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Studies on Vitamin B6 Deficiency in Rats and Mice

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FORDHAM UNIVERSITY G r a d u a t e S c h o o l o f A r t s a n d S c ie n c e s

February 1«___ 19.5.0.

This dissertation prepared under my direction by _______ Edward C. DeRenzo entitled

S tu d ie s on V itam in

D eficiency in Rats and Mic e .

has been accepted in partial fulfilment of the requirements for the Degree of..

Doctor o f Philosophy ......................................................

________ LeopoId R. Gerecedo____ ( Faculty Adviser)

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STUDIES ON VITAMIN

DEFICIENCY IN RATS AND MICE

BY EDWARD C. DE RENZO B«S* FORDHAM COLLEGE 194-5 M*S*. FORDHAM UNIVERSITY 1947

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 1950

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ProQuest Number: 10992951

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 10992951 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

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TABLE OF CONTENTS Page L i s t o f Tables * • * * * . . L i s t o f F ig u res

* • * • • . .

* * * . * . * * .

. . . * V

• • * * * * * • * * * * * * • * • • • • * • • • *

V III

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . I X I n tro d u c tio n

I

P a r t I - S tu d ie s on Vitam in C hapter I

C hapter I I

D eficiency in th e Rat • • * • • • *

- The E f f e c t o f Sulfur-Amino Acid Supplem entation on V itam in B^ D e fic ie n t Rats R eceiving a Low Casein D ie t » ^

A

.

A

- The E f f e c t o f C e rta in S u lfu r F ree Amino Acids on Rats R eceiving a Low C asein, Vitamin B^ F ree D iet* * * * * * * * * * * * * » * * * * « * * » 1 2

C hapter I I I - A Comparison o f th e E f f e c t o f C asein, M ethionine and Tryptophan on th e X anthurenic Acid E xcretio n and G eneral W elfare o f th e Vitamin B& D e fic ie n t Rat • * * * * * • * * * * * • * • * • • * * • * • • * 1 9 C hapter IV

- A Comparison o f th e E f f e c t o f C asein, L a c talbum in and F ib rin on th e Vitam in Be D e fic ie n t R a t .......................................................................................................28

C hapter V

- The E f f e c t o f Bromobenzene on th e Vitamin D e fic ie n t Rat* * * • * * • * • * » • • • • * * * • *

C hapter VI P art I I

- S tr a in D iffe ren ces in th e R esistan ce o f Rats to V itam in B^ D eficien cy • • • * * * * * * * • *

33

**51

- S tu d ie s on Vitam in B& D eficiency in th e Mouse*. * * 56

C hapter VII - The P ro d u ctio n o f th e Vitamin B^ D eficiency Syndrome in th e Mouse. . . • * . * . . . * . . * • * 5 6 Chapter V III- The E f f e c t o f Casein and Thio-Amino Acids on Mice R eceiving a Vitam in b6 Free D iet C ontaining D esoxypyridoxine . . . . . . . . . . . . C hapter IX

- A Comparison o f th e E ffe c ts o f Desoxy and M ethoxypyridoxine in th e Mouse. . . . . . . . .

63 67

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TABLE OF CONTENTS (Coni^d) Page Chapter X -

S tu d ies on th e P yridoxine Requirement o f th e Mouse and on th e I n h ib itio n Index o f D esoxypyridoxine in th e Mouse

G eneral Summary and Conclusions • . * * * * B ib lio g rap h y

73

• . . * * . * * 8 0 *• .. •.8 2

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L i s t o f Tables Page Table I

-

Composition o f th e D iets Used in S tu d ies on th e E ffe c t o f Thio-amino Acids on th e Vitam in

Table I I

-

D e fic ie n t Rat

6

Data Showing th e D e le te rio u s E ffe c t of th e Thio-amino Acids on th e Vitamin D e fic ie n t Rat

Table I I I

-

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

8

Composition o f th e D iets Used in S tu d ie s on th e E f f e c t o f S u lfu r F ree Amin Acids on th e Vitamin

Table IV

-

D e fic ie n t Rat

15

Data Showing th e E f f e c t o f Tryptophan, T y ro sin e, S erin e and Lysine on th e Vitam in B^ D e fic ie n t Rat

Table V

-

16

Composition o f th e D iets Used in S tu d ies on X anthurenic Acid E x cretio n by th e V itam in

Table VI

-

R a t ........................................... * ........................... 20

X anthurenic Acid E x cretio n o f Vitamin B^ D e fic ie n t R ats R eceiving Two D if fe r e n t Levels o f Casein and C asein Supplemented w ith e i t h e r Tryptophan o r M ethionine . , . ,

Table VII

-

X anthurenic Acid E x c retio n o f Vitamin B^ D e fic ie n t Rats R eceiving Two D if fe r e n t Levels o f C asein, and a D iet C ontaining C asein Sup­ plem ented w ith e i t h e r Tryptophan o r M ethionine „ * 23

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Page - Composition o f th e D iets Used in Comparing th e E ffe c ts o f C asein, F ib rin and Lactalbum in on th e Vitamin B6 D e fic ie n t Rat

Table IX

th e s t a r t o f th e experim ent, th e anim als receiv ed t h e i r re s p e c tiv e d i e t s supplem ented w ith 1 gamma p y rid o x in s HC1 p e r g . o f d i e t f o r a p re lim in a ry b a s a l p e rio d o f 6 days*

X anthurenic a c id

d e te rm in a tio n s were then run on th e 24 hour u rin e samples o f each group a t

th e end o f th e b a s a l p e rio d and weekly f o r 3 weeks th e r e ­

a f t e r by th e method o f M ille r and Baumann ( 2 ) .

S u ita b le re c o v e rie s

were o b tain e d b o th in p ure s o lu tio n and in r a t u rin e w ith an an aly ­ t i c a l l y pure sample o f x a n th u ren ic a c id p rep ared by th e method o f Musajo and M in c h illi - (C a lc u la te d 0-58*05, H-3*71, N-7*10 Found C-58*54> £i-3*42, N-6*84) (33) •

The food in ta k e s were measured on th e

day o f each u r in e c o lle c tio n and th e r e s u lt s a r e expressed as m icrograras o f x a n th u re n ic a c id e x c re te d p e r g ra in o f good in g ested *

This

e n t i r e experim ent was re p e a te d on a d u p lic a te s e t o f 12 r a ts * T ables VI and VII show th e d a ta o f th e s e experim ents* I t may be seen t h a t th e r a ts re c e iv in g th e 15$ c a se in d i e t supplem ented w ith m ethionine e x creted approxim ately th e same amount o f chromogen as th e unsupplem ented group re c e iv in g th e same l e v e l o f casein *

Supplem entary try ptophan caused a pronounced in ­

c re a s e in th e e x c re tio n o f t h i s u rin a ry chromogen w hile doubling th e c a s e in le v e l a ls o r e s u lte d in an in c re a se d u r in a r y o u tp u t o f xan­ th u re n ic acid *

In th e f i r s t experim ent, doubling th e c a se in le v e l

d id n o t cause -foe p ro d u ctio n o f a s much x an th u ren ic a c id as th e try p to p h an supplem ented group, w hile t h i s was re v ersed in th e d u p li­ c a te experim ent*

Except f o r t h i s l a t t e r d isc rep an cy , th e d a ta o f

b o th experim ents a re com pletely comparable* -

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T able VI (Experim ent 1)

X anthurenic a c id E x cretio n o f Vitamin

D e fic ie n t Rats

R eceiving two D if fe r e n t L evels o f C asein, and a D iet C ontaining C asein Supplemented w ith e i t h e r Tryptophan o r M ethionine (expressed a s micrograms o f X anthurenic Acid ex creted p e r g . o f D ie t Consumed on Day o f U rine C o lle c tio n ) . D iet Day

P-1M

P-3M

P-4M

P-7M

B asal P eriod*

2 8 ,0

2 9 .5

68.0

37a

7 th

2 29.6

88.0

295.9

86.5

H th

178.9

105.5

312.0

98.7

21st

179.6

119.8

460.2

89.0

Beginning Signs o f Acrodynia Day

20

> 50

>50

19

>80

38

S u rv iv a l Time Day

>80

41

* - O btained on th e l a s t day o f a 6 day b a s a l p e rio d d u rin g which tim e th e r a t s o f a l l groups receiv ed 1 gamma p y rid o x in e H C l/g. d i e t .

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Table VII (Experim ent 2)

X anthurenic Acid E x c retio n o f Vitamin

D e fic ie n t Rats

R eceiving two D if fe r e n t L evels o f C asein, and a D iet C ontaining C asein Supplemented w ith e i t h e r Tryptophan o r M ethionine (ex p ressed as micrograms o f X anthurenic Acid ex creted p e r g . o f D iet Consumed on Day o f U rine C o lle c tio n )* D iet Day

P-1M

P-3M

P-4M

P-7M

Basal P erio d *

38*5

2 5 .0

50*5

30*5

7 th

201*5

95

225.5

80

H th

250.5

85

275

89

21st

265

75*9

235-2

95.7

Beginning Signs o f Acrodynia Day

20

>50

> 50

20

>80

39

S u rv iv a l Time Day

43

>80

* - O btained on th e l a s t day o f a 6 day b a s a l p e rio d d u rin g which tim e th e r a ts o f a l l groups receiv ed 1 gamma p y rid o x in e HCl/g* diet*.

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The pronounced effect of the thio-amino acid on the defic­ iency symptoms and survival time of the deficient rats is noteworthy. On the other hand, though xanthurenic acid excretion is stimulated in the tryptophan supplemented group, the animals remained free of symptoms for a much longer period of time and survived as long as unsupplemented controls. Discussion: The data of these experiments give a quantitative interpre­ tation of the experiments with tryptophan presented in the previous chapter.

It is clear

that tryptophan supplementation causes an in­

crease in chromogen excretion without affecting the general condition of the rat.

On the other hand, methionine (and presumably other thio-

amino acids) exerts a pronounced detrimental effect on the general condition of the rat without influencing the urinary excretion of xan­ thurenic acid.

It appears therefore, that one may overload the mechan­

isms of tryptophan metabolism in Vitamin

deficiency and thus cause

a greater excretion of xanthurenic acid without affecting the general health of the animal.

It seems justifiable to conclude, therefore,

that xanthurenic acid excretion is not proportional to the severity of the vitamin

deficiency state, and its determination cannot be taken

as a measure of the gravity of the B^ deficiency state. The data also discount the possibility of tryptophan being the amino acid responsible for the toxic effect of high protein diets

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on v itam in

d e f ic ie n t r a ts *

I t would seem more l i k e l y t h a t t h i s

d e le te r io u s e f f e c t i s due m ainly to th e presence o f a d d itio n a l t h i o amino acid * The f a c t t h a t in one experim ent an a d d itio n o f try ptophan s tim u la te d a g r e a te r p ro d u ctio n o f x a n th u ren ic a c id , and in th e second experim ent a l e s s e r p ro d u ctio n than an e q u iv a le n t amount o f c a s e in , i s v e ry d i f f i c u l t to in te r p r e t*

In any ev en t, th e r e s u lts were con­

s i s t e n t w ith in th e o th e r groups*

I t must be borne in mind t h a t c o l­

l e c t i o n o f u rin e f o r a 2U hour p e rio d does n o t in s u re a tr u e ZU hour u rin e u n le ss c a th e te r iz a tio n methods a r e undertaken* tech n iq u e was n o t u t i l i z e d in th e se experim ents*

Such

I t i s p o s s ib le

t h a t t h i s would in tro d u c e an e r r o r in such an experim ent* Of i n t e r e s t in connection w ith t h i s study i s th e e n t ir e ly comparable stu d y o f M ille r and Baumann on th e mouse (.2) * These workers concluded t h a t in c re a s e s in th e e x c re tio n o f chromogen were q u a n t i t a t i v e l y s im ila r w hether L -tryptophan was fed as th e amino a c id o r as a component o f casein *

In c o n tr a s t to our experim ents

on th e r a t , th e y claim th e L -try p to p h an decreased th e s u rv iv a l tim e o f mice d e f ic ie n t in p y rid o xine*

However, t h i s e f f e c t was n o t a s

g r e a t as th e d ecrease of s u rv iv a l tim e observed when c a s e in o f equiva­ l e n t try p to p h an c o n te n t was fed*

They conclude, ^A pparently, th e r e ­

f o r e , o th e r amino a c id s th an try p to p h an a ls o c o n trib u te d to th e i l l h e a lth o f th e p y rid o x in e d e f ic ie n t mice11*

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A lso, o f i n t e r e s t in connection w ith our s tu d ie s on xan­ th u re n ic a c id e x c re tio n i s a paper by P o r te r e t a l (34)*

These

w orkers have shown t h a t x a n th u re n ic a c id e x c re tio n i s in c re a se d in vitam in d e f ic ie n c ie s o th e r than v itam in B^.

Thus, in thiam in d e f ic ie n t

r a t s , x an th u ren ic a c id was e x c re te d in g r e a te r amounts*

This would

appear to s tre n g th e n our view t h a t th e amount o f x an th u re n ic a c id ex­ c re te d i s n o t p ro p o rtio n a l to th e s e v e r ity of th e v itam in

d e fic ie n c y

s ta te * Summary and Conclusions:

(1) Vitam in B^ d e f ic ie n t r a t s re c e iv in g a 30$ c a se in d i e t o r a 15$ c a s e in d i e t supplem ented w ith an amount o f try p to p h an equiv­ a l e n t to t h a t p re s e n t in a 30 $ c a s e in d i e t , e x c re te more x an th u ren ic a c id th an th o se re c e iv in g th e 15$ c a se in d i e t supplem ented w ith m ethionine o r th e 15$ c a s e in d i e t alone* (2) This confirm s th e fin d in g t h a t x a n th u ren ic a c id ex­ c r e tio n i s p ro p o rtio n a l to d ie ta r y tryptophan* (3) D esp ite th e augmented x a n th u ren ic a c id e x c re tio n , th e try p to p h an supplem ented r a t s su rv iv ed a s long a s c o n tr o ls , w hile th e m ethionine fe d r a t s , though e x c re tin g th e chromogen in q u a n titie s s im ila r to th e c o n tr o l group, developed le s io n s more q u ic k ly and succumbed much soonder th an both th e c o n tro l and try p to p h an supplemented group* (4) This i s tak en to mean t h a t x an th u ren ic a c id e x c re tio n cannot be tak e n a s a measure of th e s e v e r ity o f th e v itam in B^ -

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d e fic ie n c y state* . (5) The harm ful e f f e c t o f m ethionine supplem entation in Bg> d e f i c i e n t r a t s i s p ro b ably n o t o p e ra tiv e v ia an im paired tr y p to ­ phan metabolism*. (6) High p r o te in d i e t s a r e n o t harm ful to

b6

d e f ic ie n t

r a t s because o f th e in c re a se d amount o f try p to p h an in g e s te d .

It

i s more p ro b ab le t h a t t h i s e f f e c t i s brought ab o u t by th e in ta k e o f la r g e r amounts o f m ethionine (o r o th e r S amino a c i d ) .

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CHAPTER IV: - A Comparison of the Effect of Casein, Lactalbumin and Fibrin on the Vitamin

Deficient Rat*

Introductions In connection with studies on the harmful effect of pro­ tein on the vitamin

deficient rat, the idea of comparing the

effects of different proteins came to mind.

Since our previous

experiments indicated a specific toxicity of S amino acids, it was particularly desirable to compare the effect of a sulfur *rich* protein and a sulfur Mpoor® protein.

Lactalbumin and casein were

suitable choices for such a study, the former containing a high percentage, the latter a small percentage of the thio-amino acids.

The e f f e c t o f f i b r i n was a ls o s tu d ie d s in c e t h i s p r o te in c o n tain s a co m p arativ ely la r g e amount o f try p to p h a n .

A ll 3 p r o te in s were fed

a t th e same l e v e l , v i z . , 15$ o f th e d i e t . Experimental and Results:

The ex p erim en tal procedure was th e same a s b e f o r e .

Only

Sherman o r W istar r a t s w eighing 30-36 g . were used in th e s e s tu d ie s . L i t t e r mates were chosen whenever p o s s ib le . stu d y a r e shown in Table V I I I .

The d i e ts used in t h i s

The amino a c id p a tte r n s o f c a s e in ,

la cta lb u m in and f i b r i n a r e shown in Table IX . Microbiological assay of the proteins used in this study by the yeast method of Atkins et al (35) gave the following results expressed as gamma per g. - Casein - 0.025;

Lactalbumin - 0.067;

Fibrin - 0 .0 5 9 .

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Table VIII Composition o f th e D iets Used in Comparing th e E ffe c ts o f C asein , F ib rin and Lactalbum in on th e Vitamin B^ D e fic ie n t R a t. C o n s titu e n t

P-3

Casein ^

15

Lactalbum in F ib rin

2

3

P-L-3

-

P-F-3

15

-

15

S a lts

7

7

Hydrogenated veg. f a t

5

C*Lj O.

3

Supplements p e r Kg. Thiamin, R ib o flav in - U mg. each Ca P a n to th e n a te - 20 mg. C holine - 1 g . 1.

Labco

2.

O btained from th e Borden Company

3-

O btained from N u tr itio n a l Biochem icals

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Table IX

Amino Acid Com position o f C asein, F ib rin and Lactalbumin*. Amino Acid

Casein

1

1 Fibrin

Methionine

3-5 ± 0 .3

2*2

3*1

Cystine

0.36 ~ 0 .0 4

1 .9 - 0 .4

4-*2

Lysine

6 .9 - 0 .7

7 .5

9*6

Tryptophan

1 .8 ± 0 .2

3 .4 i 0 J,

2*6

Threonine

3 .9 ± 0 .1

7 .9

7*4-

Leucine

12.1

14.3 - 3 .9

10*11

Isoleucine

6*5

5 .0 t 0.5

6*4-

Phenylalanine

5*2 ± 0*5

7 .0

5*4-

Tyrosine

6*4- - 0*4.

5.1 - 0 .7

4-4-

Histidine

2*5 - 0*3

2 .3 i o a

2*1

Valine

7*0

3 .9 ± 1.8

6.4-

Arginine

4-*l

6 .8 - 0.6

3*8

1*

-

0*2

2 Lactalbumin

F ig u res acco rding to Block and B o llin g “ The Amino Acid Composition o f P ro te in s and Foods®* - C harles C* Thomas P u b lish e r - 194-7*

2.

F ig u res according, to th e Borden Company - pro­ ducers o f L actalbum in.

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The r e s u lts o f th e s e experim ents a re shown in Table X*

It

may be seen t h a t c a se in and f i b r i n d isp la y e d ab o u t th e same e f f e c t on th e v itam in

d e f i c i e n t r a t*

On th e o th e r hand, th e r a ts t h a t

receiv ed la cta lb u m in a t th e same l e v e l d isp la y e d th e d e fic ie n c y sumptoms sooner and d ied more q u ic k ly th an th e p a ts o f th e o th e r two groups*

I t seems c le a r t h a t th e la ctalb u m in d i e t has th e most sev ere

e f f e c t on

d e f i c i e n t r a ts * I t i s b e lie v e d t h a t th e s e r e s u l t s b e a r o u t our previous

fin d in g s on th e d e le te r io u s e f f e c t o f th io -am in o a c id s when fe d as such*

N otw ithstanding th e f a c t t h a t th e am ino-acid p a tte r n s of th e

3 p r o te in s d i f f e r in more th an th e d i s t r i b u t i o n o f S amino a c id , we b e lie v e th e d iffe re n c e s in r e s is ta n c e d isp la y e d by th e r a t s may be v a lid ly in te r p r e te d in term s o f th e c y s tin e and m ethionine content* I f t h i s assum ption i s v a l i d , th en i t fo llo w s t h a t thio-am ino a c id s ex­ e r t a harm ful e f f e c t on th e v itam in B^ d e f ic ie n t r a t s n o t on ly in th e f r e e s t a t e b u t a ls o when th ey a r e bound in th e p r o te in m olecule* Summary and C onclusions: (1) A comparison o f th e e f f e c t o f 3 d i f f e r e n t p ro te in s on v itam in B& d e f i c i e n t r a t s i s p re s e n te d . (2). Lactalbum in i s found to have a more harm ful e f f e c t ■than c a s e in o r f i b r i n . (3) A p o s s ib le e x p la n a tio n , based on th e d iff e r e n c e o f S amino a c id c o n te n t o f th e th re e p r o te in s , f o r th e d i f f e r e n t e f f e c t s , i s d iscu ssed * -3 1 L

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Table X

Data Showing the Effects of Casein, Fibrin and Lactalbumin on the Vitamin

Deficient Rat*

# of Rats

Beginning Signs of Acrodynia (Day)

Survival Time (Day)

8

>50

>80 *

P-F-3

10

>50

>80 b

P-L-3

11

30

Diet

P-3

a*

46

6 o f th e s e r a t s were d isc o n tin u e d w ith on ly m ild ac ro d y n ia on th e 120th day*

The o th e r A su rv iv ed

beyond 79 days* b*

A o f th e se r a t s were d isc o n tin u e d w ith on ly m ild acro d y n ia on th e 320th day*

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CHAPTER V - The Effect of Bromobenzene on the Vitamin Deficient Rat*

PART 1 - S tu d ies on th e O ral A d m in istratio n o f Bromobenzene* In tro d u c tio n : The d a ta of th e experim ents o f th e p rev io u s c h ap ters p o in te d to a m etabolic i n t e r r e la t io n s h ip between vitam in B^ and th e thio-am ino a c id s*

The e x iste n c e o f such a connection le d us

to stu d y th e e f f e c ts o f a compound known to a f f e c t s u lf u r metab­ olism *

Bromobenzene serv ed th e purpose s in c e i t i s known to be

d e to x ifie d by th e an im al organism a s th e phenylm ercapturic a c id , th u s in v o lv in g i t s e l f in th e metabolism o f c y s te in e (36, 37) * The co n ju g atio n o f c y s te in e w ith bromobenzene i s d e p ic te d a s fo llo w s:

w s- ch2

S

CH,

CH3COOH

HC-NH,

>

h

COOH

I

c-itfco eH j}

COOH

Or

Br

Experimental and Results:

Experim ents were perform ed in v o lv in g both p a r e n te r a l and o r a l a d m in is tra tio n o f bromobenzene*

In th e feed in g experim ents, bromo­

benzene was fe d a t le v e ls o f 1*96 and 0*98$ o f th e d ie t*

The bromo­

benzene was r e d i s t i l l e d and th e f r a c tio n b o ilin g between 155°-156°C. -3 3 L

r

n

c o lle c te d f o r u s e .

The b a s a l d i e t used in th e s e s tu d ie s i s th e high

p r o te in d i e t shown in Table X I. Weanling r a t s o f th e Sherman and W istar s t r a i n s , weighing 3 0 -3 6 g . were p lac ed on th e v itam in

f r e e d i e t o f Table X I.

P revious

work in our la b o r a to ry (see n e x t Chapter) had shown t h a t u t i l i z a t i o n o f th e above procedure causes young r a t s to develop th e acro d y n ia w ith­ in 15 to 20 days*

I t was d ecid ed , th e r e f o r e , to supplem ent th e d i e t

on th e 13th day w ith bromobenzene.

One group receiv ed a supplem ent of

2 g . o f bromobenzene added to 100 g . o f th e b a s a l d i e t .

The bromo­

benzene was w ith h eld from a t h i r d group which serv ed a s c o n tr o ls .

All

anim als were allow ed food and w ater ad l i b . The r e s u l t s o b tain ed w ith bromobenzene prompted us to stu d y th e e f f e c t o f n ap th a len e a d m in iste re d in th e same way s in c e t h i s compound i s a ls o known to conjugate w ith c y s te in e to form a m ercap tu rie a c id ( 3 8 ).

In experim ents w ith t h i s compound, th e naptha­

le n e (m .p. 80°C ., o b tain ed from Eimer and Amend) was added to th e basal d ie t. In a second s e r i e s o f experim ents, Weanling r a t s of th e W istar s t r a i n were grown f o r a p e rio d o f 2-3 months on th e b a s a l d i e t supplem ented w ith 0 .5 to 1 gamma py rid o x in e flCl p e r g . o f d i e t .

Only

sm all amounts o f p y rid o x in e were given to th e s e r a t s to a llo w growth b u t w ith a minimal amount o f s to r a g e .

At t h i s tim e, th e y were sep­

a r a te d in to 2 groups, one re c e iv in g th e b a s a l d i e t , th e second re ­ c e iv in g th e b a s a l d i e t p lu s 10 gamma p y rid o x in e p e r g . o f d i e t (th e se

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Table XI

Composition o f th e D iet Used in S tu d ie s on th e E f f e c t o f Bromobenzene on th e Vitam in

D e fic ie n t Rat*.

D iet* C o n s titu e n t

P -l

C asein

30

Sucrose

55

S a lts

7

Hydrogenated veg* f a t

5

C*L*0*

3

Supplements added p e r Kg* Thiam in, R ib o fla v in - 10 mg* each Ca P an th o th en ate - 4.0 mg* * - D ie t

P -B ^ -l =100 g* P - l p lu s 2 g . bromobenzene

D ie t P -B ^ -l

= 100 g* P - l p lu s 1 g* bromobenzene

D ie t P -N -l

= 100 g* P - l p lu s 1 g* n ap th alen e

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r

I serv ed a s c o n tr o ls ) *

The anim als deprived o f p y rid o x in e developed

le s io n s w ith in a few weeks, l o s t much w eight, and t h e i r u rin e d is ­ played

a good q u a l i t a t i v e t e s t f o r x an th u ren ic a c i d .

?fhen th e

acro d y n ia was r a th e r se v ere in th e d e f ic ie n t group, th e anim als o f both groups were given a supplem ent o f 2 g . bromobenzene added to 100 g . o f d i e t .

The bromobenzene supplement was w ith h eld from d ef­

i c i e n t c o n tr o ls . The r e s u l t s of the f i r s t experim ent a r e summarized in T able X I I.

I t may be seen t h a t th e b e n e f ic ia l e f f e c t o f th e bromo­

benzene i s more pronounced on th e development o f th e d e fic ie n c y symptoms th an i t i s on th e s u rv iv a l tim e .

In th e m a jo rity o f c a s e s ,

th e r a t s re c e iv in g th e bromobenzene developed th e symptoms o f v i t a ­ min

d e fic ie n c y l a t e r th an c o n tr o ls , and in most c a s e s, th e acro ­

dynia was o f a s tr i k i n g l y l e s s sev ere n a tu r e .

In some c a s e s, we

have observed anim als a t d e a th e x h ib itin g a v ery m ild a c ro d y n ia , w hile i t must be ad m itted t h a t in a few c a s e s , th e bromobenzene d id n o t ap­ p e a r to e x e rt any a p p re c ia b le e f f e c t . F ig u re I shows an anim al which re ceiv ed th e b a s a l d ef­ ic ie n t d ie t.

This r a t e x h ib its th e sev ere acro d y n ia o f th e f o re

and h ind paws, s p a s tic g a i t , edema o f paws, e t c . and may be co nsidered a t y p i c a l example o f a v itam in

d e f ic ie n t r a t .

F ig u re I a ls o shows

a l i t t e r mate o f th e d e f i c i e n t r a t which receiv ed a supplem ent o f bromobenzene on th e 1 3 th day o f th e ex p erim en tal p e r io d .

These p ic tu re s

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Table XII

Data Showing th e E f f e c t o f Bromobenzene and N aptha le n e on th e Vitam in

Dl e t

#-

Rats

D e fic ie n t Rat*

Food In ta k e ( g ./r a t /d a y )

S u rv iv a l Time (Day)

P -l

10

3 .8

19

45

P-B*VL

11

3 .7

37

52

P-B1- !

T2

3 .9

45

60

P -N -l

9

3 .6

47

62

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Beginning Signs o f AcrGdynia (Day)

Figure 1.

P ic t u r e o f a young r a t which r e c e iv e d th e b a s a l d e f ic i e n t d ie t fo r 30 d ays. N ote th e hunched p o s it io n and le s i o n s o f th e fo r e and hind paws and sn o u t.

L itte r m a te o f above r a t w hich r e c e iv e d th e b a s a l d ie t f o r 12 days and th en a supplem ent o f bromobenzene f o r 18 d ays. Rat i s s tu n te d in growth b u t o th e r w ise norm al.

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were taken a f t e r both anim als had been on th e d ie ta r y regime f o r

30 days a t which tim e th e unsupplem ented, d e f ic ie n t r a t e x h ib ite d a sev ere s t a t e o f v itam in

in s u f f ic ie n c y .

The d e f ic ie n t r a t re ­

c e iv in g bromobenzene, on th e o th e r hand, d isp la y e d none o f th e s e symptoms a t t h i s tim e .

The b e t t e r g e n e ra l c o n d itio n o f t h i s r a t

i s e v id e n t. The d a ta o f Table XII a ls o show t h a t n ap th alen e adm inis­ t r a t i o n induces th e same p r o te c tiv e e f f e c t .

I t i s in t e r e s t i n g t h a t

t h i s compound fo llo w s th e same m etabolic p a th , a t l e a s t in p a r t , as does bromobenzene. O cc a sio n a lly , we have a ls o observed th e developm ent o f m ild le s io n s in th e r a t s re c e iv in g th e supplemented d i e t ab o u t th e 18 -2 0 th d a y .

This c o n d itio n , however, c le a r s w ith in a s h o r t tim e

w ith th e integum ent re tu rn in g to a normal s t a t e and may probably be a t t r i b u t e d to a p e rio d o f ad ju stm en t o f th e anim al to th e bromo­ benzene d i e t . Four p o s itiv e p a ir - f e d c o n tr o ls , re c e iv in g p y rid o x in e and bromobenzene e x h ib ite d no i l l e f f e c ts throughout th e experim ental p e rio d (85 d a y s ).

One such c o n tr o l d i e t o f s ta r v a tio n , however, on

th e 35 th d ay . In our second s e r ie s o f experim ents, we have a ls o observed a b e n e f i c i a l e f f e c t o f th e bromobenzene.

Four a d u lt r a t s given

bromobenzene a f t e r th e developm ent o f th e acrodynia p re se n te d a b e t t e r c o n d itio n o f th e sk in w ith in a p e rio d o f 2 weeks than u n tre a te d -3 9 L

-J

c o n tro ls *

In two c a s e s , th e r e was a complete r e s to r a tio n o f normal

integum ent, a regrow th o f h a ir on th e paws and co n sid e ra b le le s s e n ­ in g o f th e edema a s s o c ia te d w ith th e syndrome.

This b e n e f ic ia l e f­

f e c t could be re v erse d by re tu rn in g th e anim al to th e b a s a l d i e t , whereupon th e le s io n s a g a in a p p eared . At th e tim e th e f i r s t experim ents were perform ed, (th e re­ s u l t s o f which a r e shown in Table X II) a tte m p ts were made to determ ine th e e x te n t o f m ercap tu ric a c id s y n th e s is by th e se r a t s .

This was

done by an a ly z in g th e u r in e o f r a ts re c e iv in g bromobenzene f o r p-bromophenyl m ercap tu ric a c id by th e method o f S te k o l ( 3 9 ).

Re­

co v e rie s w ith a sample o f p-bromophenyl m ercap tu ric a c id o b tain ed from D r. S te k o l ranged from 90-96%.

These d a ta a r e shown in Table X I I I .

Our r e s u l t s in d ic a te d t h a t th e vitam in

d e f ic i e n t r a t i s capable o f

d e to x ify in g bromobenzene to th e same e x te n t a s p a ir - f e d c o n tro ls re­ c e iv in g p y rid o x in e .

F urtherm ore, in agreem ent w ith S te k o l1s fin d in g

( 4-0 ) , th e r a t s re c e iv in g a s m a lle r dose o f bromobenzene were more capable o f e lim in a tin g t h i s compound a s th e phenylm ercapturic a c i d . However, under th e s e c o n d itio n s , th e e x te n t o f d e to x ic a tio n amounts to o n ly 10 to 20 % o f th e t h e r e t i c a l based on th e amount o f in g e ste d bromobenzene ( 10% in th e case o f th e r a ts re c e iv in g 1 . 96% bromobenzene and 20% in th e case o f th e r a t s re c e iv in g 0.98 bromobenzene.

R esu lts

a r e to be tak en a s o n ly approxim ate valu es s in c e th e u rin e o f only a few r a ts in each group was a n a ly z e d ).

Table X III

Recovery of p-bromophenylmercapturic Acid* (Added to Vitamin B£ Deficient Rat Urine)

Amount Added (mg.)

Amount Determined (mg.)

% Recovery

3*18

3*05

96

1-59

1-43

90

2.385

2-21

92-5

3-18

2 .8 9

91

1 - This sample was g en ero u sly su p p lie d by D r. S te k o l. m .p . - 151°C- C a lcu lated C-41-51* H-4-01, N-4-40 Found C-41-15* H -3-77, N-4-52

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Even t h i s r e l a t i v e ly sm all e x te n t o f m ercap tu ric a c id s y n th e s is may be s i g n i f i c a n t , however*.

As n e a rly as could be a s ­

c e r ta in e d , th e food in ta k e of r a t s on th i s experim ental regime i s ap p ro x im ately 3 to A g* p er d ay .

I t may be c a lc u la te d t h a t

t h i s corresponds to a d a ily in ta k e o f 25*^1 to 33*88 mg* c y s te in e under our ex p erim en tal c o n d itio n s*

(This c a lc u la tio n i s based on

th e p resen ce o f 3 *0$ m ethionine and 0 *4$ c y s tin e in c a se in - f ig u r e s acco rd in g to th e Borden Company, m anufacturers o f Labeo c a se in and a conversion o f th e S o f th e s e amino a c id s to c y s te in e S*) Roughly, 10$ o f th e in g e ste d bromobenzene a t th e h ig h e r le v e l o f a d m in is tra tio n ( 1 *96$) was found to be d e to x ifie d a s th e p h en y lm ercap tu ric a c id which corresponds to a removal o f 6*5 mg* c y s te in e p e r day*

This means t h a t in term s o f d ie ta r y in ta k e approxim ately

20- 25 $ o f th e in g e ste d c y s te in e i s d iv e rte d from i t s normal m etab o lic p a th and removed as th e m ercapturic acid *

In th e case o f th e low er

l e v e l o f a d m in is tra tio n o f bromobenzene ( 0 *98$ ) , v alu es c lo s e r to 20$ were found to be e x c re te d a s th e m ercap tu ric a c i d .

This co r­

responds to th e same q u a n tity o f c y s te in e removed d a ily a s in th e case o f th e h ig h e r l e v e l o f a d m in istra tio n * In addition to the experiments reported above, we have also studied the effect of bromobenzene when given with cystine

to v itam in B. d e f i c i e n t r a t s re c e iv in g a low c a s e in (15$) d i e t , o Under these conditions, the same beneficial effect of the bromoben­ zene manifests itself*

r

"i

Finally, we have also studied the effect of applying bromobenzene to the paws of vitamin B^ deficient rats to determine the extent of absorption, if any, and detoxication by such route*. Our findings suggest that application of bromobenzene in that manner, exerts no beneficial effect when administered before, at the inception of, or after the appearance of the acrodynia of vitamin deficiency*. Discussion: The data of our experiments point to a beneficial effect of bromobenzene on the vitamin B^ deficient rat*. Thus, the addition of bromobenzene to the diet of rats receiving a 30$ casein vitamin B^ free diet, before the appearance of the vitamin B& deficiency syn­ drome, resulted in a prolongation of the time necessary to cause the production of these symptoms in the rat*. Figure I attests

to this

as do the data of Table Jil­ in trying to explain this protective effect of bromobenzene, the idea suggests itself that removal of cysteine as the p-bromophenylmercapturic acid is responsible for this action*

Thus, by

coupling with bromobenzene, the amount of tissue sulfur amino acid for normal metabolic processes is lessened, manifesting itself in a beneficial effect on the integument of the rat*

This hypothesis is

strengthened by our findings on the detrimental effect of sulfur amino acids on the vitamin B^ deficient rat.

The further obser-

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1

v a tio n t h a t a s im ila r b e n e f ic ia l e f f e c t i s o b tain ed when bromo­ benzene i s given w ith c y s tin e to v itam in B^, d e f i c ie n t r a ts re c e iv ­ in g a low p r o te in d i e t , a ls o s tre n g th e n s our h y p o th esis*

F in a lly ,

th e r e s u l t s w ith n ap th alen e a ls o su p p o rt t h i s co n ten tio n *

I t is

i n t e r e s t i n g , in t h i s co n nection a l s o , to n o te th e o b serv a tio n o f Moxon e t a l (41) who were a b le to in c re a s e th e o u tp u t of selenium in s e le n iz e d s te e r s by in je c tin g bromobenzene to form th e Se analogue o f th e m ercap tu ric a c id *

Here a l s o , a b e n e f ic ia l e f f e c t i s observed

w ith a d d itio n o f bromobenzene* The f a c t t h a t on ly about \0% to 20% o f th e ad m in istered bromobenzene could be accounted f o r as u r in a ry m ercap tu ric a c id , i s d i f f i c u l t to c o r r e la te w ith t h i s h y p o th e sis, however.

I t i s to

be n o te d , t h a t th e e x te n t o f m ercap tu ric a c id s y n th e s is was a s sm all in th e anim als o f a l l groups* as w e ll as d e f ic ie n t r a t s . )

( i . e . , r a t s re c e iv in g p y rid o x in e

S te k o l (42) l i s t s th e follow ing f a c to r s

as in flu e n c in g th e e x te n t o f m ercap tu ric a c id form ation from bromo­ benzene;

th e body w eight o f th e an im al, th e dose o f bromobenzene

a d m in iste re d , th e mode of a d m in is tr a tio n , and the d i e t fed*

Any d i e t

which i s incom plete f o r b u ild in g o f ti s s u e w i l l a f f e c t a d v e rse ly th e e x te n t o f s y n th e s is .

In a d d itio n , th e se i s a lo s s o f bromobenzene

in ra t* s b re a th and some i s evaporated from th e food* I t may a ls o be p o in te d o u t t h a t o r a l a d m in is tra tio n in ­ vo lv es th e p o s s i b i l i t y o f an i n t e s t i n a l r e a c tio n w ith subsequent e lim in a tio n o f th e m ercap tu ric a c id in th e fe c e s v ia th e i n t e s t i n a l — 44-— L

-i

r

“i

t r a c t ,.

This f a c t o r must a ls o be considered in an in te r p r e t a tio n o f

our experim en tal findings*. Of g r e a te r im portance in connection w ith th e s e s tu d ie s i s th e v ery rec en t fin d in g o f Spencer and W illiams (4-3)*

They found

t h a t bromobenzene fe d a t a l e v e l o f 1 *5% in a d i e t inadequate to meet requirem ents f o r grow th, was d e to x ifie d a s th e m ercapturic a c id o n ly to th e e x te n t o f 14%-

T h eir fin d in g s suggested t h a t under

th e s e c o n d itio n s , th e bromobenzene appears in th e h a i r .

D eterm ination

o f Br in th e h a i r o f th e s e r a t s in d ic a te d a very h ig h Br c o n te n t. It is to be noted that in our experiments we are dealing with a diet inadequate to meet the requirements for growth.

This

is indeed highly suggestive that the reason we could notobtain excretion values of phenylmercapturic acid inthe urinelies

greater

in the

possibility that the bromobenzene ended up inthe hair* Summary and Conclusions: (1) Oral intake of bromobenzene exerts a beneficial ef­ fect on the vitamin B£ deficient rat as judged by its effect on the B^ deficiency syndrome.

(2) A th e o ry to e x p la in t h i s b e n e f ic ia l e f f e c t , based on th e removal o f t i s s u e c y s te in e in th e form ation o f p-brom ophenylm e rcap tu ric a c i d , i s p re se n te d and d isc u s s e d .

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PART 2 - S tu d ies on th e Sub-cutaneous A d m in istratio n o f Bromobenzene» In tro d u c tio n : The r e s u l t s o f P a r t 1 on th e o r a l a d m in is tra tio n o f bromo­ benzene p o in te d to a b e n e f ic ia l e f f e c t o f t h i s compound in removing ti s s u e c y s te in e to form th e phenylm ercapturic acid *

This th eo ry could n o t be

c o r r e la te d , however, w ith our fin d in g s on th e e x te n t o f m ercap tu ric a c id s y n th e s is a s judged by u rin a ry e x c re tio n v alu es*

The m e rits and

d i f f i c u l t i e s o f th e d e te rm in a tio n under th o se c o n d itio n s were d iscu ssed in f u l l * I t was o f i n t e r e s t , th e r e f o r e , to perform experim ents where bromobenzene was in je c te d r a th e r than fed*

T his ro u te o f a d m in is tra tio n

would e lim in a te b o th th e q u e s tio n o f ev ap o ratio n from th e d i e t and a ls o th e p o s s i b i l i t y o f a d e to x ic a tio n in th e i n t e s t i n a l t r a c t w ith sub­ sequent e x c re tio n in th e fe ces* E xperim ental and R e s u lts : The same ex p erim en tal procedure a s b efo re was follow ed v iz * , w eanling Sherman o r W istar r a t s weighing 30-36 g*, were placed on th e 30$ c a s e in , v itam in

f r e e d i e t o f Table XI*

On th e 13 th day o f th e

ex p erim en tal p e rio d th e anim als were in je c te d subcutaneously w ith bromobenzene d iss o lv e d in o il*

(C o rn -o il was found to be a s u ita b le

medium and was used in some experim ents*

However, due to i t s la r g e

c o n c e n tra tio n o f u n s a tu ra te d f a t t y a c id s , and th e p o s s ib le connection between v itam in

and th e u n s a tu ra te d f a t t y a c id s ( 44 > 45 > 46 , 4 7 ), we

sw itched to th e u se o f propylene g ly c o l a s th e medium o f in je c tio n * -4 6 L

r

n Each r a t re ceiv ed 20 mg* bromobenzene p e r

day in 0 .1 m l. o f o i l .

Another group o f r a t s re c eiv ed o n ly th e 0 .1 m l. o f c o n t r o ls .

o i l and served as

A t h i r d group receiv ed no in je c tio n s a t a l l . We found t h a t d e f ic i e n t r a t s were unable to t o l e r a t e a d a ily

i n j e c t i o n o f bromobenzene f o r more th an 2 to 3 w eeks. tim e , th e r a t s became a p e th e tic and refu sed fo o d .

A fte r t h i s

C onsequently, a f t e r

2 weeks o f d a ily i n j e c t i o n , th e in je c tio n s were then given on a l t e r n a t e days u n t i l d e a th .

S te k o l (42) has experienced t h i s same d i f f i c u l t y

w ith in je c tio n s o f doses o f 350-400 mg. p er K ilo body w e ig h t. U rine was c o lle c te d f o r a 2 4 o r 48 hour p erio d a f t e r an in ­ je c tio n o f bromobenzene and analyzed f o r p-brom ophenylm ercapturic a c id by Stekol* s method ( 3 9 ) .

The d eterm in a tio n was c a r r ie d o u t on u rin e

c o lle c te d between th e t h i r d and f i f t h week o f th e ex p erim en tal p e r io d . The d a ta o f th e s e experim ents a re shown in Table XIV.

They

confirm th e b e n e f ic ia l e f f e c t o f bromobenzene o b tain ed in p rev io u s ex p erim e n ts.

More i n t e r e s t i n g was th e o b serv atio n t h a t approxim ately

40 $ o f th e bromobenzene could be accounted f o r as th e phenylm ercapturic a c id .

I t would th u s ap p ear t h a t removal o f c y s te in e does e x e r t a

b e n e f ic ia l e f f e c t which i s m an ifested by a p ro lo n g a tio n o f th e tim e o f appearance o f th e acro d y n ia o f v itam in

d e f ic ie n t r a t s .

D iscu ssio n : The subcutaneous in je c tio n o f bromobenzene in to v itam in d e f i c i e n t r a t s e x e r ts a p r o te c tiv e e f f e c t on th e tim e o f appearance

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I

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Ub °* m

to* r-

in

* sO• I> m

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Diet P-C-l is diet per kg. d i e t .

Data Showing the Effect of Sub-Cutaneous A dm in istration of Bromobenzene on the Vitamin Deficient R a t.

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o f th e d e fic ie n c y symptoms*

This confirm s our p revious fin d in g s on

th e b e n e f ic ia l e f f e c t o b tain ed when bromobenzene i s fe d in th e d ie t* When in je c te d , approxim ately J+0% o f th e ad m in istered bromobenzene ap p ears as u r in a ry p h enylm ercapturic acid * n o te t h a t th e p a ir - f e d r a t re c e iv in g vitam in

I t was i n te r e s ti n g to e x creted about th e

same amount o f p-brom ophenylm ercapturic a c id a ls o * The r e s u l t s o f th e s e experim ents, tak en in connection w ith th e r e s u l t s o f th e p rev io u s experim ents, seem to in d ic a te t h a t re ­ moval o f th io-am ino a c id by a d m in is tr a tio n o f bromobenzene e x e rts a b e n e f ic i a l e f f e c t on th e

d e f ic i e n t ra t*

In c a r e f u l c o n s id e ra tio n o f t h i s f in d in g , i t should be p o in te d o u t t h a t th e b e n e f ic ia l e f f e c t o f bromobenzene m anifested i t s e l f on th e acrodynia. o f v itam in B^ d e fic ie n c y w ith l i t t l e e f f e c t on th e p ro lo n g a tio n o f th e l i f e o f th e r a ts *

This i s in sh arp con­

t r a s t to th e e f f e c t o f th io-am ino a c id supplem entation on v itam in B^ d e f i c i e n t r a ts re c e iv in g a low c a s e in d ie t*

H ere, a d e trim e n ta l e f ­

f e c t i s o b tain ed both on th e d e fic ie n c y symptoms and s u rv iv a l tim e o f such r a ts *

This may be su g g e stiv e o f a tw o -fo ld e f f e c t o f v itam in

B^ in governing th e m etabolism o f thio-am ino a c id s , one involved p rim a rily w ith normal c e l l u l a r m etabolism and e s s e n ti a l to th e l i f e o f th e r a t , th e o th e r in volved in a secondary re g u la tio n o f normal integum entary metabolism *

I t i s to be noted t h a t bromobenzene i s ap­

p a r e n tly a b le o n ly to ease th e c o n d itio n r e s u ltin g from an in te r f e r e n c e w ith p ro p e r integum entary m etabolism , w hile i t may be e q u a lly tr u e -4 9 L

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t h a t th e s u lf u r m etabolism o f o th e r tis s u e s i s a ls o in flu en ced *

This

may be analogous to th e stu d y o f Handler and F eatherstom (AS).

These

a u th o rs have e s ta b lis h e d th e need o f e ry th ro c y te s f o r n ic o t in i c a c id , a phenomenon which i s pro b ably g e n e ra l and a p p lic a b le to a l l c e lls * However, i t was on ly due to th e ra p id r a te o f tu rn o v e r of th e e ry th ro c y te s which p e rm itte d th e p a r t ic u la r o b se rv a tio n s o f t h e i r study*

So to o , th e o b se rv a tio n s o f our study a r e probably much de­

pendent upon th e e s s e n t i a l i t y o f c y s tin e metabolism in th e sk in w hile i t may be no l e s s tr u e t h a t o th e r c e l l s a re a ls o involved* Summary and Conclusions:

(1) Subcutaneous a d m in is tra tio n o f bromobenzene e x e rts a b e n e f ic ia l e f f e c t on th e v itam in

d e f ic ie n t r a t*

(2) A pproxim ately J+0% o f th e in je c te d bromobenzene was ac­ counted f o r as u r in a ry phenylm ercapturic acid* (3) In agreem ent w ith experim ents where bromobenzene was fe d , t h i s b e n e f ic ia l e f f e c t was ap p aren t only on th e d e fic ie n c y syn­ drome and n o t on th e s u rv iv a l tim e o f such r a ts * (A) A th e o ry , in v o lv in g a d u al ro le o f v itam in governing th e m etabolism o f thio-am ino a c id s , i s p resen ted *

in

CHAPTER VI - S tr a in D iffe re n c e s in th e R e sistan ce o f R ats to V itam in

Deficiency*.

In tro d u c tio n s During th e course o f s tu d ie s on Vitamin B, d e fic ie n c y in 6 th e r a t , i t became ap p aren t t h a t c e r ta in r a ts d isp la y e d an a b i l i t y to r e s i s t th e B^ d e fic ie n c y s t a t e .

I t was th e r e f o r e th o u g h t im­

p o rta n t to compare th e perform ance o f d i f f e r e n t s t r a i n s o f r a t s on a v itam in B^ d e f ic ie n t regime*

A p re lim in a ry re p o rt from our

la b o ra to ry in d ic a te d t h a t Sprague-Dawley r a ts were more r e s i s t a n t th an W istar r a t s to v itam in B^ d e fic ie n c y (4-9)* S tr a in d iffe re n c e s in vitam in d e f ic ie n c ie s have been ob­ serv ed b e f o r e .

S tr a in d iff e r e n c e s in th e requirem ent o f chicks f o r

th iam in e and r ib o f la v in , r e s p e c tiv e ly , have been re p o rte d ( 50 , 51)* A s im ila r o b se rv a tio n w ith re g a rd to th e requirem ent o f mice f o r r ib o f la v in has been made by Fenton and Cowgill ( 5 2 ).

In th e case

o f th e r a t , i t has been shown th a t d i f f e r e n t s tr a i n s vary in t h e i r need f o r th iam in e (53) and f o r c h o lin e (54-, 55)*

The p re s e n t d isc u s s io n

i s concerned w ith th e e f f e c ts o f v itam in B^ d e fic ie n c y on fo u r s tr a i n s of ra ts . Experim ental and R e s u lts : The anim als u sed belonged to th e W istar, Sprague-Dawley, Evans-Long and Sherman s tr a in s *

They were p laced a t weaning on

th e b a s a l d i e t (P -l) shown in Table XL.

Only anim als w eighing 30-36 g .

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were used so a s to e lim in a te th e f a c to r o f s to ra g e (56)* to th e la c k o f v itam in

R esista n c e

in th e d i e t was measured by (a) tim e o f

o n se t o f th e a c ro d y n ia , (b) th e g ain in w eight d u rin g th e e x p e ri­ m ental p e rio d and (c) th e s u rv iv a l tim e* th e d a ta o b tain ed in th e s e s tu d ie s *

Tables XV and XVI show

I t i s ev id en t t h a t th e Sprague-

Dawley r a t s were more r e s i s t a n t t o a la c k o f v itam in th an th e o th e r s t r a i n s .

in th e d i e t

The reason fo r th e d iff e r e n c e i s s u s c e p ti­

b i l i t y shown by th e s e s t r a i n s i s unknown* D iscu ssio n : The f a c t t h a t s t r a i n d iff e r e n c e s to v itam in B^ d e fic ie n c y e x i s t i s im p o rtan t in connection w ith s tu d ie s on th e b io lo g ic a l a c tio n o f t h i s vitam in*

P revious s tu d ie s in t h i s la b o ra to ry (56, l )

have shown t h a t in th e r a t (a) th e c a p a c ity to s to r e p y rid o x in e and (b) th e p r o te in l e v e l in th e d i e t , in flu e n c e th e time.*of o n se t and th e s e v e r ity o f th e acrodynia in v itam in B£ d e fic ie n c y *

The

p re s e n t stu d y shows t h a t a t h i r d f a c t o r , namely, th e s t r a i n o f r a t s u sed , should be tak en in to account when stu d y in g th e b io lo g ic a l a c tio n o f t h i s v ita m in . The difference in susceptibility of strains to B^ def­ iciency may also explain certain discrepancies in the literature.

In comparing th e e f f e c ts o f vitam in B^ d e fic ie n c y in r&ts and m ice, M ille r and Baumann (2) found th a t mice were more s u s c e p tib le to a la c k o f t h i s vitam in*

In our ex p erien ce, however, r a ts give more

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Table XV

Data Showing th e E f f e c ts o f Vitamin

D eficien cy on

Four S tra in s o f Rats*

Strain

No* of Rats

Average Time Of Survival Onset of Acrodynia Time ______(Day)________ (Day)

Sherman

16

18

44

W istar

13

17

44

Evans-Long a

11

18

47

Sprague-Dawley

14

29

69 ^

a.

A d dition o f 10 mg* o f p te ro y lg lu ta m ic a c id and 100 gamma o f b io tin p e r K ilo o f d i e t gave s im ila r r e s u l t s w ith 3 anim als o f t h i s s tr a in *

b.

5 anim als in t h i s

group were s t i l l a l i v e a f t e r

110 days on th e d i e t , and showed o n ly s l i g h t symptoms o f acrodynia*

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Table XVI

Data showing the Effect of Vitamin B£ Deficiency on the Gain in Weight of Rats During the First Four Weeks of the Experimental Period (Results expressed as the average gain in grams per rat per day). Week of Experiment Strain

No. of Rats

1

2

1

k

Sherman

16

1J

1 .1

0.43

0.13

Wistar

13

1 .2

1 .2

0.55

Evans-Long

11

1 .2

1 .1

0.38

0.17

Sprague-Dawley

14

1 .1

1 .1

1.0

1 .0

- 0 .3

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c o n s is te n t r e s u l t s and e x h ib it a more pronounced e f f e c t o f th e d e f i­ c ie n c y .

I t may be no ted t h a t th e se workers used Sprague-Dawley r a ts

in t h e i r ex p erim en ts. Summary and C onclusions: (1) Four s t r a i n s o f r a ts were s tu d ie d and t h e i r su sc e p t­ i b i l i t y to v itam in

d e fic ie n c y compared*

(2) The Sprague-Dawley r a t s were found to d is p la y a g r e a te r r e s is ta n c e to a la c k o f t h i s vitam in th an W istars, Sherman and Evans-Long r a t s .

The l a t t e r 3 s tr a in s d isp la y e d th e same re­

s is ta n c e . (3) The Im portance o f t h i s fin d in g in connection w ith s tu d ie s on th e b io lo g ic a l a c tio n o f v itam in

L

i s d isc u s s e d .

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1

PART I I - STUDIES ON VITAMIN B6 DEFICIENCY IN THE MOUSE* CHAPTER VII - The P ro d u ction o f th e Vitamin

D eficiency

Syndrome in th e Mouse* In tro d u c tio n : C ontrary to t h a t which i s observed in th e case o f th e r a t , sim ple d e p riv a tio n o f v itam in

in th e d i e t o f th e mouse i s

in cap ab le o f producing a s p e c if ic s e t o f d e fic ie n c y symptoms* This fin d in g was f i r s t re p o rte d from t h i s la b o r a to ry (57) and was confirm ed by l a t e r workers (58, 2 )*

The mice e v e n tu a lly d ied

b u t d id n o t d is p la y any o f th e symptoms a s s o c ia te d w ith v itam in B^ d e fic ie n c y in th e r a t , e*g*, s c a ly d e r m a titis , s p a s tic g a i t , rin g t a i l , etc*

I t seemed w orthw hile to atte m p t to induce th e

v itam in B^ d e fic ie n c y syndrome in th e mouse* S ev e ral methods o f approach were a v a ila b le , based on p rev io u s work in th e l i t e r a t u r e , to produce th e d e fic ie n c y symptoms* These in clu d ed th e fo llo w in g : (a)

To p la c e a l a c t a t i n g mother on a v itam in B^ d e f ic ie n t

d i e t and th en grow th e young weaned under th e s e c o n d itio n s on a d e f i c i e n t d ie t*

This pre-w eaning d e p le tio n m ight conceivably in ­

duce a g r e a te r s t r a i n on th e young which m ight m an ife st i t s e l f in th e p ro d u ctio n o f th e B^ d e fic ie n c y syndrome*

This method o f ap­

proach i s based on th e re p o rt o f Cerecedo and Foy (56) who ob­ serv e d , in th e case o f th e p y rid o x in e d e f ic ie n t r a t , t h a t th e previous

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nutritional history of the animal influences the time of ap­ pearance of the skin lesions* (b) To place young mice on a

deficient but otherwise com­

plete diet with a sulfa-drug to eliminate the possibility of intestinal synthesis as a source of the missing vitamin*

In­

clusion of a sulfa-drug has been used by various investigators to induce deficiencies of vitamin factors which need not be supplied exogenously (59, 60). (c) Place weanling

mice on a

b

quate diet containing thyroid powder*

deficient but otherwise adeThis method of experimental

approach was feasible in the ligpt of Drill*s finding (6l) that an increased need of vitamin B^ is produced when rats are subjected to experimental hyperthyroidism.

It seemed logical to conceive of

a greater strain induced in mice which received thyroid powder which might manifest itself in the appearance of the deficiency syndrome. (d) An antivitamin could be administered to B^ deficient mice in the hope that a more severe strain under these conditions would result in the production of the deficiency symptoms*

Desoxypyridox-

ine, an analogue of pyridoxine, having the formula of 2, 4 dimethyl 3 hydroxy - 5 hydroxymethylpyridine, had previously been shown to exert a pronounced anti-pyridoxine activity in the chick (62) * That antivitamins are capable of inducing vitamin deficiencies is well-known from the work of many authors (63, 64., 65).

r

Experimenta l and R e s u lts : Each o f th e above l i s t e d methods was attem pted*

Two b a s a l

d i e t s were used in th e s e s tu d ie s ; one c o n ta in in g 25 %, th e o th e r 30% casein *

However, use o f th e l a t t e r d i e t was d isco n tin u ed s in c e

more unifrom r e s u l t s were o b tain ed w ith th e 25% c a se in d ie t*

This

d i e t i s d esig n a te d as d i e t C-28-C and i t s com position i s shown in Table XVII.

When p y rid o x in e i s in clu d ed in t h i s d i e t and given to

m ice, i t g iv es e x c e lle n t growth and f a i r r e s u l t s in rep ro d u ctio n and la c ta c tio n ( 66 ) . When procedure (a) o f above was follow ed and la c ta c tin g m others given th e vitam in

d e f ic i e n t d i e t d u rin g l a c ta tio n

follow ed by placem ent o f th e weaned young on th e d e f ic ie n t regim e, i t was n o t p o s s ib le to produce th e d e fic ie n c y syndrome.

S im il­

a r l y , th e in c lu s io n o f e i t h e r th y ro id powder (Thyroid Glands, D esic ca ted , P ark e, Davis & C o., D e tr o it, Michigan) a t a le v e l o f

0 . 2 % o r 0 . 6%, o r o f s u lfa s u x id in e a t a l e v e l o f 2 % in th e B5 d ef­ i c i e n t d i e t , was w ith o u t a f f e c t on th e appearance o f th e d e fic ie n c y syndrome.

(When th e s u lfa -d ru g was in clu d ed in th e d i e t , 10 mg. o f

p te ro y lg lu ta m ic a c id and 100 X o f b io tin were a ls o added to th e d e f i c i e n t d i e t shown in Table X V II). Under th e s e ex p erim en tal c o n d itio n s , th e r e f o r e , we were u n ab le to induce th e appearance o f th e v itam in B^ d e fic ie n c y syn­ drome in th e mouse.

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Table XVII

B asal D iet C-28-C Used in S tu d ies on Vitamin

D eficien cy

in Mice* C o n s titu e n t

D ie t C-28-C

Casein

25

Sucrose

53

S a lts

5

Ruffex

2

Hydrogenated veg* f a t J

10

Lard

5

Supplements added p e r Kg* Thiamin, R ib o fla v in - 10 mg* each Ca P a n to th en ate - 100 mg* A lp h a-to co p h ero l - AO mg* Vitam in D - 2500 u n its B eta-C arotene - 20 mg* Choline - 1*5 g*

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The final method was then resorted to.

The basal def­

icient diet was supplemented with 20 mg. desoxypyridoxine per kg.

(We a re in d e b ted to Merck & Co. f o r a generous supply o f th e desoxy and raethoxypyridoxine used in th e se s tu d ie s .)

This d i e t was fed

to w eanling a lb in o mice o f th e Rockland, Swiss-W ebster and Fordham s tra in s .

Under th e s e c o n d itio n s th e d e fic ie n c y symptoms were pro­

duced in th e mouse.

A fte r 3 weeks on th e experim ent, mice developed

th e c h a r a c t e r i s t i c acro d y n ia o f th e f o re and hin d paws w hile th e f u r was unkempt and th e anim als hunched.

In some c a s e s , th e s o -c a lle d

* rin g -ta il® c o n d itio n developed and a s p a s tic g a i t was e v id e n t. anim als su rv iv ed a b o u t 30 d a y s.

The

A ll symptoms d isap p eared on th e

a d d itio n o f p y rid o x in e to th e d i e t .

L i t t e r mate c o n tro l anim als which

re ce iv e d th e unsupplem ented d e f ic ie n t d i e t e x h ib ite d none o f th e symptoms o f p y rid o x in e d e f ic ie n c y .

Growth was b e t t e r a ls o in th e

c o n tr o l group as i s e v id e n t from th e growth curves shown in F ig u re I I . Discussion: It has been amply demonstrated that there is a consider­ able difference in the response of differenc species to deficiencies of members of the vitamin B complex. the case of vitamin

An example of this is seen in

deficiency in the rat and mouse.

While a

diet deficient in vitamin B^ but otherwise adequate will cause the production of a specific set of symptoms in the rat, this is not true in the case of the mouse.

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