith young Sherman rats using low r a tio s o f thiam ine/oxythiam ine.

The manometric method o f

Irebs and H en seleit (4 ? ) was employed in t h is work fo r follow in g urea sy n th e sis.

The lim ita tio n o f the manometric method as to the number

o f determ inations that could be run at one tim e, led to the adoption o f the xanthydrol co lo rim etric procedure for determining urea.

This

procedure had been developed by Engel and Engel (48) for q u an titative work for use w ith t is s u e s .

It was compared w ith the manometric

method, and the two a n a ly tic a l procedures were found to g iv e com­ parable r e s u lt s .

For use in t is s u e work the xanthydrol method was

modified w ith regard to the prelim inary removal of p ro tein .

Engel

and Engel found that tu n g stic acid p r e c ip ita tio n of the p rotein was s a tis fa c to r y fo r th is purpose.

In th is work 10$ tr ic h lo r a c e tic acid

was employed and found to give good r e s u lt s .

Standard so lu tio n s with

known concentrations o f urea were s e t up in the same manner as the experim ental tu bes. g e r ie s A.

Orea Synthesis w ith Eat Liver S lic e s

74

Weanling Sherman Rats were put on the thiamine d e fic ie n t C-28-A d ie t u n til th e ir growth curves in d icated a severe thiamine d efic ie n c y . At t h is time the animals were s a c r ific e d and t is s u e s li c e s prepared. The liv e r s l i c e s were suspended in a l l cases in Krebs-Binger phosphate b u ffer o f pH 7*4 to which was then added the sub strate and t e s t sub­ stance or substances under examination.

The f i r s t experiments with

t h is enzyme system were made w ith low r a tio s o f thiam ine-oxythiamine. In a s e r ie s o f 20 experim ental tu bes, employing r a tio s of l / 3 and l / 6 , at most only a s lig h t e f fe c t of oxythiamine was observed.

N evertheless,

the observation ©f Leuthardt (46) w ith d e fic ie n t rat liv e r s lic e s was confirmed.

Severe thiamine d e fic ie n c y in the rat caused a s ig n ific a n t

lowering o f urea sy n th esis as compared to that shown by controls* Addition o f 100 gamma o f thiamine chloride or thiamine iodide augment­ ed the urea sy n th esis by about 30^.

The thiamine iodide employed in

th is work was synth esized by the method o f Gerecedo and Tolpin (4 9 ), and was found to be b io lo g ic a lly a c tiv e as the chlorid e sa lt* It was apparent from th ese experiments that optimum cond ition s were not being used.

In a fu rth er s e r ie s of experiments the le v e l of oxy­

thiamine was increased to r a t io s o f 1/40 and both normal and d e fic ie n t rat liv e r s l i c e s were employed. With the manometric method fo r determining urea, 7 experimental Warburg v e s s e ls were se t up w ith d e fic ie n t liv e r s li c e s in th e f o l­ lowing manner:

L

75 r

-I

Y essel Ho*

Contents

3

Thermobarometer-3• 5 sal* o f Water

5

Blank-3.5 ml.

2

T.S.-f 3*0

ml. Buffer-t-0.5

ml. Substrate

2a

T.S.-+-2.5

ml* B u ffers-C .5

ml. Substrate+BiCl

1

T.S.+- 2 .5

ml. Buffer-V 0*5 ml. Substrate+B^I

U

T .S .+ -2 .0

ml. B u ffe r -*-0*5 ml. Substrate+BiCl+OB^

2b

B uffer pH 7A

___________ T .S .4 -2 .0 ml. Buffer + 0*5 ml. Substrate-vBi I-H3Bi_____

Two separate runs were se t up employing t h is technique in which thiamine ch lorid e and iodide were used at le v e ls at a le v e l of 100 gamma per Warburg v e s s e l.

Oxythiamine was added at a concentration o f 4 .0 mg.

per Warburg v e s s e l.

The f in a l volume in a l l cases was adjusted to

3*5 ml.

These suspensions were incubated d ir e c tly in Warburg v e s s e ls

at a 38°C fo r two hours. to constant w eight.

The t is s u e s lic e s were then removed and dried

Immediately follow in g removal of the tis s u e s l i c e s

the urea was determined w ith the aid of a p u r ifie d preparation o f urease. X 7II.

The r e s u lts obtained in two runs are summarized in Table The data are expressed in terms o f cu. mm. o f COg obtained in

each case per mg. o f dry li v e r t is s u e .

L

76 r

~\ Table XVII

Urea Synthesis in D eficien t Rat Liver S lic e s and I ts In h ib itio n by Oxy____________________thiam ine._______________ __________________

. Substrate:

Ammonium Chloride (.0066M) and Pyruvate (.0 0 9 $ ) 0. C0g/mg. dry weight

Exp. Bo.

Bo B]_

SjCl

B il

BjCli-aB].

B^I*V- ©B^

1

4 .7

14.2

130

5*6

6 .9

II

4 .2

1 4 .2

_ _12,3....

2L ................

6 .6

Employing the more p r a c tic a l colorim etric method, th ese stu d ies were continued and extended w ith both normal and d e fic ie n t rat liv e r s lic e s *

The experiments were carried out with young Sherman ra ts as

described b efore.

In ad d ition to oxythiamine, in th ese experiments

the ro le o f c y ste in e and methionine was studied sin ce th ese su lfu r amino acid s were reported by Leuthardt (50) to in h ib it t h is syn th esis w ith normal ra t l i v e r s l i c e s . Two id e n tic a l s e r ie s o f experiments were set up w ith the two types o f t is s u e s .

In a l l cases the substrate was ammonium chlorid e and sodium

pyruvate which gave the b est r e s u lt s .

The manner in which the experi­

mental tubes were set up i s shown i n t h e follow in g scheme: 1)

T.S. 4-Substrate

2)

T.S*4- Substrate 4*4.0 mg. Q3±

3)

T.S. 4- Substrate 4-3 0

Cysteine

4)

T.S. -4 Substrate 4-30

Methionine

5)

T.S. 4* Substrate «i-100Y B^

6)

T.S.

7)

T.S. 4-S u b strate4-100 VB^+Cysteine (30 ;oM)

4-

Substrate +100 JfBi+OBi (4 .0 mg.)

8*} T.S. 4- Substrate + 100 VB^M ethionine (30 uH)

77 r

-i In a l l th erefo re, 16 tubes were se t up, 8 w ith normal tis s u e s and

8 w ith d e fic ie n t t is s u e s ,

The tubes were incubated at 38°C fo r one hour

and f i f t y minutes a fte r which time the liv e r s li c e s were removed and d ried in an oven*

Two ml, o f each o f the incubation mixtures was placed

in cen trifu ge tubes and 2 ,0 ml. of 10$ tr ic h lo r a c e tic acid added.

This

was allowed to stand at room temperature for 3® minutes, and the p rotein cen trifu ged down.

Two ml. o f the supernatant in each case was taken

and treated w ith two ml. o f g la c ia l a c e tic acid and then 0 .2 ml. of xanthydrol so lu tio n . 24 to ^8 hours.

The tubes were then put in the ic e box fo r

The p r e c ip ita te which formed in each tube was ce n tr i­

fuged down and washed according to the method o f Engel and Ih g el u n t il the washings gave a negative t e s t fo r excess xanthydrol w ith 50$ s u lfu r ic a cid .

A fter the f in a l washing the tubes were drained and

the color produced w ith 10 ml. o f 50$ s u lfu r ic acid .

The readings

were made in an Evelyn p h o to e le c tr ic colorim eter w ith a kZO f i l t e r . The r e s u lts obtained w ith both d e fic ie n t and normal liv e r s lic e s are summarized in Table XVIII.

For the sake o f convenience abbrevia­

tio n s are used in the ta b le fo r oxythiamine, cy stein e and methionine. Table XVIII Urea Synthesis in D eficien t and Normal Hat Liver S lic e s . Substrate:

Ammonium Chloride (.0Q66M) and Pyruvate (.009M) Gamma Urea E/mg. Dry Weight No By OBi

Cys.

Met.

B1

D eficien t S lic e s

1 2 .6

10.6

12.1

12.1

18.5

15 .8

17.9

18.0

Normal S lic e s

16.1

8 .0

1 1 .0

10.3

16. h

15 .8

12.1

11.5

L

Bi+OBi

Bi+Gys.

Bi+Met.

D iscussion The ad d ition o f thiamine to normal liv e r s l i c e s i s found to have l i t t l e or no e f f e c t on urea sy n th e sis,

This i s to he expected sin ce

c e l l s are b eliev ed to syn th esize only enough coenzyme to saturate the apoenzyme.

The ad d ition o f 12 pH o f oxythiamine to the normal liv e r

s l i c e s brought about a 5®$ decrease in urea sy n th esis as compared to the normal valu e.

Cysteine and methionine reduced the amount o f urea

by 31 and 36$ r e s p e c tiv e ly when added at a le v e l o f JO jolM* Addition o f further thiamine had an in s ig n ific a n t e f f e c t in overcoming t h is in h ib itio n . When thiamine d e fic ie n t rat liv e r s li c e s were used, the p ictu re was somewhat d iff e r e n t .

The importance o f thiamine in t h is mechanism

i s seen in the fa c t that a severe thiamine d e fic ie n c y o f i t s e l f , re­ duced the amount o f urea in comparison to normal liv e r s l i c e s , by 21$. We fin d a lso that the e f fe c t o f a l l three in h ib ito r s i s l e s s pro­ nounced.

With d e fic ie n t s l i c e s , oxythiamine, when added at a le v e l of

12 pH was found to produce on ly a 16$ in h ib itio n .

Cysteine and

methionine under th ese con d ition s had an in s ig n ific a n t e f f e c t . Comparative r e s u lts were obtained w ith oxythiamine w ith the manometric method fo r urea. found in th is case.

L

An average figu re of 6 l$ in h ib itio n was

79 r S eries B.

i Urea Synthesis in Mouse Liver S lic e s

Sim ilar experiments w ith hoth normal and d e fic ie n t mouse liv e r s l i c e s demonstrated that a lik e phenomenon occurs w ith t h is sp e c ie s. Prelim inary to studying the e f f e c t o f thiamine in mouse liv e r s , several determ inations were made to a scerta in the thiamine content o f the liv e r s o f hoth normal and d e fic ie n t mice.

The thiamine determ inations were run

hy the complete thiochrome method of Hennessy (51) and Cerecedo and Hennessy (5 2 ),

Young Rockland mice were used in th ese experiments.

Two animals were kept on stock d ie t, and two animals were placed on the thiamine d e fic ie n t d iet u n t il they developed a moderate thiamine d e fic ie n c y .

A fter the mice were s a c r ific e d , the whole liv e r was

removed, washed w ith d i s t i l l e d water, dried w ith f i l t e r paper, and weighed.

The l i v e r was then homogenized w ith 0.1K HgSOjj,.

The f in a l

volume was brought to 75 ffll* w ith 0.1H HgSO^ and the ex tra ctio n car­ ried further hy treatment in a pressure s t e r i l i z e r for 30 minutes. The liv e r suspensions were allowed to cool and 0.1 g. each o f takad ia sta se and papain in 7 ml. of 2.5M sodium a ceta te was added to give a pH o f ahout 4 .6 .

The enzymic d ig e stio n was carried out at t h is pH

at 54 °G fo r three hours.

The liv e r suspensions were then transferred

to 100 ml. volum etric fla s k s and brought to volume w ith water*

A fter

mixing, the so lu tio n s were f ilt e r e d and 50 ®1* o f the clea r f i l t r a t e were passed over Decal so. the Decalso treatm ent.

At the same time a strandard was run w ith

The procedure follow ed from th is point was as

in the regular thiochrome method. For the normal mice on stock d ie t , valu es of 13 and 15 gamma of thiamine per gram o f wet l iv e r was found.

Mice maintained on the

(5728-A d ie t u n t il thiamine d e fic ie n t gave valu es of 1.5 and 2 .0 gamma/g.j

80 r wet liv e r .

These valu es are in agreement w ith the few valu es reported

in the lite r a tu r e fo r mouse liv e r .

I

Soodak (3)* reported fo r normal mice,

valu es ranging from 1 0 .2 to 15*5 gamma per gram wet w eight.

Singher

(5 3 ) reported v alu es obtained w ith three mice on a stock d ie t and found values o f 4 .5 to 16.1 gamma o f th iam in e/g/ wet w eight.

In con­

n ectio n w ith th ese stu d ies o f urea sy n th esis, i t i s apparent that the resid u a l amount o f thiamine remaining in an approximately 10 mg. liv e r s l i c e would he very small* In these experiments w ith normal and d e fic ie n t Rockland mice, the experimental co n d ition s fo r the xanthydrol method were the same as described p rev io u sly .

The l e v e l of thiamine was held constant at

100 gamma, w hile the concentration of oxythiamine was varied .

In a l l

ca ses sodium pyruvate and ammonium ch loride served as su b stra te.

The

normal liv e r s were run sim ultaneously w ith the d e fic ie n t ones and a s e t o f 20 experim ental tubes were set up.

The scheme and general

plan in which the experiment was se t up was as follow s:

L

-1

81 i

D eficien t Mice

Normal Mice 1)

Control

13 )

Control

2)

OB^ ( 1*5 m g.)

14)

B^Cl (100 Gamma)

3)

OB^ ( 2*5 mg* )

15)

B^I (100 Gamma)

4)

OB^ (4*0 mg* )

16)

B^-t-OB^ ( 1*5 mg*)

5)

Cysteine (30 piM)

1?)

( 2 .5 mg* )

6)

Methionine (30 jiM)

18)

B1 +OB1 (5»0m g*)

( j 7)

i v u vGamma) s m c i/ Br \(100

19)

21*4- Cysteine (30 pM)

8)

Bi-4-OBi (1*5 mg. )

20 )

B^ + Methi onine (3 0 jaM)

9)

Bj4-G©2. (2«5 mg.)

1 0 ) %4-OBi ( 4 .0 mg.) 1 1 ) B i +-Cysteine (30 jiM) 12 ) Bx +• Methionine (30 pM) The tubes were incubated fo r one hour and 45 minutes, the liv e r s l i c e s removed and dried to constant w eight.

Two ml. a liq u o ts were

taken, trea ted w ith 2 .0 ml. o f 10$ tr ic h lo r a c e tic acid and urea deter­ mined as Before hy the xanthydrol method.

The data obtained w ith hoth

typ es o f liv e r s l i c e s are shown in Table XIX.

l

j

82

r

I Table XIX Urea Synthesis in D eficien t and Normal Mouse Liver S lices* Gamma Urea N/mg. Dry Weight No B^

B1

B il B1+OB1 1 .5

D efic ien t 10.5 15.3 14 .9 Mice No B^ OBi Gys.

13*5

BvVOB^ B^+OBx B^Cys. B^Met. 2 .5 Big* 4 Big. 9 .5

Met.______ B-)

8 .4 Bj4 0B-| Bi-vQys.

15*0

14. 9

B]+-Met.________

Normal 13*9 6 .3 1 4 .2 12.8 14.6 13.7 14.9 14 .4 ___________________________________________________________________ Mice The ad d ition of thiamine gave no s ig n ific a n t increase over that o f the normal v alu e.

A lso,

in t h is case cy stein e and methionine showed at

most a s lig h t e f f e c t on the urea

syn th esis by normal s l i c e s . On the

contrary, oxythiamine was found to have a pronounced e f f e c t on th is sy n th e sis.

At the three l e v e ls employed, oxythiamine was found to give

25, 55* and 70$ in h ib itio n ^ , r e sp e c tiv e ly . 4s

in the case

w ith rat T issu e, the behaviour of the d e fic ie n t

s l i c e s was somewhat d iffe r e n t.

With d e fic ie n t mice the e f fe c t o f oxy­

thiamine was considerably l e s s pronounced and th is might be expected. At the concentrations used, oxythiamine gave 12, 38, and 45$ in h ib itio n s w hile cy stein e showed no e f f e c t , and methionine at most only a s lig h t e ffe c t. D iscussion Although the general observation o f Leuthardt with respect to the r o le o f thiamine in urea sy n th esis was confirmed in th is work, i t s fu nction in t h is system must be relegated to i t s s p e c if ic action on pyruvate.

It i s in t h is lig h t that the role o f oxythiamine can be

in terp reted .

In th is resp ect, p a r tic u la r ly s ig n ific a n t i s the work

o f Froilman and la y (3 2 ) w ith oxythiamine in r a ts .

Such animals re­

ce iv in g oxythiamine together w ith thiamine were incapable o f metabo­ l i z i n g pyruvate as judged by the marked r is e o f pyruvate in the blood. This o f course, in d icated that pyruvate metabolism was in some way sev erely blocked. In vitro^ work w ith liv e r s li c e s would bring us to the same conclusions and i t would th erefore be reasonable to expect that in vivo ra ts re ceiv in g oxythiamine would show a s ig n ific a n t drop in blood urea.

This i s further evidence that oxythiamine may in h ib it

tile v i t a l fu n ction in g o f thiamine or cocarboxylase as regards pyruvate metabolism, by e ith e r blocking the phosphorylation o f thiamine or by in h ib itio n o f cocarboxylase in the form of oxythiam ine-diphosphate.

84 r Chapter XI.

Studies on the Hole of Oxythiamine and Neopyrithiamine on

i

the Clam and Carp Thiamine Destroying Factor It was soon a fte r the work of Green, Carlson and Evans (54, 55) k&d shown that Chastek p a r a ly sis was a r e s u lt o f thiamine avitam in osis, that many workers in th is f i e l d took up the study to fin d how c erta in raw f i s h rendered thiamine u n availab le.

Mich of the work was carried out

in vivo w ith ch ick s, foxes and other sp ecies hut e s p e c ia lly s ig n ific a n t were the r e s u lts o f in v itr o experim ents.

Woolley (5&)* was able to

obtain a very a c tiv e extract from carp by ex tra ctin g the fresh tis s u e w ith 1 0 sodium ch lo rid e.

The nature of the in v itr o in a c tiv a tio n of

thiamine was studied by Sealock and coworkers (57)*

These early

stu d ies of Sealock in d icated that the in a c tiv a tio n o f thiamine by carp t is s u e was an enzymatic rea c tio n .

This concept received further sup­

port from the work o f Krampitz and Woolley (5 8 ), who is o la te d the products 4 -met h yl-5-be t ahydroxy eth y l th ia z o le and 2-me thy l~4~amino-5-hy dr oxmet hyl pyrimidine o f thiamine breakdown from a thiamine free mixture o f carp t is s u e incubated w ith thiam ine. The thiaminase a c t iv it y o f carp was found to be com p etitively in h ib ited by 0-am inobenzyl-4-m ethyl-thiazolium ch lorid e, which to a certa in extent i s s tr u c tu r a lly sim ilar to thiam ine.

In t h is work by

Sealock (59)» i t was observed that i f the amino group was su b stitu ted by a n itro group the r e s u ltin g compound lo s t i t s cap acity to in h ib it . A very important observation concerning the ro le or n e c e s sity of the amino group fo r in h ib itio n by such 11thiam ine-1 ik e ” compounds was made by Soodak and Gerecedo (2 7 ).

These workers, employing oxythiamine,

found that th is d eriv a tiv e a lso in h ib its the thiaminase o f carp.

On

four runs w ith one enzyme preparation, these workers found that 33* 46,

85 r

50 and 60$ in h ib ition s o f thiamine "breakdown were produced by the oxy­ thiam ine. Clams have a lso been found to contain a thiaminase fa cto r which i s capable o f attack in g both added thiamine and d ieta r y thiam ine.

Melnick

and coworkers ( 6 0 ) have shown that a d ie t containing raw clams markedly reduced the a v a ila b ilit y of d ieta ry thiamine as measured by the thiamine ex cretio n o f human su b jects. Hennessy and coworkers (61, 62) have shown that in the case o f the clam a sim ila r phenomenon may occur as in the carp as fa r as the s p l i t ­ tin g of thiamine i s concerned.

These workers have is o la te d the pyri­

midine d eriv a tiv e which i s formed.

As y et no d e t a ils o f th e ir work have

been reported. Experimental In t h is work the a ctio n o f oxythiamine and neopyrithiamine was stu d ied w ith both the fa cto rs present in clams and in carp.

Experi­

ments were f i r s t set up w ith an acetone dried clam preparation ob­ tain ed from Mr. Tenmatay o f the Organic Chemistry Department.

This

preparation was found in i n i t i a l experiments w ith Just thiamine to be very a c tiv e . S eries A The a c tiv e p r in c ip le was extracted from 2 .5 grams o f the clam acetone d ried powder by 5 ex tra ctio n s w ith water in a hand homogenizer.

The

f in a l volume was adjusted to 100 ml* so that k ml. of t h is extract was equivalent to 100 mgs. o f the o rig in a l powder.

This was the amount

used in a l l o f the incubation and in h ib itio n stu d ies w ith the clam p rin cip le*

This amount o f ex tra ct was incubated w ith 100 gamma -e£

86 o f thiamine in the presence o f phosphate "buffer of pH 7*4.

The experi­

mental scheme used i s summarized "below: Tube #1 4 ml* of Extract

4 ml. o f Extract

4 ml. o f Extract

Phosphate Buffer pH 7 .4 (0.2M)

Phosphate Buffer pH 7*4

Phosphate Buffer pH 7*4

100 gamma B^

100 gamma 3^

100 gamma B^ 400 gamma QB^

This rep resen ts the general plan in which the experiments were se t up w ith the clam p r in c ip le .

The le v e l o f oxythiamine used in those

stu d ie s was 400 gamma or at a le v e l o f 1 /4 in the presence o f 100 gamma o f thiam ine.

This le v e l o f r a tio was found to be very a c tiv e w ith the

carp p rin c ip le by Soodak and Cerecedo. for two hours at 37°C*

The tubes were then incubated

A fter t h is incubation period, 100 gamma of

thiamine was added to tube #2 which served as a co n tro l.

Five ml. o f

10$ tr ic h lo r a c e tic acid was added to a l l the tubes and they were per­ m itted to stand fo r 3© minutes.

A fter cen trifu g a tio n an appropriate

a liq u o t o f the supernatant was taken for a resid u a l thiamine a n a ly sis. In a l l cases the thiamine determ inations were made by the rapid th io ­ chrome method.

A 2 ml. a liq u ot was taken fo r the thiochrome an alysis

which represented 13*33 gamma o f o r ig in a l thiam ine.

The readings were

made in the P fa ltz and Bauer photoflourom eter w ith the follow in g galvanometer readings: Tube # 35

1

2

3 73

36

Ehder th ese con d ition s we fin d that a fte r 2 hours incubation, s lig h t ly over 5®$ or $0 gamma of the thiamine was destroyed by the

clam factor*

A lso, th at oxythiamine does not in h ib it the thiamine

d estru ctio n at the concentration used and under th ese con d ition s.

In

th ese experiments w ith the clam p r in c ip le , i t i s s ig n ific a n t that not a l l o f the thiamine i s destroyed, and th erefo re, the lack o f in h ib i­ tio n on the part of oxythiamine at the r a tio used i s tr u ly represent­ a tiv e . These experiments were repeated and neopyrithiamine was a lso includ­ ed, representing an amino pyrimidiia^ d eriv a tiv e in contrast to oxy­ thiam ine.

The experim ental procedure was the same as before and neo­

pyrithiam ine was included at a l e v e l o f l/A on a molecular b a s is .

In

t h is case the tubes were incubated fo r 3 hours at 37°C in stead of two hours*

Two ml. o f the supernatant was taken in each case for the t h io -

chrome a n a ly sis . Incubated B^

Control

20

72

j

Bi 4- OBi

B^4*0PB^

2k

over 100

A fter 3 hours incubation 72$ or 72 gamma o f the thiamine was destroyed by the clam fa c to r .

The observation w ith oxythiamine was

confirmed, although i t i s q u ite p o ssib le that much higher concentra­ tio n s o f oxythiamine and longer incubation periods would show an in­ h ib itio n o f thiamine d estru ctio n .

The r e su lt obtained w ith neopyri­

thiamine i s very in te r e stin g and c le a r ly shows that neopyrithiamine g iv e s the thiochrome test*.

This fa c t was then esta b lish ed with pure

so lu tio n s o f neopyrithiam ine, although no study was made to determine i f the compound could be q u a n tita tiv ely determined in th is way. observation was independently made by Tenmatay o f the Organic Chemistry Department.

This

88 r A fter completion o f t h is work, Sealock ( 6 3 ), in studying the e f fe c t o f neopyrithiam ine on the carp fa c to r , has found that the thiochrome t e s t could he q u a n tita tiv e ly used to determine t h is an tagon ist.

He made

the further observation that neopyrithiamine does not give the azoco lo r t e s t of Prebluda and McCollum (6h, 65) given by thiam ine, nor does i t in te r fe r e with th is t e s t . Due to t h is in terferen ce th erefore, i t was thought advisable to carry out the stu d ie s w ith lower concentrations o f neopyrithiam ine, and to run blank v a lu es on pure so lu tio n s o f th is compound.

Further ex­

periments were th erefore set up in which the amount of neopyrithiamine was reduced to le v e ls of 300 $ 200, and 100 gamma per tube.

In a l l

cases a blank was run on a pure so lu tio n of neopyrithiam ine, w ith amounts that would be equivalent to that present in the actu al t e s t . In th ese cases one ml. o f the supernatant was used for determining resid u a l thiamine and th is amount was equivalent to 6.66 gamma o f thiamine and m u ltip les o f t h is o f meopyrithiamlne.

Oxythiamine was

again included in th is s e r ie s o f experim ents. liable XX The E ffect o f Oxythiamine and Neopyrithiamine on the Thiamine D estroying Factor o f Clam Substance Tested

Concentration gamma

$ In h ib itio n

Oxythiamine

hoo

0

Neopyrithiamine

100

30

Neopyrithiamine

200

Neopyrithiamine

L

70

89 r

The concentration o f thiamine in a l l cases was maintained constant at 100 gamma per tube, and the readings were made immediately sin ce le s s in ter feren ce i s found from neopyrithiam ine when the flu orescen ce i s read immediately.

However, at high concentrations a s ig n ific a n t blank value

i s n ev erth eless obtained under th ese con d ition s.

The flu orescen ce due

to neopyrithiamine in creases and reaches a maximum, only a fte r standing fo r sev era l minutes.

Thus in a l l o f these c a lc u la tio n s the blank value

due to neopyrithiamine was subtracted from the t o t a l reading.

However,

th ese blank v alu es were made on amounts o f neopyrithiamine equivalent to the amount used in the a liq u o t taken for thiamine a n a ly sis .

This,

th erefo re, represents a minimum in h ib itio n and i t i s very probable that the in h ib itio n w ith neopyrithiamine reported in Table XX may be somewhat higher.

The reason fo r t h is i s that during the incubation

some o f the neopyrithiamine i s destroyed together w ith the thiamine w hile i t was in h ib itin g the thiamine d estru ction .

The blank valu es

would obviously not take t h is amount into con sid eration . D iscussion In t h is work, oxythiamine employed at a le v e l found to be very a c tiv e w ith carp, was found not to in h ib it the clam p r in c ip le .

This

would in d ica te that the amino group i s o f importance in such compounds fo r obtaining in h ib itio n .

However, recent work by Hennessy and

Tenmatay ( 6 6 ) Has shown that oxythiamine i s s p lit by the clam p rin cip le but very slow ly.

Thus i t becomes more probable that under the proper

con d ition s an in h ib itio n by oxythiamine could be obtained, but cert­ a in ly not to the same degree as the amino-pyrimidine d eriv a tiv e s that have been employed. L

The in h ib itio n by neopyrithiamine i s found in th is work to be

90 r q u ite pronounced.

This observation has sin ce been confirmed by the

work o f Sealock (63)*

L

ji

91 r S eries B.

The E ffec t o f Oxythiamine on the thiamine Destroying Factor o f Carp,

The only work appearing in the lite r a tu r e which d eals s p e c if ic a lly w ith the e f fe c t o f oxythiamine on the enzyme o f carp is th at o f Soodak and Cerecedo (2?)•

Their observations have been checked and extended

in the present study. The technique employed in th ese experiments was e s s e n t ia lly that o f Sealock (5?)*

In the case o f carp, which i s extremely more a c tiv e

than clam, the concentration o f thiamine used in a l l o f the exp eri­ ments to be described was 2 ,5 micromoles ( 8^2*5 gamma). An i n i t i a l experiment was carried out to a scerta in a number o f p ertin en t fa c to r s such as lo s s due to a lk a lin it y , blank flu orescen ce, and lo s s through adsorption.

An a c tiv e extract of acetone dried

carp powder was prepared from 2.5fi* o f the powder.

This quantity

was extracted 5 tim es w ith 10$ sodium ch loride in Q.2M phosphate b u ffer o f pH 7.4'.

The supernatants were combined and d ilu te d w ith

b u ffer to a f in a l volume o f 50 ml.

Two ml. o f t h is extract was

equivalent to 100 mg, o f the o r ig in a l powder and was the amount used in a l l o f the experiments which were se t up w ith the fo llo w in g general plan: Tube Ho, 1

2

2 ml. Ext.

2 ml. Ext.

2 ml. Ext.

3 ml. Ext.

2 ml. Water

2 ml. Water

3 ml. Water

1 ml. Buffer 1 ml. Buffer

2 .5 uM

2 .5 uM Br Blank a fte r incubatio n .

L

3

h

2 .5 uM B..

5 3 ml. Water

2 .5 uM B

92

r

t

The general plan of experim entation employed, even in the in h ib itio n stu d ie s was that described above*

The tubes were incubated for two

hours at 37*5°C s-ud a fte r t h is period 5 nl* o f

tr ic h lo r a c e tic acid

was added and the tubes allowed to stand fo r 30 minutes*

four ml* of

supernatant in each case was taken and d ilu te d to 100 ml*

Two ml* of

the d ilu ted sample was used for running the thiamine an alyses.

This

two ml. sample was equivalent to 6.7 4 gamma o f thiam ine. 1

2

3

4

5

10

39

6

38

41

The galvanometer readings obtained in t h is prelim inary experi­ ment showed that the blank value on the carp powder was qu ite sm all. The follow in g fa c ts were e sta b lish e d . 1)

There i s l i t t l e or no flu orescen ce due to the carp it s e lf *

2)

A fter

3)

There i s l i t t l e or no d estru ction o f the thiamine due to

2 hours incubation most o f the thiamine wasdestroyed.

the pH a fte r 2 hours incubation. 4)

Hone o f the thiamine i s lo s t by absorption on the p reci­

p ita te d p ro tein a fte r cen trifu g a tio n . The main experiments w ith carp were therefore se t up. thiam ine, in t h is work, was employed at two l e v e l s .

Qxy-

The thiamine in

a l l o f th ese experiments was used at the same le v e l, 2*5 uil per tube. The oxythiamine was employed in concentrations of 20 x 10” 10~Ss.

and 582c

This rep resen ts le v e ls of thiam ine/oxythiam ine o f 1 /4 and 1 /1 0 .

It i s the purpose here to show how the r e s u lts obtained depend en tir e ­ ly on the exact manner and con d ition s in which the experiment i s se t up.

In t h is f i r s t experiment the tubes were incubated for three

jaours and the concentrations mentioned above were used*

A 0.2 ml.

93 r a liq u o t o f the supernatant was taken fo r the thiochrome t e s t which re­ presen ts a le v e l o f 16.85 gamma, o f thiam ine.

The readings obtained

under these con d ition s were the follow in g: Incubated Bj^

Control with 31

3

92

( 20x10^ ) 3

Bx+OBi ( 50x1 7

It would appear, th erefo re, that a lso in the case o f carp, oxythiamine at 20x10

M when incubated fo r three hours does not in h ib it thiamine

d estru ctio n .

At ten times the concentration o f thiamine the oxy­

thiamine has a s lig h t e f fe c t amounting to about 8^ in h ib itio n .

However,

th ese r e s u lts a lso show that in the case o f carp the a c t iv it y of th is fa c to r i s such, at le a s t in our preparation, that a l l of the thiamine i s destroyed.

Under such con d ition s a true in h ib itio n study w ith oxy­

thiamine or with any other in h ib ito r could not be carried out.

It i s

obvious that to a rrive at any true value some o f the o r ig in a l thiamine must be p resen t. To check t h is extreme a c t iv it y o f the carp preparation, a further experiment was s e t up e x a ctly as above and in th is case much larger a liq u o ts taken fo r the thiamine a n a ly sis .

I t was quite p o ssib le that

i f a small amount o f resid u a l thiamine did remain, i t may not have been d etected w ith the a liq u o ts used.

To check t h is p o in t, two

experiments were set up e x a c tly as above, but in n eith er case could thiamine be found. It was p e r fe c tly obvious that the exact cond itions of the ex­ periment would have a great deal to do w ith the r e s u lts obtained and the conclusions drawn.

The important fa c to r s which had to be taken

in to account were the amount and a c t iv it y of the p a rtic u la r carp preparation on hand, the concentration o f thiamine and oxythiamine

94

employed, and, th ir d ly , the exact time and temperature of incubation# With t h is firm ly e sta b lish e d , the experiments were repeated with oxythiamine and the incubation period was cut down to one hour.

The

experiment was se t up as b efo re. A 0 .2 ml. aliq u ot was taken in th is p a rtic u la r ease fo r determining the resid u a l thiam ine.

Two id e n tic a l

runs were made w ith oxythiamine under th ese cond itions at concentra­ tio n s o f l / 4 and 1 /1 0 (o f thiam ine/oxythiam ine.

In the f i r s t case,

the aliq u ot taken for a n a ly sis was 0 .2 m l., and in the second se t a larger aliq u ot of 0 .4 ml. was taken as a check.

The. r e s u lts of

th ese experiments firm ly e sta b lish ed that oxythiamine was a potent in h ib ito r o f the thiaminase o f carp.

This i s brought out in the

fo llo w in g ta b le . Table 3X1 The E ffe c t o f Oxythiamine on the Thiaminase of Carp. Experiment Ho. I

II

Cone. Oxythiamine

$ In h ib itio n

20 x ICT^M

43

50 x lCf^M

57

20 x 1(H*H

42

50 x icH*m___________________*>k Under the con d ition s o f these experiments th erefore, oxythiamine i s an in h ib ito r o f thiamine d estru ction by the carp p r in c ip le .

The

in h ib itio n found in t h is work i s considerably l e s s pronounced than th at reported by Soodak (3)*

The wide range o f r e s u lts obtained by

t h is worker under sim ila r cond ition s can be a ttrib u ted to a v a r ia tio n o f the experim ental con d ition s which i s o f primary importance. The r e s u lts o f t h is work were further sub stan tiated by e s ta b lish ­ in g that carp has the capacity of r e a d ily destroying oxythiamine in

95 r the absence o f thiam ine.

This point i s of obvious s ig n ific a n c e since i t

would in d ica te the manner in which oxythiamine a cts as an in h ib ito r .

To

study t h is problem the color t e s t o f Prebluda and McCollum served very n ic e ly .

This t e s t which i s given by oxythiamine as rea d ily as by

thiamine gave a method of fo llo w in g the fa te o f th e oxythiamine on in­ cubation with the carp p r in c ip le . The oxythiamine was used at the same le v e l as the thiamine in the previous experim ents, that i s , at 2 .5 micromoles per tube. ment was run in d u p licate at 3?°C fo r three hours. made in the Evelyn colorim eter w ith 520 f i l t e r .

The experi­

The readings were

A control w ith un­

incubated oxythiamine was s e t up sim ultaneously. Oxythiamine Control

Incubated Oxythiamine

1)

89

26

2)

92

2k

The evidence d e f in it e ly shows that oxythiamine i s r e a d ily des­ troyed by the carp fa c to r .

In a three hour incubation period ?Q$

o f the oxythiamine employed (2 .5

was destroyed by the equivalent

o f 100 mgs. o f the o r ig in a l powder.

This observation perm its an

in t e llig e n t in te r p r eta tio n o f the data, and o ffe r s an explanation for the in h ib itio n o f the carp thiam inase by oxythiamine.

r

L

i

^

Chapter XII

A Study on the Carboxylase Enzyme of Yeast

A s e r ie s o f In v itr o experiments w ith the carboxylase enzyme system o f yeast was set up w ith the in ten tio n o f making a complete study o f th is system as regards the mechanism of carboxylase a ctio n ,

A great deal o f

work has appeared in the lite r a tu r e by a number of authors who have made a study of t h is enzyme both in a crude and p u rifie d s ta te , as regards to the manner in which cocarboxylase ex erts i t s function as a coenzyme.

In the ea rly work of Ochoa (6 ? ), i t was observed that thiamine

although i t could no replace eoearboxylase, did exert in i t s presence a marked stim ulatory e f f e c t on pyruvate decarboxylation.

In h is work

Ochoa employed an a lk a lin e washed preparation of yeast as a source of the apoenzyme.

Later Westenbrink (68) demonstrated that bakers yeast

contains a phosphatase capable o f destroying cocarboxylase and that the stim ulatory e f fe c t o f thiamine could be a ttrib u ted to i t s capacity to act as an in h ib ito r o f t h is phosphatase.

Lipton and Elveh^em (69)

working w ith both bakers and brewers yeast arrived at a second explan­ a tio n .

These workers maintained that a crude preparation o f the yeast

carboxylase contains an in a c tiv e p rotein capable o f combining with cocarboxylase to give a couplex which was not a c tiv e on the pyruvate system.

The r o le o f thiamine as an a ctiv a to r was a ttrib u ted to i t s

cap acity to combine w ith the in a c tiv e p rotein and thereby allow ing cocarboxylase to combime with the true apoenzyme. More recent work has shown that when a p u r ifie d carboxylase preparation was employed no .stim ulatory e f fe c t on the part o f thiamine was observed.

The presence o f cocarboxylase as such was th erefore

found to be necessary fo r coenzyme a c t iv it y .

This has been thoroughly

e sta b lish e d "by Weil-Malherbe (70) and more recen tly by C hevillard and coworkers (71, 7 2 ), In a recent paper d ealin g w ith the d iss o c ia tio n of carboxylase, Westenbrimk (7 8 ) showed that the complete enzyme system in li v e or dried yeast did not behave in the same manner as that which i s formed when cocarboxylase i s added to a lk a lin e washed y e a st. h is stu d ie s, he proposed the follow in g hypothesis:

On the b a sis o f

In whole yeast

we are d ealin g w ith an u n d issociated enzyme, that i s , one in which the ooenzyme i s firm ly bound to the apoenzyme.

On the b a sis o f h is

d is s o c ia tio n stu d ie s, festen b rin k p ostu lated a twofold lin k age, a strong bonding o f the amino group o f cocarboxylase to an a c id ic group o f the p ro tein , and a weak or h igh ly d isso c ia b le bonding o f the pyro­ phosphate group o f cocarboxylase to a b asic group of the p ro tein . In whole y ea st both o f th ese lin k ages are present to give the op­ timum or most a c tiv e enzyme system.

When cocarboxylase i s added

to the apoenzyme a fte r a lk a lin e washing, then only one linkage i s p ictu red as being formed, the one through the pyrophosphate group. The firm ly bound linkage through the amino group i s apparently not formed.

For t h is reason t h is enzyme system only shows a fr a c tio n

of the a c t iv it y in pyruvate decarboxylation which i s obtained w ith whole yeast without a lk a lin e washing.

This work o f Westenbrink was

in part confirmed by the in h ib itio n stu d ies o f Buchman and Heegaard (7*0.

These workers employing the th ia z o le pyrophosphate moiety of

cocarboxylase, found that cocarboxylase was stron gly in h ib ited by t h is d e r iv a tiv e .

The evidence suggested that the coenzyme formed a

d isso c ia b le lin k age through the pyrophosphate grouping.

This con-

gept i s , o f course, supported by the fa c t th at cocarboxylase as such

98

and not thiamine or thiamine-monophosphate i s required by t h is enzyme for a c t iv it y ,

The reverse was a lso found to he the case in the in h ib i­

tio n stu d ies o f Buchman.

Although the th ia z o le pyrophosphate moiety

of coearboxylase was very in h ib ito r y , the th ia z o le alcoh ol and the th ia z o le monophosphate did not in h ib it in t h is system. The work reported here represents the f i r s t a p p lica tio n o f s p e c ific antithiam ines to the study of the carboxylase problem.

The i n i t i a l

work was carried out w ith an a lk a lin e washed yeast carboxylase system and the la te r work w ith a p u r ifie d carboxylase preparation from brewers y e a s t.

A v a r ie ty of compounds were s e le c te d fo r study with

t h is enzyme system, in clu d ing oxythiamine, bromoxythiamine, oxythiaminemonophosphate, oxythiam ine-diphosphate, thiazole-pyrophosphate, and neopyrithiam ine.

The experimental procedure in t h is phase of the work

was e s s e n t ia lly that o f Buchman and Heegaard (7^)* w ith the exception o f the method o f preparation o f the a lk a lin e washed y e a st.

The a lk a lin e

treatment o f Westenbrink was adopted as a source of the cocarboxylase free apoenzyme.

The fr ee thiazole-pyrophosphate employed in one run

was synthesized by the method o f W eijlard (9 ) and obtained in aqueous so lu tio n containing 82 gamma per 0.1 ml. Experimental In a l l o f th ese experiments the usual Warburg manometric tech­ nique was follow ed.

Each v e s s e l contained 0 .2 g. o f dried bakers

y ea st which had been washed w ith a lk a lin e phosphate b u ffer to remove the coearboxylase.

The y ea st preparation was then suspended in 0.2M

phosphate b u ffer of pH 6 .2 to which was added 0.5 ml* o f Q.Q5M sodium pyruvate which contained 0.1 mg, o f magnesium and manganese per 0.5 jln the sid e ara o f each v e s s e l was placed 0.1 ml. so lu tio n o f cocar-

hjI .

99 r 1 boxylase containing 4 gamma of the coenzyme, and varying amounts o f the in h ib ito r .

These so lu tio n s were tipped in to the main v e s s e l a fte r

temperature e q u ilib ra tio n had been reached. The r e s u lts o f two ty p ic a l experiments employing oxythiam ine-diphosphate at varying concentrations in the presence of a constant amount o f cocarboxylase are shown in Table XXII* Table XXII In h ib itio n o f the Carboxylase Enzyme System by Gxythiamine-Diphosphate. Exp. Ho. I

Cone, o f OB^DiPOZj, Gamma 0

II

p i o f COg

$ In h ib itio n

155.6

0

48 .5

106*3

31

8 7 .0

81.9

47

174.5

3 5 .6

77

262

1 5 .0

90

0

141*5

0

4 8 .5

103.9

26

8 7 .0

77.3

45

174*5

26*8

81

262___________________ 2*5_____________ ...93.

......

A pronounced in h ib itio n o f cocarboxylase a c t iv it y i s observed at a l l the le v e ls used, and t h is in h ib itio n in creases w ith in creasin g concentrations o f oxythiamine-dipho sphat e .

This point was checked

fu rth er, employing the thiazole-pyrophosphate in one run.

When th is

moiety was used in a sim ila r type experiment at a concentration o f 82 gamma per Warburg v e s s e l, i t reduced the amount of carbon dioxide to 8 .6 $ o f the co n tro l v alu e.

This i s in agreement with the observation

100

r

o f Buchman and Heegaard (? h ).

More s ig n if ic a n t, however, i s the r e su lt

found w ith oxythiamine-diphosphate sin ce t h is compound was shown to he a s p e c if ic and to x ic thiamine antagonist in mice*

The higher concentra­

t io n o f oxythiamine-diphosphate represents a molecular r a tio to co­ carboxylase o f 6 0 /1 .

At t h is le v e l, on f iv e separate runs, in h ib itio n s

o f 97, 93, 90, 93» and 9*$ were obtained* It i s r e a liz e d that the carboxylase system represents only one is o la te d enzyme but i t i s reasonable to expect that a lik e phenomenon w ill occur w ith other systems and t h is w ill be elaborated upon in the fo llo w in g section s* In sim ila r experiments w ith oxythiamine, oxythiamine-monophosphate, hromoxythiamine, and neopyrithiam ine, no in h ib itio n o f coearboxylase a c t iv it y was found w ith any o f th ese d e r iv a tiv e s.

Oxythiamine in very

high o®ncentrations o f 1/500 and 1/1000 ex h ib ited no in h ib ito ry power towards the carboxylase system. P a r tic u la r ly in te r e s tin g are the r e s u lts w ith ©xythiamine-monophosphate, in which t h is d eriv a tiv e was found not to in h ib it the car­ boxylase system.

The oxythiamine-monophosphate was employed in th is

experiment at three le v e ls o f 1/lG , 1/20, and l/^O.

Even at the

high est concentration, the monophosphate did not in h ib it pyruvate decarboxylation.

The COg ev olu tion at a l l le v e ls was comparable to

the control w ith ju st cocarboxylase.

Apparently the monophosphate

can not compete w ith cocarboxylase for the apoenzyme, and th is i s in accord w ith the corresponding observation of Buchman w ith the th ia z o le monophosphate.

We must conclude, th erefore, th at to obtain in h ib itio n

of cocarboxylase attachment to the apoenzyme, the pyrophosphate

101 r grouping i s e s s e n t ia l,

fu rth er, sin ce the r a tio s employed p a rtic u la rly

1

w ith oxythiamine, were very high, i t can lo g ic a lly he concluded that the pyrimidine ring or hydroxyl group does not seem to p lay an a c tiv e part in th is in h ib itio n .

This concept i s supported by the in h ib itio n obtained

w ith th ia z o le pyrophosphate which in d ica tes that the pyrimidine ring i s not required to obtain in h ib itio n .

However, t h is reasoning ap p lies

only to th ese in v itr o stu d ies with yeast sin ce the same obviously does not apply in vivo w ith mice. These observations w ith thiamine an tagon ists lead to the same conclusions that were arrived at by other workers a§ regards:? the nec­ e s s i t y o f having the pyrophosphate grouping for a c t iv it y .

Thus, as

has been mentioned, coearboxylase i s necessary in t h is system and in most other systems fo r a c t iv it y whereas, thiamine or i t s monophosphate e s te r are in a c tiv e . The r e s u lts obtained w ith bromoxythiamine agreed w ith those found w ith oxythiamine and i t s monophosphate.

In t h is case the bromo deri­

v a tiv e was employed at k l e v e l s . Concentration of Bromoxythiamine Control o l C02

9^.1

1/10 99.6

1/2 0 96.3

l/W

1/60

95.6

90.3

In one experiment the high le v e ls o f the bromo d eriv a tiv e showed no tendency towards in h ib itio n . The a v a ila b ilit y of neopyrithiamine offered a means o f further studying the e f f e c t o f an amino pyrimidine d eriv a tiv e on the carboxy­ la s e system.

Using the a lk a lin e washed yeast preparation as described

by Westenbrink ( 6 8 ), neopyrithiam ine was found to have a pronounced jjtimulatory e f f e c t in pyruvate decarboxylation in the presence o f co-

102

r carboxylase*

Concentration of Neopyrithiamine

p i co2

Control

1/10

1 /20

1/40

1/60

108

189

193

183

179

119

180

19©

173

186

This stim u lation by neopyrithiamine i s in lin e w ith the observation o f Ochoa (67) w ith thiam ine.

This augmenting e f f e c t i s only obtained

w ith neopyrithiamine and was not observed in those experiments w ith oxythiamine or oxythiamine-monophosphate* It was n ecessary in t h is case to elim in ate the p o s s ib ilit y that the augmenting e f f e c t on the part o f neopyrithiamine was due to a c a t a ly tic a ctio n o f th is compound.

This was e sta b lish ed in an experi­

ment in which neopyrithiam ine was employed at low le v e ls in the absence o f any cocarboxylase. Concentration o f Neopyrithiamine— Camma

yOL

C02

Control W C oB , 12G

4__________ 4

IQ

10

1 .2 2

1 .0

-3 .0

- 4 .0

There i s l i t t l e doubt that neopyrithiamine can not replace co­ carboxylase as a coenzyme in t h is system.

Although, as has been

reported in the b io lo g ic a l and m icrobiological se c tio n s, neopyri­ thiamine i s very to x ic to mice and microorganisms, w ith the is o la te d carboxylase system i t does not act as an in h ib ito r . These stu d ie s on the carboxylase system of yeast would appear to support the hypothesis advanced by lesten b rin k on the d is s o c ia tio n of t h is enzyme system.

It i s reasonable to expect that i f the amino

linkage between the cocarboxylase and the apoenzyme were necessary £or a c t iv it y in v it r o , neopyrithiamine would in h ib it th is sybtem.

1Q3 r

-]

Other evidence in the lite r a tu r e a lso tends to support th is hypothesis. In p a rtic u la r the work o f Stumpf (75) w ith the pyruvic oxidase system o f Proteus v u lg a ris e sta b lish ed that the pyrithiam ine o f Tracy and E ld e r fie ld (25) did not in h ib it cocarboxylase when added to the co­ carboxylase free apoenzyme. I f t h is lin e of thought i s correct, then the important question as to whether oxythiamine-diphosphate in the absence of any cocarboxylase has any c a t a ly tic a c t iv it y in the carboxylase system, remains to be answered.

I f the pyrophosphate grouping was a l l that i s e s s e n tia l for

a c t iv it y , i t i s quite p o ssib le that in low concentrations oxythiaminediphosphate would show a coenzyme a c t iv it y .

To answer t h is question an

experiment was se t up w ith oxythiamine-dipho sphat e in low concentrations. The diphosphate was employed in concentrations of 2, 4, and 8 gamma per Warburg v e s s e l. Failure o f Qxythiamine-Diphosphate to Act as a Goenzyme in the Carbo­ xylase system Blank jjlL

CCL

Controls 2VC0Bi 4Y CoBx

24___________ 56

Concentration o f OBjBiPO^-G-amma 2 4 8

105________ 22_________ 24_______ 21_________

The r e s u lts show that oxythiamine-diphosphate by i t s e l f does not have any coenzyme a c t iv it y w ith t h is system.

Although

the pyrophos­

phate grouping i s present fo r attachment to the apoenzyme, i t appears that an amino group i s s t i l l e s s e n t ia l in ad dition to the pyrophosphate group to obtain a c a ta ly tic e f f e c t . To further su b sta n tia te the observations made in t h is work with an a lk a lin e washed bakers y ea st preparation, i t was decided to employ a p u r ifie d carboxylase preparation from brewer*s y e a st. l

In these stu d ies j

r the p u r ific a tio n procedure o f Weil-Malherbe ( 7 0 ) was adopted for is o la tio n and fr a c tio n a tio n o f the p u r ifie d enzyme system.

This system

o ffered an ad d ition al one fo r studying the mechanism of oxythiamine in ­ h ib itio n . The experiments in t h is phase o f the work were designed in an attempt to answer the fo llo w in g questions? 1)

Do compounds o f the type o f thiamine and neopyrithiam ine have any

stim ulatory e f f e c t in the presence o f coearboxylase? 2)

Can oxythiamine-diphosphate d isp lace cocarboxylase in a semi-

p u r ifie d enzyme preparation which s t i l l contains some of i t s cocarboxy­ la se content? 3)

Httiat i s the e f f e c t of oxythiamine-diphosphate on the pure enzyme

system as regards pyruvate decarboxylation? The a c tiv e preparation was prepared from fresh brewer fs yeast which gave a sem i-p u rified preparation of 8 grams from 5© grams of a ir dried y e a st.

This sem i-p u rified fr a c tio n i s an acetone fra ctio n a ted

and dried preparation which was q u ite sta b le in the d esicc a to r.

The

cocarboxylase fr e e p ro tein i s prepared from t h is preparation on the same day that i t i s to be used fo r i t i s not very sta b le .

The fin a l

ammonium s u lfa te fra ctio n a ted p ro tein i s e s s e n t ia lly free of cocarboxy­ la s e , and i s very a c tiv e when coearboxylase is . added to the system. The acetone fra ctio n a ted preparation represen ts about a f iv e fo ld p u r ific a tio n and s t i l l contains a considerable amount of cocarboxy­ la s e . 1)

In an attempt to answer the f i r s t question an experiment was

se t up w ith the cocarboxylase free enzyme system, using thiamine and neopyrithiamine at t«w con cen trations.

105

r V essel No. Contents

1

5

2

4

4 V Cocarboxylase---------------------Control

lOVBx

2a ■--------

IOYnPBx

The concentrations o f thiamine and neopyrithiamine represent le v e ls w ell ahove that required to m anifest a stim ulatory e f f e c t .

One ml. of

the yeast preparation, which contained about one mg. of the a c tiv e p ro tein was used in each Warburg v e s s e l.

Nitrogen was used as the

second gas phase and the readings were taken a fte r ten minutes. Table XXIII The E ffect of Thiamine and Neopyrithiamine on the P u rified Carboxylase Enzyme of Brewer*s Yeast V essel No.

Contents

3

Yeast Blank

jal COg 20

1

Control- 4 Gamma o f Coearboxylase

178

5

4 Gamma CoB^ +• 10 Gamma B^

177

2

4 Gamma CoB^ + 40 Gamma B]_

174

4

4 Gamma CoB^ + 10 Gamma NPB^

l 6l

2a

4 Gamma CoB^

40 Gamma NPB^____________ 166

The r e s u lt s show that in th is case the stim ulatory e f fe c t on the part o f thiamine or neopyrithiam ine i s not obtained in the presence of cocarboxylase.

This point i s of primary importance sin ce i t elim in ates

the p o s s ib ilit y o f f a ls e high r e s u lt s due to the stim u lation of the decarboxylation by the presence of some excess of thiam ine. 2)

The problem as to the displacement of cocarboxylase from the

surface o f the apoenzyme by oxythiamine-diphosphate was stud ied in two s e ts o f experim ents.

It Is evident th a t, fo r t h is aspect of the

106 r problem, the o r ig in a l yeast preparation would not serve too w ell be­ cause of i t s very high cocarboxylase content and the presence of other p r o te in s.

Nor could the p u r ifie d carboxylase system be used because

o f i t s la ck of cocarboxylase.

It was decided th erefore, that a semi-

p u r ifie d preparation obtained by the acetone fra ctio n a tio n would serve most s a t is f a c t o r ily .

This preparation i s quite sta b le and s t i l l

contains a considerable amount o f i t s coearboxylase content. was esta b lish ed in two runs w ith th is preparation using 5° y ea st per Warburg v e s s e l.

This fa c t of

It was obviously o f importance to have some

idea as to how much cocarboxylase was present per u nit weight o f the y ea st preparation.

For such an experiment as th is the optimum con­

d itio n would be that in which the coearboxylase was lim itin g .

From

th ese prelimary assay runs w ith the preparation on hand i t was found that there was about 4 .5 gamma of cocarboxylase present in 50 Eg* of the y e a st. In the displacement stu d ies i t was decided to use 20 mg. of the y ea st preparation per Warburg v e s s e l, t h is amount corresponding to about 1 .8 gamma of cocarboxylase.

The oxythiamine-diphosphate was

used at 4 le v e ls o f 50, 100, 500, and 1000 gamma per v e s s e l.

L

J

T able XXIV A

F ailu re o f Chcythiamine-Diphosphate to D isplace Coearboxylase From the Apo carb oxylas e Contents

V essel Ho.

p i co2

3

Control containing 20 mg. o f Yeast

196.8

5

50 V Oxythiamine-Dipho sphat e

191.9

2

1OOVQxyt hiamine-Dipho sphat e

196.0

4

5 00YQxyt hiamine-Dipho sphat e

188.5

2a

1 OOOYQx.ythiamine-Dioho sohat e

19.0.1

Even at the h igh est concentration of oxythiamine-diphosphate used there was no apparent in h ib itio n or displacement of the cocarboxy­ la s e from surface of the apoenzyme.

It was th erefore decided to make

the con d ition s o f the t e s t more strin gen t by using s t i l l l e s s o f the p rotein preparation and higher concentrations o f oxythiamine-diphos­ phate.

In t h is experiment i t was decided to employ.only one mg. of

the yeast per Warburg v e s s e l. Table XXIV B F ailu re o f Oxythiamine-Diphosphate to D isplace Coearboxylase From the Apocarboxylase Under More Stringent Conditions. V essel Ho.

Contents

> 1 CO;

3

Control containing one mg. o f Yeast

43.7

1

50Vgamma o f oxythiamine-diphosphate

3 8 .8

5

lOOVgamma o f oxythiamine-diphosphate

41.4

2

500 gamma o f oxythiamine-diphosphate

32 .2

4

1000 gamma of oxythiamine-diphosphate

39 .0

2a

10.000 gamma o f oxythiam ine-diohosohate

36.4-

108 r Even when the con d ition s are made very strin g en t, oxythiaminediphosphate can not d isp la ce coearboxylase in th is system,

This work

should he in terp reted in the lig h t o f Westenhrink, s observations dealing w ith the d iss o c ia tio n o f t h is same system.

The data which were found in

th ese experiments would again favor the concepts advanced by th is worker p ertain in g to the d ifferen ce in the enzyme systems obtained from whole y ea st and that obtained when cocarboxylase i s added to the apo­ enzyme a fte r a lk a li washing. In view of these r e s u lt s , i t was decided to check the role of oxy­ thiam ine-diphosphate on the cocarboxylase fr e e p u r ifie d enzyme system. The experimental procedure was that employed p reviou sly w ith t h is system, w ith the exception that one mg. o f the p u r ifie d preparation was used in each Warburg v e s s e l,

The sid e arm of each v e s s e l contained

4 gamma of cocarboxylase and varying amounts o f oxythiamine diphosphate. A con trol and blank were s e t up at the same tim e. Table XXV The E ffect o f Qxythiamine-Diphosphate on the P u rified Carboxylase of Brewers l e a s t V essel Ho.

L

Contents

p i CO.

3

Blank-one mg. o f Yeast Preparation

1

Gontrol-4 Y CoBx

156.0

5

4 Y CoBx+ 4 8 .5 Y OBxDiPO/,,

121.3

2

4 YCOB-J+ 8 ? .0 YOBxDiPO^

95-3

4

4 Y CoBx + 174 V QBxBiPO/,,

58 .0

2a

4 VCoB-, +• 262 YQBnDiPOA

__ 36».g_.

26.5

These r e s u lts check f a ir ly w ell w ith those found p rev io u sly w ith the bakers y ea st a lk a li washed system. The oxythiamine-diphosphate was found to be s ig n if ic a n tly in h ib ito r y at the ls v e ls used.

Expressing

th ese r e s u lts in terms o f per cent in h ib itio n we fin d: 1/10-------------------- 22$ in h ib itio n 1 /2 0 -------------- 3 9 $

1/60

77$ D iscussion

In any attempt to in terp ret the fin d in gs in th is phase o f the work, the fa c ts reported in the lite r a tu r e must obviously be taken in to account.

The carboxylase system lik e many other complete enzymes

i s recognized as a firm ly bound or u n d issociated enzyme.

The lite r a tu r e

a ls o t e s t i f i e s to the fa c t that many other substances in h ib it th is system.

Thus Barron (76) has shown that as an SH enzyme i t i s r ea d ily

in h ib ited by SH reagents.

Other SH in h ib ito r s such as quinones and

ce rta in a r se n ic a ls can a lso ex h ib it t h is in h ib itio n .

In a l l o f these

ca ses, however, i t i s to be recognized that we are d ealin g w ith a s t i l l fu rth er p ortion o f the enzyme surface.

These in h ib itio n s are

obviously not com petitive although to a large extent they can be reversed by reducing agents such as cy stein e and g lu tath ion e. For complete a c t iv it y th erefore, the in te g r ity of the SH group in a reduced fo r must be m aintained.

A sim ilar phenomenon a lso

e x is t s , in the case o f a complex enzyme system, w ith regard to the SH group o f the coenzyme.

In t h is resp ect, the n e c e s sity o f a re­

duced SH system was e sta b lish e d by the work of P eters (77) w ith co-

110 carboxylase d is u lf id e . In ad dition to t h is one aspect o f the enzyme, the evidence report­ ed here in d ica tes that the in te g r ity of the pyrophosphate group i s e s s e n t ia l.

The stu d ies w ith oxythiamine-diphosphate and oxythiamine-

monophosphate stron gly suggest the ex isten ce o f a d isso c ia b le linkage through t h is grouping.

The displacement stu d ies with oxythiamine-

diphosphate suggest fu rth er th at there i s s t i l l a further point o f attachment in t h is complex enzyme system to be considered.

The

u n d issociab le linkage through the amin0 group o f cocarboxylase as proposed by Westenbrink i s supported by the fin d in g s o f t h is work.

L

I ll

Part V P h o s p h o ry la tio n S tu d ie s W ith Thiam ine aad th e Hole o f O xythiam ine and

Ne opyri t hiamine. Chapter X III

Enzymatic Phosphorylation o f Oxythiamine by Bakers and Brewers Y east.

The sy n th esis o f oxythiamine-diphosphate, accomplished by chemical means, was attempted in th is work enzym atically.

The further altern a­

t iv e as to the manner in which oxythiamine may exert i t s e f fe c t s t i l l remained to be stu d ied .

vivo

It was q uite p o ssib le that although oxy­

thiam ine-diphosphate was a potent in h ib ito r in v it r o , in viv o the actu al point o f in h ib itio n was prelim inary to i t s phosphorylation.

At s u f f i­

c ie n t ly high con cen tration s, oxythiamine may w ell In h ib it or block the phosphorylation o f thiamine and thereby prevent i t from becoming fu n c tio n a l.

An attempt to p ictu re the p o in ts of p o ssib le a ctio n of

oxythiamine i s shown in the follow in g schemes ATP B]_-------------------------------- -— ->CoBi------------------ -— ^Apocarboxylase _ ATP 0BX------------------------- ^OBjDiPOij,

Pyruvate C02+CH3CH0

It i s w e ll esta b lish ed that the a c tiv e form o f thiamine in the t is s u e s i s mainly the diphosphate and t h is i s the predominant form. The enzymatic sy n th esis o f cocarboxylase has been e sta b lish ed by a number o f workers.

Ochoa (7 8 ), working w ith li v e r s lic e s from

thiamine d e fic ie n t pigeons, observed that such s li c e s were able to ra p id ly convert added

thiamine to cocarboxylase.

tis s u e fromthiamine d e fic ie n t l e s s a c tiv e than l iv e r . L

pigeons

Brain and muscle

werefound to be considerably

Tauber (?9# 80, 81), has succeded in phos-

112 r i phorylating thiamine by means of a dried duodenal mucosa preparation from pigs*

In h is work Tauber a lso obtained phosphorylation w ith an a lk a li

washed yeast preparation.

The system employed by Tauber was the simp­

l e s t o f those used by other workers but at the same time was probably not optimum, sin ce he obtained only a 60$ conversion o f thiamine to cocarboxylase.

This enzymatic sy n th esis o f cocarboxylase has a lso

been accomplished by other workers with dried brewerfs y e a s t.

Kinnersley

and P eters (82, 83), llvehjem and P otter (8 4 ), and Weil-Malherbe (85) were among the e a r lie s t o f the workers to e s ta b lis h the phosphorylation of thiamine in v it r o .

Cocarboxylase has a lso been obtained from thiamine

by b a c te r ia l sy n th e sis, u sing Prooionibacterium oentosaceum. by Silverman and Werkman ( 86).

A ll o f th ese enzymatic processes occur

only in the presence o f the apocarboxylase, the s p e c if ic p ro tein of the carboxylase, and the rea ctio n i s stopped when the p ro tein i s part­ l y or f u ll y saturated ( 8?, 8 8 ). I t would be o f some value i f an enzymatic phosphorylation of oxythiamine could be e sta b lish e d .

This would not t e l l us whether oxy-

t h i amine s p e c if ic a lly in h ib ited the phosphorylation o f thiamine but i t would stron gly point in th is d ir e c tio n .

That oxythiamine i s phos-

phorylated by y ea st i s the subject matter of t h is s e c tio n .

The more

important question o f thiamine in h ib itio n i s taken up in the follow in g sect ion. In these experiments brewers and bakers yeast preparations were used prepared as described by Tauber.

The b a sis fo r a scerta in in g

whether oxythiamine was phosphorylated was the color t e s t o f Prebluda and McCollum as m odified by Melnick and F ield (89, 9©)*

Since the

fnethod o f approachvAs a co lo rim etric one, Tauber*s se t up was s e le c te d

113 r because o f i t s s im p lic ity . E xperim ental An a l k a l i washed d rie d brew ers y e a st p re p a ra tio n was employed in th e f i r s t experim ents.

To two grams o f dry y e a s t in a 250 ml* c e n tr i­

fuge b o t t l e th e re was added 100 ml. of 0.1M disodium phosphate a t 30°C and shaken f o r 12 m in u tes.

The procedure was re p e a te d once more.

A fter washing w ith d i s t i l l e d water the yeast was suspended in 20 ml. o f phosphate b u ffer at pH 6 .7 , and t h is suspension was used in th ese experiments. To one ml. o f a n eu tra lized oxythiamine so lu tio n containing exact­ l y one mg. at pH 6 .7 . 5 El* o f the yeast suspension was added. le d water was added to make a t o t a l volume o f 8 ml.

D is til­

The sample was

kept at 30°C fo r 20 hours, a fte r which time i t was placed in a beaker o f b o ilin g water fo r 4 minutes and then cen trifu ged .

This served as

the t e s t so lu tio n and together with th is there was se t up a con trol and a blank.

A sample o f dry washed yeast suspension without any

oxythiamine was trea ted sim ila r ly and to a th ird sample of 5 ®1* of y ea st suspension, one mg. o f oxythiamine was added ju st before b o ilin g . In a l l th erefo re, 4 tubes were se t up: 1 ) A stan d a rd oxythiam ine s o lu tio n c o n ta in in g 25 gamma/10 ml. 2) A t e s t s o lu tio n as d e sc rib e d above. 3 ) A c o n tro l which had oxythiam ine added ju s t b efo re b o ilin g . 4 ) A blank containing no oxythiamine. A fter incubation, tubes 2, 3 . and 4 were d ilu ted so that a l l so lu tio n s now contained 25 gamma of oxythiamine/10 ml. Ten ml. in each case was taken fo r the color t e s t .

114 r

The readings werfe made in the Evelyn colorim eter w ith a 520 mu f ilt e r *

The readings obtained in terms of o p tic a l d en sity were as

fo llo w s; Standard O.1549

Test

Control

Blank

0.1115

0.1702

0.0177

When compared to the standard the t e s t so lu tio n showed a free oxythiamine content o f 18 gamma and t h is would in d ica te a 28$ phos­ phorylation o f the oxythiamine.

It w ill he n oticed that the blank

value in th ese experiments i s quite s ig n ific a n t and must be a ttrib u ted to the small amount o f thiamine th at remains in the a lk a li washed y e a st. When the blank i s subtracted from the values obtained, as i t should be, i t i s found that 40$ o f the oxythiamine was phosphorylated.

Sim ilar

experiments were carried out w ith an air dried bakers yeast preparation and i t was found that 26$ o f the oxythiamine was not in the free form. On the b a sis o f th ese experiments i t would appear th a t oxythiamine was phosphorylated in much the same manner as thiamine only not as r e a d ily .

However, on the b a sis of some recent work by Hochberg and

Melniek ( 9 1 ) on the absorption of thiamine by y ea st, a seriou s ob­ je c tio n may be ra ise d again st th ese experiments.

It i s q u ite pos­

s ib le that part o f the oxythiamine added to the yeast preparation was absorbed and l o s t when the tubes were cen trifu ged.

This would a lso

account fo r the production o f l e s s color in the t e s t so lu tio n as compared to the standard. r e s u lts obtained by Tauber.

I t may w e ll account a lso fo r the low It was decided th erefo re, to carry out

the ex tra ctio n fo r one h a lf hour w ith 0. 1N s u lfu r ic acid rather than by Tauber*s method. l

In a second se t o f experiments i t was decided to check t h is p o i n t . j

115 r

The experimental d e t a ils were e s s e n t ia lly the same as before w ith the exception of the ex tra ctio n procedure# Two t e s t so lu tio n s were se t up in t h is case as a check and in a l l f iv e experimental tubes were s e t up as before# Standard

Test #1

0.2509

0.1788

Test #2

Blank

Control

0.1805

--------

0.2509

In t h is experiment the yeast blank served as the colorim eter blank. When pure xylene was used as blank, the yeast blank gave a reading o f

96% transm ission which in d icated again that some color production takes place w ith the a lk a li washed y e a s t. On the b a sis o f these r e s u lt s approximately 14$ phosphorylation of the oxythiamine took p la ce.

It further demonstrates that the technique

o f Tauber can not v a lid ly be employed for such a study. D iscussion It i s recongnized that in such work as t h is there are several handicaps which are d i f f i c u l t to overcome#

Thus a colorim etric method

on such a complex system lends i t s e l f to high blank values which in any co lo rim etric method i s a decided disadvantage,

fu rth er, i t i s not

im possible that on such prolonged incubation the molecule of oxythiamine may be s p l i t by the y e a st, although th is does not appear very lik e ly . The p o s s ib ilit y o f b a c te r ia l growth on 20 hours incubation must a lso be considered e s p e c ia lly on a medium such as y e a s t.

The b a c te r ia l

sy n th esis o f cocarboxylase and i t s in h ib itio n by oxythiamine i s con­ sidered in the next chapter* In view o f th e n a tu re o f the experim ent th e re is more than ju s t an in d ic a tio n t h a t oxythiam ine i s phosp h o ry lated in v i t r o and may w ell iJ>e so in v iv o * r""

A much more s a tis fa c to r y method of approach would be

th* ji

116 r

manometric technique*

In t h is manner the e f f e c t o f such compounds as

oxythiamine and neopyrithiamine could he studied w ith respect to th e ir ro le on the enzymatic sy n th esis of cocarhoxylase hy a number o f systems in vitro*

L

117 r C h ap ter XIV

P h o s p h o ry la tio n S tu d ie s w ith Thiam ine and th e Hole o f

Oxythiamine and Neopyrithiamine in the Enzymatic Synthesis o f Cocarboxylase. In t h is phase of the work a m icrob iological system and a p u rified system from brewer*s yeast were se le c te d for in v e stig a tio n .

Whether

oxythiamine s p e c if ic a lly in h ib ite d or blocked the phosphorylation o f thiamine as was depicted in the scheme ahove was only ind icated by the r e s u lts on the enzymatic phosphorylation of oxythiamine.

This problem

was now s p e c if ic a lly attacked and the r o le o f neopyrithiamine was a lso stu d ied .

It was quite p o ssib le that the very to x ic neopyrithiamine was

involved in a sim ila r mechanism.

However, as w ill be brought out in

t h is se c tio n , neopyrithiamine does not seem to be involved in a sim ilar manner as oxythiamine.

The evidence in d icated rather that the p yri­

dine analogue i s concerned w ith a d iffe r e n t mechanism than that which has been proposed for oxythiamine. S eries A. In a previous paper by Silverman and Werkman (@6 ), d ealin g w ith the enzymatic sy n th esis o f cocarboxylase, i t was shown that P rooionibacterium nentosaceum was capable o f phosphorylating thiam ine.

This

system was in v e stig a te d employing the experimental procedure of Silverman and Werkman. P. nentosaceum was obtained from the American Type Culture C o llectio n .

The a c tiv e c e l l preparation was prepared by incubation

fo r 72 hours at room temperature (30-32°C ).

The c e l l s were harvested

by c en trifu g a tio n and washed tw ice w ith d i s t i l l e d water.

The organ­

ism was maintained on a stock yeast extract peptone broth and was l

j

118 r

-i

tran sferred from t h is stock medium to the h asal medium o f Tatum, Wood, and Peterson (9 2 ).

Three tr a n sfe r s were made from the stock medium to

the hasal medium at 24 hour in te r v a ls , in each case in to 300 ml. o f the h asal medium.

An a ctiv e p aste was made o f the organisms a fte r 3 days

incubation w ith a small amount of d i s t i l l e d water.

This p aste o f the

organism was used in the phosphorylation s tu d ie s. Experimental The incubation stu d ies fo r phosphorylation and in h ib itio n were set up sim ultaneously.

Two in dividu al runs were made w ith th is enzyme

system set up in an id e n tic a l manner.

These runs were made on d if ­

feren t occasions w ith a fr e sh cu ltu re o f the organism. #1

#2

#3

0 .2 ml. c e l l p aste

0 .2 ml. c e l l paste

0 .2 ml. c e l l paste

1 ml. water

1 ml. water

1 ml. OB^ ( 5^0 gamma)

1 ml* B^

1 ml. P0^ Buffer

1 ml.

1 ml. P0j^ B uffer

(10 gamma)

1 ml. P0j^ Buffer

Three experimental tubes were set up for each run in the manner described above.

The blank served to show not only that phosphory­

la t io n took p lace but a lso to elim in ate the p o s s ib ilit y that the cocarboxylase a c t iv it y a fte r incubation was not due to a stim ulation o f cocarboxylase a c t iv it y on the part o f the excess thiamine remaining* The experiment was set up in cen trifu ge tubes which were then incubated in a ir at 30°C w ith frequent shaking for 4 hours.

A fter

t h is period the tubes were put in a b o ilin g water bath fo r 5 minutes. One ml, o f the thiamine so lu tio n containing 10 gamma was added to the second tube at the time o f b o ilin g .

A fter 5 minutes at 100°0, the

ifubes were cen trifu ged down and one ml. of the supernatant in each case j

119 r assayed fo r cocarboxylase.

-i

Assay The cocarboxylase assays were carried out in Warburg v e s s e ls at 30°G under an atmosphere o f nitrogen*

The data obtained in two runs are shown

in Table XXVI. Table XXVI Oxythiamine In h ib itio n o f Cocarboxylase Synthesis by P. •pentosaceum Exp. Wo. Flask Contents I

II

jilCOg $ In h ib itio n by OBi

Blank-Bi at b o ilin g

1 8 .4

Test-B^ incubated 4 hours

115*7

Bi incubated w ith OB^

42.7

Standard-2 gamma CoB^

87.4

St andard-4-gamma GoBi

182

Blank-Bi a t b o ilin g

17*0

Test-B^ incubated 4 hours

83*7

B*^ incubated w ith 0B^

60

Standard-2 gamma CoB^

101

63$

28

From th ese fig u r e s there i s no doubt that sy n th esis o f cocarboxy­ la s e did occur.

In the case where the vitam in was not incubated w ith

the b a c te r ia l c e l l s there i s almost no c a ta ly s is (1 8 .4 and 17.0 ^1 CO2 ).

A very s ig n ific a n t in crease in carbon dioxide i s n oticed in the

case in which the thiamine was incubated with the c e l l suspension for 4 hours.

On the b a sis of the con trols se t up in each experiment we

fin d that in one case approximately 2*5 gamma of cocarboxylase was synthesized whereas, in the second case s lig h t ly over one gamma was |y n th esized .

Likewise, the in h ib itio n s on the part o f oxythiamine are

120 r

somewhat fa r apart.

In two runs at the same le v e l o f oxythiamine, in­

h ib itio n s o f 63 and 28$ were found.

Q u a lita tiv ely , the r e s u lts o f the

two experiments are in agreement and in d ica te that oxythiamine i s cap­ able o f in h ib itin g not only as the diphosphate but may a lso in h ib it the enzymatic sy n th esis of cocarboxylase.

As the cocarboxylase assays are

carried out, a p o s s i b i l it y a lso e x is t s that the decreased carbon dioxide production may be due to in h ib itio n of cocarboxylase by oxythiam ine-diphosphate formed by phosphorylation of oxythiamine. presupposes that .oxythiamine i s phosphorylated.

This of course,

This i s in general

agreement w ith the observations made in t h is work.

Further, in view o f

the q u a n tita tiv e data on the phosphorylation o f thiamine, the amount of oxythiamine that could be phosphorylated must be very sm all.

In stu d ies

w ith the yeast system, as w ill be pointed out la te r , the amount of oxyt hiai*iine-diphosphate needed to in h ib it pyruvate decarboxylation i s much greater than that which could be accounted foa. by the phosphory­ la tio n o f oxythiamine. It appears most l i k e l y at t h is point w ith theinform ation on hand that oxythiamine in h ib its in both ways, being i t s e l f phosphorylated in the process o f in h ib itin g the phosphorylation o f thiamine and then in the form of oxythiam ine-diphosphate. With neopyrithiam ine at the same concentration, no in h ib itio n of cocarboxylase sy n th esis was found.

It appeared th erefore, that th is

antithiam ine did not block the phosphorylation o f thiam ine. S eries B.

Phosphorylation Studies With a P u rified Enzyme %stem From Brewers Y east.

The r e s u lts obtained w ith the m icrob iological sy n th esis of coLcarboxylase and the ro le o f oxythiamine and neopyrithiamine on th is

121 r sy n th esis were sub stan tiated in further work with a p u rified enzyme system from brewers y e a s t,

The method of p u r ific a tio n employed for ob­

ta in in g the enzyme system was that o f Weil-Malherbe (70).

In the work

w ith P. pentosaceum. an inherent d ifferen ce between the action of oxy­ thiamine and that o f neopyrithiamine was observed.

To further extend

t h is work, sim ila r experiments were now set up with these an tagon ists. Experimental Thiamine was employed at a le v e l o f 10 gamma per Warburg v e s s e l. Oxythiamine was used at three high le v e ls in th is case, 5° 0» 1000, and 5000 gamma per Warburg v e s s e l. The ATP used in th is work was from N u trition al Biochem icals, Cleveland, Ohio.

A hydrolyzable and t o ta l phosphorus a n a ly sis on the

dibarium s a lt showed the compound to be about 92$ pure.

The barium

s a lt was converted to a sodium s a lt and the f in a l concentration made approximately 0.05M. The experiment was set up as follow s: 12

3

le a s t h X Blank CoB^

2a

5

1 0 Vbx lO Y ;^ Control Control

2

k

11

10YB1 10 IOYBx 0.5mg. QB^ l.Omg. 0B^ 5 ®g*

One ml. o f the yeast preparation was employed in each case, equivalent to one mg. o f the p ro tein . cases 2 .8 mg. per Warburg v e s s e l.

The le v e l o f ATP was in a l l

Air served as the second gas

phase and the experiment was run at 30°G*

L

t

122 r T able XX?II

Oxythiamine In h ib itio n of Cocarboxylase Synthesis by Yeast Plask No*

Contents

j il CO2 $ In h ib itio n

3

Control-4-Y CoB^

195.9

2a

Control-10 Yb^

189.0

5

Control-10Y Bx

186.4

2

1QY Bi+0*5 mg. OB^

117.9

37

4

10Y B3+I.O mg. QBX

53.3

72

11

IOY Bi+5 .0 mg. 0BX

15.5

91

A s ig n ific a n t in h ib itio n o f t h is sy n th esis i s found w ith oxythiamine at the three high le v e ls employed*

A yeast blank or rather the blank

value obtained w ith the p u r ifie d enzyme system amounted to 27*3 p i of carbon dioxide and in d ica te s that a s a tis fa c to r y removal o f cocarboxy­ la s e was accomplished.

On the add ition o f 4 gamma of cocarboxylase,

the a c t iv it y o f the enzyme becomes very marked, w ith a production of

195*9 p i o f carbon d ioxid e.

The phosphorylation o f thiamine in the

presence o f ATP in a 15 minute incubation period almost approaches t h is v a lu e.

With the concentration of enzyme employed approximately 4

gamma or s l i g h t l y l e s s than th is was synthesized. In the presence o f oxythiamine, the sy n th esis o f cocarboxylase was markedly reduced.

A 37. 72 and 91$ in h ib itio n was obtained at the

three concentrations used.

Under the con d ition s of the t e s t , approxi­

m ately 4 gamma o f cocarboxylase was synthesized*

Prom the work o f

Weil-tyalherbe (70) we know that 6 to 7 gamma o f the coenzyme is s u f f ic ie n t to satu rate the apoenzyme.

As in the case o f P. uentosaceum.

the p o s s ib ilit y o f in h ib itio n through the diphosphate i s a secondary fa c to r .

The concentrations o f oxythiamine-diphosphate that could be

synth esized -under th ese con ditions could not account for the extent of in h ib itio n of cocarboxylase synthesis* B)

The E ffect o f Neopyrithiamine on the Synthesis of Cocarboxylase by Brewer's Yeast E s se n tia lly the same type o f study with brewer's yeast was set up

w ith neopyrithiamine*

The on ly change in th is work was that the neo­

pyrithiam ine was employed at lower concentrations but more than suf­ f ic ie n t to ex h ib it an in h ib itio n i f i t did in h ib it the sy n th esis of cocarboxylase* In t h is case the data o f Table XXVIII in d ica te that as in the case of P* -pentosaceum. neopyrithiamine does not block the phosphorylation o f thiam ine. Table XXVIII Failure o f Neopyrithiamine to In h ib it Synthesis o f Cocarboxylase by Brewer's Yeast Flask No*

Contents

p i co2

3

C ontrol-4- Y CoB^

202.6

2a

Gontrol-10 YBx

174*5

5

Control-10 Y b^

179.5

2

10 Y Bj+iOO Y Neopyrithiamine

175.4

4

10Y B1+5OOY Neopyrithiamine

176.8

11

IOY Bj+lOOOYNeopyri thiamine

183.4

Very comparable r e s u lts are obtained on repeated runs in the absence o f any in h ib ito r , as regards the amount o f cocarboxylase sy n th e sis.

A uniform preparation and constancy o f experim entation i s

indicated*

Neopyrithiamine which was found to be considerably more

12k r

to x ic to animals than oxythiamine, i s apparently involved in a d iffe r e n t mechanism than oxythiamine.

The explanation fo r i t s e f f e c t in vivo may

he a ttrib u ted to a greater cap acity for storage on the part of neopyri­ thiam ine.

The in h ib itio n in t h is case may be brought about by the d is ­

placement o f thiamine or cocarboxylase in the tis s u e s w ith or without e ffe c t in g phosphorylation in v iv o .

I f the hypothesis o f Westenbrink

i s accepted, then t h is d iffe ren ce can be explained on the b a sis o f the d ifferen ce o f the enzyme systems obtained in v itr o and that in v iv o . It may w e ll be and i s even probable that in the animal body neopyrican compete w ith the vitam in, only in t h is case through the amino group to form a firm ly bound and in a ctiv e complex.

L

125 r

G eneral Summary I t has been found th a t in a b u ffe re d medium cocarboxylase can he deam inated w ith o u t d ep h o sp h o ry latio n .

A ttem pts to i s o l a te th e r e s u l t ­

in g oxythiam ine-diphosphate were u n s u c c e ss fu l.

The p re p a ra tio n of oxy-

th iam in e-d ip h o sp h ate was c a r r ie d out by a m o d ific a tio n o f K a rre r’s procedure and o b ta in e d in about 3Qfo y ie ld .

A study o f the chem ical

p r o p e r tie s of oxythiam ine-diphosphate re v e a le d th a t i t behaved a s d id cocarboxylase tow ards H HG1 a t 100°C and tow ards s u l f i t e .

S u lf ite

s p l i t oxythiam ine-diphosphate to g iv e th e o x y su lfo n ic a c id fragment and th e th ia z o le pyrophosphate fragm ent. The b eh av io r o f oxythiam ine-diphosphate towards N HC1 a t 100°C a ffo rd e d one pathway to th e p re p a ra tio n of oxythiamine-m onophosphate. An impure monophosphate was a ls o p rep ared by the deam ination o f co­ carb o x y lase in an aqueous medium. A new method f o r th e p re p a ra tio n o f th e th ia z o le a lc o h o l of thiam ine has been worked out w ith th e a id o f th e new reag en t LiAlH^. The th ia z o le a lc o h o l was o b ta in e d in about 50$ y ie ld and u ltim a te ly employed in th e s y n th e s is o f a new p y rim id in e -th ia z o le d e r iv a tiv e . S tu d ie s c a r r ie d out w ith mice showed th a t oxythiam ine-diphosphate and oxythiamine-monophosphate had about the same potency as thiam ine a n ta g o n is ts , as oxythiam ine i t s e l f .

In c o n tra s t to th ese hydroxy

p y rim idine d e r iv a tiv e s , neo pyrithiam ine was found to be approxim ately 50 tim es as to x ic to mice as oxythiam ine.

The in h ib itio n index f o r

neo p y rith iam in e was determ ined and found to be about 1.

The evidence

o b tain ed on a d m in is tra tio n o f neopyrithiam ine a t v ery low le v e ls in ­ d ic a te d t h a t th e extrem e p o tency o f neopyrithiam ine was due to a cu m u lativ e e f f e c t .

Bromoxythiam ine and th e new u r a c il- th ia z o le d e riv a -

126 r ~i t iv e were found not to be t o x ic to mice when adm inistered at high le v e l s . M icrobiological stu d ies were carried out w ith these thiamine ant­ a g o n ists, employing two organisms requiring thiamine as an e s s e n tia l growth fa c to r .

The a ctio n o f oxythiamine, oxythiamine-diphosphate

and neopyrithiamine on the growth o f Staph. aureus, and L, fermentum. was studied*

Oxythiamine and i t s diphosphate were found to be s ig n i­

f ic a n t ly more to x ic to Staph, aureus than neopyrithiam ine.

When the

a ctio n o f these d e r iv a tiv e s on the growth of L, fermentum. was studied, neopyrithiamine was fo$nd to be the most to x ic .

However, oxythiamine

and oxythiamine-diphosphate were found to be markedly in h ib ito ry to ­ wards t h is organism which in d ica ted that an amino group was not es­ s e n tia l fo r in h ib itio n . A study o f the e f f e c t o f thiamine an tagonists on cer ta in enzyme systems was made.

In v e stig a tio n s on urea syn th esis in normal and

thiamine d e fic ie n t mouse and rat liv e r s lic e s showed that thiamine was a contributory fa cto r in t h is mechanism.

The add ition o f thiamine

to d e fic ie n t s l i c e s augmented urea syn th esis s ig n if ic a n tly .

This

e f f e c t o f thiamine was in h ib ite d markedly by high le v e ls of oxythiamine. The sig n ific a n c e o f th ese r e s u lt s i s d iscu ssed . In h ib itio n stu d ies on the thiaminase o f clam and carp were car­ ried out with oxythiamine and neopyrithiam ine.

Oxythiamine was

found not to in h ib it the fa c to r present in clam ex tra cts under the con d ition s o f the t e s t .

Neopyrithiamine was found to be markedly

in h ib ito ry under the same co n d itio n s.

Under the proper con d ition s,

oxythiamine was found to be in h ib ito ry to the thiaminase o f carp. This antithiam ine was found to be r e a d ily destroyed by t h is fa c to r .

127 r

1

The regenerated carboxylase enzyme system o f yeast was found to be

in h ib ited by oxythiam ine-diphosphate.

Employing an a lk a li washed

yea st preparation as the source o f the enzyme system, a le v e l o f co­ carboxylase/oxythiam ine-diphosphate of l /6 0 produced about a 9Q$> in h ib itio n of pyruvate decarboxylation.

Oxythiamine, oxythiamine-

monophosphate, bromoxythiamine, and neopyrithiamine were not in h ib ito ry under th ese co n d itio n s.

In contrast to th is in h ib itio n , when a complete

enzyme system was. employed from whole yeast without a lk a lin e washing, oxythiamine-diphosphate was found not to in h ib it . Prelim inary in v e stig a tio n s on the enzymatic phosphorylation of oxythiamine by bakers and brewers yeast suggest that on prolonged in­ cubation, oxythiamine may have been about 1Wfo phosphorylated under the, con d ition s o f the t e s t .

Employing ana enzyme preparation from Prouion-

ibacterium nentosaceum. the enzymatic sy n th esis of cocarboxylase was found to be s ig n if ic a n tly in h ib ited by oxythiamine but not by neopyri­ thiam ine.

Sim ilar stu d ies were carried out w ith a p u r ifie d enzyme

preparation from brewers y e a st.

As In the former case, the sy n th esis

o f cocarboxylase from thiamine was found to be la r g e ly in h ib ited at a le v e l o f thiam ine/oxythiam ine o f 1/ 500.

Heopyrithiamine f a ile d to

in h ib it the enzymatic phosphorylation of thiam ine. of the data obtained i s d iscu ssed .

The sig n ifie a n e

A p o ssib le mechanism for the

in h ib itio n on the part of oxythiamine in vivo i s proposed on the b a sis of the r e s u lt s obtained.

L

-1

128 r

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-J

r Y ita Hame

Albert J . Eusebi

Date o f B irth

June 1, 1920

Elementary School

Public School 48 Brooklyn, H. I .

Graduated Junior High School Graduated High School Graduated Baccalaureate Degree Date Other Degree Date

Shallow Jr. High School Brooklyn, N. Y. June, 1935 Hew Utrecht High School Brooklyn, N. Y. June, 1938 Eordham U n iversity June, 1942 H.S.

Eordham U niversity

June, 1947

P u b lication s Anti thiamine E ffect o f Oxythiamine and Heopyri thiam ine. A Comparative Study. Albert J. Eusebi and Leopold R. Cerecedo Science, 110. 162, 1949 A Hew Method for the Preparation of the Thiazole Moiety of Thiamine. Albert J. Eusebii E l l i s Y. Brown and Leopold R. Cerecedo J . Am. Chem. Soc. 2L» 2931* 1949 In viv o and in v itr o Studies w ith Oxythiamine and Heopyrithiamine. Albert J. Eusebi and Leopold R . Cerecedo Federation Proc. % 169, 195© F ailure o f F o lic in Mice and Rats Albert