VOL. 16
(1955)
THE
BIOCHIMICA ET BIOPHYSICA ACTA
DISAPPEARANCE
AND a-KETOGLUTARIC
219
OF PYRUVIC DECARBOXYLASE
DECARBOXYLASE
ON T H I A M I N E - D E F I C I E N T
FROM PIGEON
MUSCLES
DIETS*
by
C. H. M O N F O O R T
Laboratory/or Physiological Chemistry, The University, Utrecht (Netherlands)
The aim of m a n y investigations carried out in this laboratory is to find quantitative
relations between the stage of thiamine deficiency and the occurrence of thiaminedeficiency symptoms on the one hand and the disappearance of thiamine pyrophosphate
(TPP) and the T P P dependent enzymes from the tissues on the other hand. By determining this coenzyme and these enzymes in various tissues in the course of the time
during which animals are confined to a thiamine-free diet we hope to contribute to our
knowledge of the development of the symptoms and the immediate cause of death.
We have also included in this programme investigations on tl{e so called thiaminesparing action of fat. According to several authors, symptoms and death occur much
earlier when a thiamine-free diet contains a high percentage of carbohydrate than when
the carbohydrate in the diet is replaced b y fat. According to EVANS AND LEPKOWSKY 1,
who have coined the expression "vitamin B 1 sparing action of fat", rats on a thiaminefree high fat diet grow much better than rats on a thiamine-free high carbohydrate diet.
As pointed out by WESTENBRINK2 and by GRUBER3 "sparing" m a y in this connection
mean "to use less of" as well as "to dispense with".
GRUBER4 has proved that on a diet rich in fat the organism uses up its store of
T P P at a lower rate, for the amount of T P P in the tissues diminishes much more rapidly
on the thiamine-free high carbohydrate diet than on the thiamine-free high fat diet
(experiments with liver, cerebrum, breast muscle and heart muscle of the pigeon). Moreover GRUBER has shown that, immediately after death induced by withholding thiamine,
in animals consuming the carbohydrate diet the T P P content of heart muscle and liver
is lower, while the T P P content of breast muscle and cerebrum is higher than in animals
receiving the fat diet. In these experiments the pigeons on the carbohydrate diet died
in the mean after 16 days of thiamine deficiency, those on the fat diet after 43 days.
We ourselves 5 have reported already on our experiments on the disappearance of the
pyruvic decarboxylase and the a-ketoglutaric decarboxylase* from breast muscle, heart
* This w o r k forms p a r t of the i n v e s t i g a t i o n s on the m e t a b o l i s m and physiological action of
t h i a m i n e b y H. G. K. WESTENBRINK and collaborators.
** We h a v e called t h e e n z y m e s responsible for the anaerobic d e c a r b o x y l a t i o n of p y r u v a t e and
a - k e t o g l u t a r a t e : decarboxylases. We m i g h t also h a v e designed t h e m b y p y r u v i c dehydrogenase and
a-ketoglutaric dehydrogenase, for the p u r e s t p r e p a r a t i o n s of these e n z y m e s p r e p a r e d from animal
m a t e r i a l (pigeon b r e a s t muscle a n d pig h e a r t respectively), which are h o m o g e n e o u s b o t h in the
electrophoretic and ilt the gravitational field, are active in d e h y d r o g e n a t i o n as well as in straight
Re/erences p. 228.
15
220
c . n . MONFOORT
VOL. 16 (I955)
muscle and legmuscle of pigeons during the first 12 days on the same diet, free of thiamine
and rich in carbohydrate, as was employed by GRUI~ER. We have now continued these
experiments over longer periods of deficiency and have carried out similar determina
tions in breast muscle and heart muscle of pigeons on the diet free of thiamine and rich
in fat, thus extending our own work as well as that of GRUBER (breast muscle and heart
muscle belong to the two groups of organs that are distinctly different as regards the
T P P content at the time of death; see above). This work has also been carried out
with pigeons, as they are very suitable for forced feeding. Animals soon lose their
appetite on a thiamine free diet. Therefore the difference between a carbohydrate diet
and a fat diet is less distinct when the animals are allowed to eat ad libitum.
The pyruvic decarboxylase was determined by measuring the amount of acetoin
formed from added pyruvate in a muscle homogenate under anaerobic conditions, the
a-ketoglutaric decarboxylase by measuring the amount of succinic semiald~'~hyde formed
from added a-ketoglutarate under similar conditions.
EXPERIMENTAL
The n o r m a l pigeons c o n s u m e d ad l i b i t u m a m i x t u r e of cereals (this is called diet A). The
thiamine-free diets B and C contained sucrose and an isocaloric a m o u n t of pea n u t oil respectively.
The pigeons confined to these diets were forcibly fed t h r o u g h a glass t u b e which was i n t r o d u c e d
into the stomach. A pigeon on diet B received 2 g casein, 18 g sucrose, o. 3 g of a salt m i x t u r e , a n d
o.o9 g of a v i t a m i n m i x t u r e daily, a pigeon on diet C 2 g casein, 8 g pea n u t oil, o. 3 g of the salt
m i x t u r e and o.o 9 g of t h e v i t a m i n m i x t u r e . The salt m i x t u r e was composed according to JANSEN
AND "vVEsTENBRINK~, the v i t a m i n m i x t u r e contained all k n o w n v i t a m i n s (in the p u r e s t form obtainable) with the exception of t h i a m i n e (see GRUBER4).
After a v a r y i n g n u m b e r of days on these diets, the pigeons were killed b y decapitation. I m mediately after death p a r t of the large b r e a s t muscle (m. pectoralis major) and the h e a r t were excised
and cooled in an isotonic p h o s p h a t e solution of p H 7.4. The p r e p a r a t i o n of the h o m o g e n a t e s was
carried out in the cold r o o m (2 ° C). 2 g of t h e b r e a s t muscle and 2 g of the left ventricle of the h e a r t
were homogenized in 18. 4 ml of o.i M p o t a s s i u m s o d i u m p h o s p h a t e buffer, pt-I 6.2, containing
o.ooi M MnC12, w i t h a homogenizer as described b y CAMPBELL et aLL W i t h this homogenizer the
tissues were perfectly homogenized in I minute. D u r i n g this time the t u b e in which the process
took place was cooled w i t h ice and water, so t h a t t h e t e m p e r a t u r e of the h o m o g e n a t e n e v e r increased
to more t h a n 4 ° C. The p H r e m a i n e d c o n s t a n t d u r i n g this procedure.
2 ml samples of the h o m o g e n a t e s were p i p e t t e d into the m a i n c o m p a r t m e n t of cooled W a r b u r g
flasks. The center well contained a piece of p h o s p h o r u s in order to r e m o v e the last traces of oxygen,
still p r e s e n t after gassing w i t h the p u r e s t q u a l i t y of nitrogen. 2. 5 mg of s o d i u m p y r u v a t e or 3.9 mg
of s o d i u m a - k e t o g l u t a r a t e were tipped in from the side bulb. After 3 h o u r s at 37.5 ° C 5 ml of a IO %
m e t a p h o s p h o r i c acid solution were added to the reaction m i x t u r e s in t h e W a r b u r g flasks. After
centrifuging the d e t e r m i n a t i o n of acetoin or succinic semialdehyde was carried o u t in the clear
supernatants.
Acetoin was determined according to WHITE, KRAMPITZ AND W E R K M A N 8, succinic s e m i a l d e h y d e
in the presence of a - k e t o g l u t a r a t e according to a m e t h o d based on the principle published b y OCHOA9
(see MONFOORT10). The production of CO 2 and the a m o u n t s of p y r u v a t e and a - k e t o g l u t a r a t e utilized
were also determined, b u t these d a t a have no i m p o r t a n c e in connection w i t h the p r o b l e m discussed
in this paper. T h i a m i n e p y r o p h o s p h a t e was d e t e r m i n e d according to }VESTENBRINK AND STGYNPARVI~n. T h e sodium p y r u v a t e was a p r e p a r a t i o n p u r c h a s e d from H o f f m a n n - L a Roche (97 % pure),
d e c a r b o x y l a t i o n and are generally called d e h y d r o g e n a s e s in view of the preponderance of deh y d r o g e n a t i o n in metabolism. H o w e v e r , it has not yet been decided w h e t h e r the same prosthetic
group is responsible for b o t h actions. REED AND DE BUSK la h a v e obtained strong evidence in s u p p o r t
of the view t h a t in E. coli T P P would be responsible for anaerobic d e c a r b o x y l a t i o n and lipothiamide
p y r o p h o s p h a t e for aerobic decarboxylation and d e h y d r o g e n a t i o n . T h o u g h tile animal dehydrogenase
p r e p a r a t i o n s mentioned above contain lipoic acid, it has n o t y e t been d e m o n s t r a t e d t h a t it is combined w i t h T P P to form lipothiamide p y r o p h o s p h a t e . Therefore it is possible t h a t in muscle the
same T P P molecule combined w i t h t h e e n z y m e p r o t e i n is acting u n d e r aerobic as well as u n d e r
anaerobic conditions. At a n y rate, as these questions have n o t yet been decided, we believe it is
advisable to speak a b o u t decarboxylases in connection w i t h our e x p e r i m e n t s .
Re[erences p. 228.
16 (i955)
VOL.
DISAPPEARANCE
OF
DECARBOXYLASES
FROM
MUSCLES
221
the a-ketoglutaric acid a preparation from the Nutritional Biochemicals Corporation, Cleveland
(at least 98 % pure). Racemic acetoin (95 % pure) was prepared according to DIELS AND STEPHAN12,
succinic semialdehyde (at least 96 % pure) according to CARRI~REla.
RESULTS
T a b l e I shows t h a t a d d e d acetoin a n d succinic s e m i a l d e h y d e are c o m p l e t e l y recovered after 3 hours' i n c u b a t i o n w i t h muscle h o m o g e n a t e s u n d e r the a c t u a l e x p e r i m e n t a l
conditions. Hence t h e y m u s t be considered t o be final p r o d u c t s u n d e r these conditions.
W e m a y therefore conclude t h a t t h e r a t e of p r o d u c t i o n of these c o m p o u n d s from a d d e d
p y r u v a t e a n d a - k e t o g l u t a r a t e can be r e g a r d e d as a measure of t h e a c t i v i t y of p y r u v i c
d e e a r b o x y l a s e a n d a - k e t o g l u t a r i c d e c a r b o x y l a s e respectively.
TABLE
I
RECOVERY OF ACETOIN (RACEMIC) AND SIJCCINIC SEMIALDEHYDE ( G S A ) ADDED TO MUSCLE
HOMOGENATES ( 2 0 0 m g MUSCLE) AFTER 3 HOURS' INCUBATION AT 3 7 . 5 ° C
Added
Muscle
Exp.
Breast
I
II
Heart
III
400 ~g
ace~oin
450 t*g
SSA
.
+
Breast
1V
Recovered
(l*gJ
__
43
456
413
--
--
+
--
373
408
+
--
+
--
781
__
+
__
--
+
+
+
+
39 °
798
.
+
.
--
_
.
Determined
(t~g)
__
.
.
6 I*g
TPP
--
.
.
2 mg
pyruvate
.
.
_
.
.
+
408
io
_
_
_
_
-
-
_
_
.
406
396
o
465
465
I n T a b l e s I I a n d I I I " s e v e r e s y m p t o m s " m e a n s t h a t according to our experience
t h e a n i m a l s w o u l d h a v e d i e d in a few hours.
T a b l e I I shows t h e decline of pyTuvic d e c a r b o x y l a s e a c t i v i t y of b r e a s t muscle a n d
h e a r t muscle in t h e course of t h i a m i n e d e p r i v a t i o n .
Breast muscle. D u r i n g t h e first 12 d a y s of deficiency t h e difference b e t w e e n t h e
c a r b o h y d r a t e diet a n d t h e fat diet is not significant. As on t h e average t h e pigeons live
m u c h longer on t h e l a t t e r diet t h a n on t h e former i t m i g h t be expected, p r e s u m i n g
d e c a r b o x y l a s e a c t i v i t y would continue t o be lost on t h e fat diet, t h a t t h e final decrease
on t h i s d i e t would be m u c h g r e a t e r t h a n t h a t on t h e c a r b o h y d r a t e diet. This was i n d e e d
observed. Hence t h e severe s y m p t o m s i m m e d i a t e l y preceding d e a t h occur at a m u c h
h i g h e r p y r u v i c d e c a r b o x y l a s e a c t i v i t y of t h e b r e a s t muscle on t h e c a r b o h y d r a t e diet t h a n
on t h e fat diet.
Heart muscle. N o w we see t h a t t h e r e is a significant difference b e t w e e n t h e effects
of t h e diets after 12 d a y s : t h e a c t i v i t y of t h e p y r u v i c d e c a r b o x y l a s e has d i m i n i s h e d m u c h
m o r e on t h e c a r b o h y d r a t e diet t h a n on t h e fat diet. So t h e decline on t h e fat diet is
slower, b u t as t h e deficiency t a k e s more t i m e to develop on this diet, t h e r e is no more
Re#fences
p. 228.
222
c.H. MONFOORT
VOL. 16 (1955)
a significant difference between the pyruvic decarboxylase activities of heart muscle on
both diets when the pigeons have incurred severe symptoms.
TABLE
II
ACETOIN PRODUCTION IN MUSCLE ItOMOGENATES
D i e t A = m i x t u r e o f c e r e a l s ; D i e t B = c a r b o h y d r a t e d i e t , f r e e of t h i a m i n e ; D i e t C = f a t d i e t ,
f r e e o f t h i a m i n e . A l l s t a n d a r d d e v i a t i o n s m e n t i o n e d a r e s t a n d a r d d e v i a t i o n s of t h e m e a n s ( s t a n d a r d
e r r o r s ) . P - v a l u e s c a l c u l a t e d a c c o r d i n g t o \VILcOXON ~4.
Diet
A
B
C
Conditions o] pigeons
normal
Number
o/pigeons
6 pg T P P
added
8
4 d. d e f . ,
no symptoms
S
12 d. d e f . ,
no symptoms
to
I8 d. d e f . in m e a n
(I1 d. t o 25 d.),
severe symptoms
I3
20 d. d e f . in m e a n
(17, 18 a n d 2 6 d. def.),
severe symptoms
3
12 d. d e f . ,
no symptoms
io
2 6 d. d e f . ,
no symptoms
lO
33 d. d e f . i n m e a n
(29 d. t o 37 d . ) ,
severe symptoms
9
51 d. d e f . in m e a n
(44, 4 4 a n d 4 8 d. d e f . ,
severe symptoms ;
5 9 a n d 6 o d. d e f . ,
no symptoms)
5
Breast muscle
"
p M acetoin produced in 3 h
Breast muscle
Heart muscle
4.3 ~:o.2
2. 3 ~=o.l
I
:~
5.2±0.2
3.6 ! - o . i
t6
~
3.5 ± 0 . 3
4 . 8 ± o.4
1.8 ± o . i
3.8 ± o. t
2
2
>
-'.4 ± o . 2
5 . o ± o.4
o. 3 ± o . o 2
3.2 ± o.2
33+
t-7 :t=°.3
0.2 ± 0 . 0 3
4 "
~-
1.6 ± 0 . 2
3.6 ± 0 . 2
o.i ±o.oz
1.8 ± 0 . 3
55+
~
'.7 ±0.2
4.5 ± o . 3
i . i -~20.l
3.t ±o.2
06!
- !
o.8 +o.i
2.9 ± o.3
o.3 ± o . o 3
2. 3 ± o . t
7
7+
o.4--0.o 3
o.: ±o.o2
8
o. 3 ± o . o 2
2. 7 ± o.2
o1 ±o.o2
1. 5 ± o . 2
09+.
-
~
Heart muscle
P(1--2-) = o.oi;
P ( I - 3--) < O.OO1;
P(1 4 ) ( o . o o I ; P(1 6 ) ( 0 . 0 0 1 ;
P(1 -7-) < 0 . 0 0 I , P(1 - s - ) < 0 . 0 0 I .
t'(1--_2-- ) = O.O2; P(I
3-) < o . o o i ;
1)(1--4-) • 0 . 0 0 i ; l)(t - 6 - ) < o . o o i ;
P(1---7 ) < o . o o I ; I)(i - 8-) < o . o o I .
P(1+_2 +) > 0.1;
[)(1 + 3+) > 0. i ;
P(1+_5 +) < 0.001 ; P(l+_.6+) > 0.15
P ( 1 + - 7 +) < 0 . 0 0 I ; P ( l + - 9 + ) < 0 . 0 0 I .
P ( I + 2+) :> 0 . t ;
P(1 + 3+) > o . I ;
I3(1+._5+) < o . o o I ; P(l+_6+) > 0. t ;
[ ' ( 1 + - 7 + ) < 0 . 0 0 I ; P(1+_9 +) < o . o o I .
P(3--~-) < °'°°1; P(i--5-) >°'1;
P(6 - ~ - ) < 0 . 0 0 I ; P(7 - s - ) < 0 . 0 0 1 ;
P(8 9-) ~> o . I ;
P(4 8 ) • 0 . 0 0 [ .
P(3- 4 )>0.I;
P(6--7-) < O.OOI;
]9(8 -9-) ) o . I ;
Re/erences p. 228.
IndiceS/or
P
at loot
o/table
PQ-_5-) > o.i;
-- 0"004;
P(4 - 8 - ) ) 0.I.
P(7--8-)
VOL.
16 (1955)
DISAPPEARANCE
OF D E C A R B O X Y L A S E S
TABLE
SUCCINIC
(ssa)
SEMIALDEHYDE
FROM MUSCLES
223
III
PRODUCTION
IN
MUSCLE
IIOMOGENATES
D i e t A = m i x t u r e of c e r e a l s ; D i e t B = c a r b o h y d r a t e d i e t , free of t h i a m i n e ; D i e t C = f a t d i e t ;
free of t h i a m i n e . All s t a n d a r d d e v i a t i o n s m e n t i o n e d a r e s t a n d a r d d e v i a t i o n s of t h e m e a n s ( s t a n d a r d
e r r o r s ) . P - v a l u e s c a l c u l a t e d a c c o r d i n g t o ~VVILCOXON14.
Diet
Number
ol pigeons
Condition ol pigeons
0 tig T P P
added
Breast muscle
Heart muscle
Indices
lo r p
at loot
ot table
tiM SSA produced in 3 k
A
normal
7
-+
7.3 ± 0-5
9.0 _4:_0.4
5.8 ± 0. 4
6.3 ~ 0.3
ii+
B
4 d. def.,
no symptoms
9
-+
4.6 ± 0. 4
9.2 ± 0.3
6.0 ± 0. 5
7.0 ~ o.3
22+
12 d. def.,
no symptoms
9
+
4 .8 ~ o - 4
8.8 ~ 0. 4
4.5 ~ o . i
5.8 + 0. 3
33+
13
--
4-7 ± 0.2
3.0 ± 0.2
4-
3
--+
5-4 -- 0.3
8.1 ~ o . 5
3.2 ± 0.6
6. 5 ~ o . 5
55+
12 d. def.,
no symptoms
lO
--+
6.0 ± 0. 4
9.2 ± 0.4
6.2 ± 0. 4
6.7 ± 0.3
6
6+
26 d. def.,
no symptoms
Io
-+
3.o-t-o. 4
7.1 :t= 0.5
4.1 :t:o.2
5.5 ~= 0-3
7-
18 d. def. i n m e a n
(11 d. t o 25 d.),
severe symptoms
20 d. def. i n m e a n
(17, 18 a n d 26 d. def.),
severe symptoms
C
33 d. def. i n m e a n
(29 d. t o 37 d.),
severe symptoms
9
--
2.9±o.1
4.4±0.2
8-
51 d. def. i n m e a n
(44, 44 a n d 48 d. def.,
severe symptoms;
59 a n d 6o d. def.,
no symptoms
5
-+
2. 3 ± 0 . 2
4 .6 ± 0 . 3
3.2 ± o . i
4.5 ~ ° . 4
9-
Breast muscle
P ( 1 - - 4 ) < o . o o i ; P ( 1 - - 6 - ) > 0.1;
P ( I + - 2 +) ~> o . I ;
P(1+_5+) = 0.07;
P(l+_7 +) = o . o i ;
P(1 + z+) ;> 0 . I ;
P(l+_s+) > 0.I;
P ( l + _ 7 +) = o . o I ;
P(1 + a+) > o . I ;
P(1+_6+) > o . I ;
P(l+_a+) < o . o o i .
P(4----5--) > 0.1;
P(~--7-) < 0.00I; P(7--s-) > 0.i;
P(s--9-)
Table
= 0.02;
II further
P(4--s-) < o.ooi.
shows
the
in the case of normal
Re#fences p. 228.
9+
Heart muscle
P(I--=-) < 0,00I; P(2--3-) > o.!;
10(2----4-- ) > 0.1;
P ( 1 - - 6 - ) = 0.02;
P(1----7--) ( O.OO1; P ( 1 - - s - ) < o . o o I .
P ( a - - 4 - ) > O.I;
observed
7.t-
V(1 -= ) > o.1;
P ( x - - v - ) <~ 0 . 0 0 I ; P ( 1 - - s - ) < o . 0 0 I .
There
P ( l + - a + ) = o.o9;
P(l+_6+) > o . I ;
P(l+_9 +) < o . o o i .
P(a--4-) < 0.00I; P(4--s-) > 0.I;
P(6 --2 ) < O.OOI; P ( l - - 8 - ) > o . I ;
P(s--9 ) < o.ooi; P(8--4-) < o.ool.
effect of adding
muscles.
P(a--a-) < o.ooi;
TPP
in vitro. A s m a l l e f f e c t i s a l r e a d y
explanations
f o r t h i s , viz.
are two possible
224
c.H. MONt~OORT
W)I.. 16 (I055)
a small part of the pyruvic decarboxylase is present in the tissue i~t situ as the apoenzyme,
even when the body is abundantly supplied with thiamine, or part of the prosthetic
group is split off in the course of the preparation of the homogenate. We believe the
latter to be the most probable.
It further appears that in both muscles and on both diets the apoenzyme is maintained during the first 12 days of thiamine deficiency, for the same level is reached
upon adding T P P to the homogenates of the muscles of the 12 days' deficient pigeons
as in the case of llomogenates of the muscles of normal pigeons. When the deficiency
lasts longer the apoenzyme is partly lost, for in the later stages addition of T P P does
not restore the pyruvic decarboxylase activity to its maximal level.
The data concerning the a-ketoglutaric decarboxylase activity of the muscle
homogenates are assembled in Table I I I .
Breast muscle. We see that on the carbohydrate diet the activity declines during the
first 4 days of deficiency, but then remains practically constant until the occurrence of
severe symptoms, which would have been followed b y death in a couple of hours. The
activity is then still rather high.
The decline on the fat diet is smaller in the first 12 days, but it gradually increases
as thiamine deprivation is prolonged. In the last stage of deficiency the a-ketoglutaric
decarboxylase activity is significantly lower on the fat diet than on the carbohydrate diet.
Heart muscle. Now we see that the activity remains constant for 4 days on the
carbohydrate diet; after 12 days on this diet the activity has significantly decreased,
and this decrease continues unLil severe symptoms are observed.
On the fat diet, however, the activity has not yet diminished after I2 days; after 26
days a significant decrease is observed. The activity remains more or less on this level
until severe symptoms are observed, though there is some tendency to decrease still
further with prolonged time of survival. In most cases the a-ketoglutaric activity is
higher at death on the fat diet than on the carbohydrate diet.
On the carbohydrate diet the apo-a-ketoglutaric decarboxylase appears to be
maintained both in breast muscle and in heart muscle until the lase stage of deficiency
(on the average I8 days).
On the fat die~, however, on which the time of survival is much longer, there is a
definite decrease of the amount of apoenzyme in the stages when symptoms occur
(compare the activities of normal and deficient homogenates in the presence of
added TPP).
To compare the decrease of pyruvic decarboxylase and a-ketoglutaric decarboxylase
activities with progressive deficiency Figs. I and 2 m a y be consulted. In these figures
the activities, expressed in percentages of the activity of the homogenates of the normal
muscles, are plotted against the period of thiamine deprivation. These figures show that
under similar conditions, viz. carbohydrate or fat diet, both in breast- and heart muscle a
much higher percentage of active a-ketoglutaric decarboxylase remains in the tissue as
compared to pyruvic decarboxylase. Thus the former enzyme always appears to lose its
prosthetic group less readily than the latter, with one exception only: in the breast
muscle during the first 4 days of deficiency on the carbonhydrate diet; in that case the
breast muscle loses its a-ketoglutaric decarboxylase activity with an extreme rapidity.
As mentioned above the pigeons with severe symptoms were in a condition from
which it could be predicted that they would die within a few hours. In general death
was much more acute on the carbohydrate diet than on the fat diet, and on the latter
Re[erences p. 228.
VOL.
16 (i955)
temporary
225
DISAPPEARANCE OF DECARBOXYLASES FROM MUSCLES
remissions
thiamine-deficient
were
often
observed.
But
we
had
sufficient
experience
pigeons to be able also to predict the rapid approach
with
of death in the
c a s e of t h e f a t - f e d p i g e o n s . T h e r e f o r e w e m a y c o n c l u d e t h a t b o t h e n z y m e s n e v e r d i s a p p e a r
completely
from the muscles examined.
"/OC "~
$00
\
80
~50
~, 6C
%
"%,
\xx
\~2
N x ~ Xx' ~ - \~ " o
"~"o,\\\
•~
~
- - ×. . . .
~\
"-
x-,,
O
2o
2o
o
;
+
~;}o 2; :5-
Days of deficiency
I
6
I
Days of deficiency
Fig. I. P e r c e n t u a l decrease of t h e e n z y m e activities in b r e a s t muscle. I. a - K e t o - g l u t a r i c decarb o x y l a s e , c a r b o h y d r a t e diet; 2. a - K e t o g l u t a r i c
d e e a r b o x y l a s e , f a t diet; 3. P y r u v i c d e c a r b o x ylase, c a r b o h y d r a t e diet; 4- P y r u v i c d e c a r b o x ylase, f a t diet.
Fig, 2. P e r c e n t u a l decrease of t h e e n z y m e activities in h e a r t muscle. I. a - K e t o - g l u t a r i c decarb o x y l a s e , c a r b o h y d r a t e diet; 2. a - K e t o g l u t a r i c
d e c a r b o x y l a s e , f a t diet; 3. P y r u v i c d e c a r b o x ylase, c a r b o h y d r a t e diet; 4. P y r u v i c d e c a r b o x ylase, f a t diet.
TABLE
IV
THIAMINE PYROPHOSPHATE CONTENT OF MUSCLES
Diet A = m i x t u r e of cereals; D i e t B = c a r b o h y d r a t e diet, free of t h i a m i n e ; D i e t C = f a t diet,
free of t h i a m i n e . " S t a n d a r d e r r o r s " .
Breast muscle
1) ie~
Condition o] pigeons
.X
B
Heart muscle
Number
of pigeons
pg T P P
per g
Number
o] pigeons
i~g T P P
per g
normal
2o
7.3 ~- O. I
20
6. 3 ± O.2
4 d. def.,
no s y m p t o m s
21
4.9 j c o . 2
19
3.9 4-o.1
12 d. def.,
no s y m p t o m s
24
3.2 ~=o.i
20
1. 7 ~ o . i
18 d. def. in m e a n
( i i d. to 25 d.),
severe symptoms
13
2.7 dco.I
13
1. 4 :t=o.o6
12 d. def.,
no s y m p t o m s
io
4.9 =E o.I
IO
3.7 q- o.I
26 d. def.,
no symptoms
io
1. 7 i o . o 8
io
1.6 -t-o.o2
9
1.8 dco.I
9
33 d. def. in m e a n
(29 d. to 37 d.),
severe symptoms
Re#fences p. -'26'.
1. 7 4-o.1
2z6
c.H. MONFOORT
V()L. 16 (I~)55)
Table IV summarizes the results of all T P P determinations carried out during this
investigation. In general they confirm G~UBER'S results. Furthermore the remarkabh~
fact that the decrease of the TPP content in the later stages of deficiency is only very
small or nil (comp.: carbohydrate diet, pigeons after 12 days of deficiency without
symptoms and pigeons with severe symptoms; fat diet, pigeons after 26 days of deficiency without symptoms and pigeons with severe symptoms) permits the following
conclusion. Presuming that enzymes wittl T P P as a prosthetic group, other than the
two enzymes studied, occur in the normal muscles in not too small amounts these must
also lose their prosthetic group to a large extent long before the symptoms occur, for
otherwise the T P P content of the muscles would still decrease considerably in the last
stages.
DISCUSSION
According to GRUBER4 T P P disappears less rapidly from the tissues, when the
thiamine-free diet contains fat instead of carbohydrate. Actually, this diminished rate of
utilization of T P P is not due to an effect of the fat in the diet, but to the absence of
carbohydrate. This appears from an experiment by the same author a, in which he
administered various amounts of carbohydrate, but no fat. The feeding of a small
amount of carbohydrate provoked a less rapid disappearance of T P P than the feeding
of a larger amount. Hence in the discussion of our experiments the presence or the absence of carbohydrate in the diet should be emphasized and not the presence of fat.
The curves in Figs. I and 2 representing the change of pyruvic decarboxylase and
a-ketoglutaric decarboxylase of breast muscle and heart muscle of pigeons on the fat diet,
are thus characteristic for diets containing no carbohydrate. As these figures show, the
phenomena appear to be much more complicated than one could expect from the results
of the determination of total TPP, as carried out by GRUBER. During tile first I8 or
20 days there is not much difference in breast muscle between tile rates of inactivation of
pyruvic decarboxylase on both diets, but tile difference is very pronounced in the case of
heart muscle {after I2 days of thiamine deprivation on the carbohydrate diet the pyruvic
decarboxylase activity has already decreased to IO % and on the diet free of carbohydrate
to only 45 % of the normal value). The difference between the effects of the two diets is
very remarkable for the a-ketoglutaric decarboxylase : in breast muscle the inactivation
is much more pronounced on the carbohydrate diet than on tile diet containing no
carbohydrate in the first 4 days ; during the following days, however, no further inactivation takes place on the former diet, while tile inactivation gradually proceeds on the
latter. In heart muscle the activity remains constant during the first 4 days on the
carbohydrate diet and during the first 12 days on the diet containing no carbohydrate.
After these initial periods the decrease sets in at about equal rates on both diets.
Altogether we see that the determination of total TPP, which is not only the
prosthetic group of the two enzymes studied, but very probably also that of other
enzymes, gives only a very incomplete picture of thiamine metabolism during thiamine
deprivation. However, summarizing various conclusions from Tables II and III, to
be found in the Experimental Part, we see that at death both kinds of muscle tend
to show the same differences between their enzyme activities on the two diets as
between their total T P P contents, as described by GRUBER. This is schematically shown
in Table V.
References p. 228.
VOL. 1 6
(1955)
DISAPPEARANCE OF DECARBOXYLASES FROM MUSCLES
227
TABLE V
TPP
CONTENTS
AND
ENZYME
ACTIVITIES
AT
DEATH
Breast muscle
T P P c o n t e n t at d e a t h
l ' y r u v i c decarb, a c t i v i t y at d e a t h
a - K e t o g l u t a r i c decarb, a c t i v i t y at d e a t h
on diet B ~> on diet C
on diet B ~> on diet C
on diet B > on diet C
Heart muscle
on diet B < on diet C
on diet B = on diet C
on diet B < on diet C
As we have seen, the decarboxylases can be easily determined in muscles, as acetoin
and succinic semialdehyde appear to be final products under anaerobic conditions. We
have obtained evidence that it is not so easy in other tissues. If the conception of REED
AND DEBuSK that the T P P responsible for anaerobic decarboxyoation is also responsible
for the oxidation of pyruvate and a-ketoglutarate by artificial electron acceptors as
ferricyanide also applies to the animal enzymes, these decarboxylases may possibly be
determined in other tissues by measuring the rate of oxidation of these substrates by
ferricyanide.
However this may be, in our opinion the results of this investigation indicate, that
the determination, during the progress of thiamine deficiency, of all enzymes which have
T P P or a TPP-lipoic acid complex as prosthetic group in the largest possible number of
tissues promises to give much more insight into the origin of the symptoms of thiamine
avitaminosis, which finally depend on a derangement of metabolism.
SUMMARY
I n h o m o g e n a t e s of b r e a s t muscle and h e a r t muscle acetoin and succinic semialdehyde are final
p r o d u c t s of the anaerobic m e t a b o l i s m of p y r u v a t e and a - k e t o g l u t a r a t e respectively. Therefore the
p y r u v i c and a-ketoglutaric decarboxylase activities of these muscles could be m e a s u r e d b y determ i n i n g the r a t e s of p r o d u c t i o n of these c o m p o u n d s from p y r u v a t e and a - k e t o g l u t a r a t e added to
the h o m o g e n a t e s .
D u r i n g t h i a m i n e d e p r i v a t i o n b o t h activities decreased according to tile curves s h o w n in Figs.
i and 2. P r o n o u n c e d differences exist b e t w e e n b o t h kinds of muscle regarding the r a t e s of decline
of the activities as well as the activities r e m a i n i n g at death. The presence or absence of c a r b o h y d r a t e
in the thiamine-free diet also h a s a m a r k e d influence on b o t h p h e n o m e n a .
I n general the a-ketoglutaric decarboxylase activity decreases less rapidly t h a n the p y r u v i c
decarboxylase activity.
At d e a t h the p y r u v i c decarboxylase a c t i v i t y of b r e a s t muscle is m u c h higher on the c a r b o h y d r a t e diet t h a n on the fat diet, while in this regard no difference a p p e a r e d to exist in h e a r t muscle.
Regarding the a-ketoglutaric decarboxylase a c t i v i t y at death, this is also higher on the c a r b o h y d r a t e
diet t h a n on the fat diet in b r e a s t muscle, while in m o s t cases the reverse was o b s e r v e d in h e a r t muscle.
As c o m p a r e d to the n o r m a l a c t i v i t y of b r e a s t muscle or h e a r t muscle the final p e r c e n t a g e
decrease of the p y r u v i c decarboxylase a c t i v i t y is a l w a y s - m u c h g r e a t e r t h a n t h a t of the a - k e t o g l u t a r i c
decarboxylase activity, b u t the f o r m e r also is never completely lost.
The a p o e n z y m e s are c o m p l e t e l y m a i n t a i n e d in the muscles d u r i n g m o s t of t h e period of deficiency; t h e y are only p a r t l y lost in the last stages, w i t h the exception of the a-ketoglutaric decarboxylase on the c a r b o h y d r a t e diet.
RI~SUMI~
Les p r o d u i t s finaux du m6tabolisme ana~robie du p y r u v a t e et de l ' a - c 6 t o g l u t a r a t e d a n s des
h o m o g 6 n a t s de muscle pectoral ou de muscle cardiaque s o n t r e s p e c t i v e m e n t l'ac6toine et la semialdehyde suceinique. P a r cons6quent on p e u t m e s u r e r les activit6s p y r u v i q u e et a-c6toglutarique
d~carboxylasiques de ces muscles en d 6 t e r m i n a n t les vitesses d ' a p p a r i t i o n de ces corps g p a r t i r du
p y r u v a t e et de l ' a - c 6 t o g l u t a r a t e ajout6s a u x homog6nats.
La diminution des deux activit~s au cours d ' u n e carence en t h i a m i n e est repr6sent6e p a r les
Figs. i et 2. Les vitesses de d i m i n u t i o n des activit6s ainsi que les activit6s r6siduelles au m o m e n t
ReJerences p. 2z8.
228
c . H . MONFOORT
VOL. 16 (1955)
de la m o r t s o n t tr~s diff6rentes selon le muscle envisag6. L a pr6sence ou l ' a b s e n c e de glucides d a n s
le r6gime s a n s t h i a m i n e infiue 5 g a l e m e n t b e a u c o u p s u r ces d e u x p h d n o m ~ n e s .
E n g6n6ral l'activitd d 6 c a r b o x y l a s i q u e vis ~ vis de l ' a - c d t o g l u t a r a t e d i m i n u e m o i n s r a p i d e m e n t
que l'activit6 vis 5~ vis du p y r u v a t e .
Au m o m e n t de la m o r t , l'activit6 de la p y r u v i q u e d 6 c a r b o x y l a s e d u m u s c l e pectoral est b e a u c o u p
p l u s dlev6e p o u r u n r~gime glucidique que p o u r u n r6gime lipidique; cette difference ne s ' o b s e r v e
p a s d a n s le cas du m u s c l e c a r d i a q u e .
L ' a c t i v i t 6 de l ' a - c 6 t o g l u t a r i q u e d 6 c a r b o x y l a s e v a r i e de la m ~ m e fa~on en fonction du r 6 g i m e
d a n s le m u s c l e pectoral, alors que, d a n s la p l u p a r t des cas, elle v a r i e en s e n s i n v e r s e d a n s le cas
du muscle c a r d i a q u e .
Le p o u r c e n t a g e final de d i m i n u t i o n d'activit~, p a r r a p p o r t it l'activit6 n o r m a l e , de la p y r u v i q u e
d ~ c a r b o x y l a s e e s t t o u j o u r s b e a u c o u p plus g r a n d q u e celui de l ' a - c d t o g l u t a r i q u e d 6 c a r b o x y l a s e ;
c e p e n d a n t la p e r t e d ' a c t i v i t 6 du p r e m i e r e n z y m e n ' e s t i a m a i s totale.
L e s a p o e n z y m e s p e r s i s t e n t d a b s les m u s c l e s p e n d a n t la plus g r a n d e p a t t i e de la carence; ils
ne s o n t p e r d u s q u e p a r t i e l l e m e n t d a n s les derniers stades, s a u f d a n s le cas de l ' a - c 6 t o g l u t a r i q u e
d 6 c a r b o x y l a s e et d ' u n rdgime glucidique.
ZUSAMMENFASSUNG
I n H o m o g e n a t e n des B r u s t - u n d H e r z m u s k e l s sind Acetoin u n d t 3 e r n s t e i n s S m r e h a l b a l d e h y d
E n d p r o d u k t e des a n a e r o b e n Stoffwechsels der B r e n z t r a u b e n s ~ u r e bezw. der a - K e t o g l u t a r s X u r e .
D a h e r k o n n t c n die Aktivit~tten der B r e n z t r a n b e n s f i u r e - u n d der a - K e t o g l u t a r s / i u r e - D e c a r b o x y l a s e
g e m e s s e n werden, i n d e m die G e s c h w i n d i g k e i t der P r o d u k t i o n dieser V e r b i n d u n g e n n a c h Z u s a t z v o n
P y r u v a t u n d a - K e t o g l u t a r a t zu d e n H o m o g e n a t e n b e s t i m m t wurde.
Bei V i t a m i n B 1 Mangel n a h m e n die Aktivit~iten e n t s p r e c h e n d d e n K u r v e n der Fig. I u n d 2 ab.
ILs b e s t e h e n a u s g e s p r o c h e n e U n t e r s c h i e d e zwischen d e n beiden M u s k e l a r t e n , w e n n der G e s c h w i n d i g keitsabfall der A k t i v i t ~ t e n u n d die Aktivittttsreste b e i m T o d b e t r a c h t e t w e r d e m Die G e g e n w a r t oder
A b w e s e n h e i t v o n K o h l e h y d r a t bei V i t a m i n B 1 freier Di~t h a t e i n e n m e r k b a r e n Einfluss a u f beide
PhSa~omene.
IIn a l l g e m e i n e n fiel die a - K e t o g l u t a r s / i u r e - D e c a r b o x y l a s e - A k t i v i t ~ t w e n i g e r schncll ab, als die
der BrenztraubensS~ure-Decarboxylase.
Beinl T o d ist i m B r u s t m u s k e l (tie P y r u v a t - D e c a r b o x y l a s e A k t i v i t / i t n a c h K o h l e h y d r a t d i g t viel
h 6 h e r als n a c h Fettdifit, w S h r e n d im H e r z m u s k e l keine solche V e r s c h i e d e n h e i t zu b e s t e h e n scheint.
E b e n s o ist im B r u s t m u s k e l die a- K e t o g l u t a r a t - D e c a r b o x y l a s e AktivitS~t b e i m T o d n a c h K o h l e h y d r a t digit gr6sser als n a c h Fettdifit, wtthrend in vielen Fallen i m H e r z m u s k e l d a s u m g e k e h r t e b e o b a c h t e t
wurde.
Verglichen m i t der n o r m a l e n A k t i v i t g t v o n B r u s t - u n d H e r z m u s k e l ist der finale p r o z e n t u a l e
Abfall der P y r u v a t - D e c a r b o x y l a s e Aktivit~it i l n m e r viel gr6sser, als d e r der a - K e t o g l u t a r s ~ u r e D e c a r b o x y l a s e , a b e r a u c h die erstere g e h t n i e m a l s vollst~ndig verloren.
Die A p o e n z y m e der M u s k e l n bleiben w'ghrend des g r 6 s s t e n Teiles der M a n g e l p e r i o d e n volls t ~ n d i g e r h a l t e n . E i n teilweiser V e r l u s t t r i t t allein in d e n l e t z t e n S t a d i e n ein, m i t A u s n a h m e der
a-IKetoglutars/i, u r e - D e c a r b o x y l a s e bei K o h l e h y d r a t d i S t .
R1LFERILNCES
1 ]7I. M. EVANS AND S. LEPKOWSKY, J. Biol. Chem., 83 (1929) 2 6 9 .
2 ~I. O. K. WESTENBRINK, Arch. nderl, physiol., 19 (1934) 94.
3 1~]. ORUBER, Thesis, U t r e c h t (1952).
4 M. GRUEER, Acta Physiol. PharmacoI. Neerl., 2 ( i 9 5 i / 5 2 ) 411 ; Biochim. Biophys. Acta, IO (1953) 136.
5 C. H. MONFOORT, Biochim. Biophys. Acta, 9 (1952) 331, 572; 14 (1954) 291.
6 I3. C. 1°. JANSEN AND H . O. K. WESTENBRINK, ,~cta Brevia Neerl., 3 (1933) 9.
7 j . CAMPBELL AND I. ~¢V. I ~'. DAVIDSON, J . Lab. Clin. Med., 34 (1949) lO278 A . G . C. W H I T E , L . O. KRAMPITZ AND C. H . WERKMAN, Arch. Biochem., 9 (1946) 229.
9 S. OCHOA, J'. Biol. Chem., 155 (1944) 87.
10 C. H . MONFOORT, Thesis, U t r e c h t (1953)n H . G. K . WESTENBRINK AND }~. I~. STEYN-I°ARV£, Intern. Rev. Vitamin Research (Z. Vitamin[orsch.),
21 (195 ° ) 461.
13 O . DILLS AND E . STFPI-IAN, Ber., 4 ° (19o7) 4336.
13 E. CARRIERE, Ann. Chim., 17 (1922) 41.
1 4 ]-I. P~. v. D. VAART, Rapporl S 32 (M4) Statist. A/d. 2dathem. Centrum, A m s t e r d a m (195o).
is L. J. REED, Physiol. Rev., 33 (1953) 544Received September 2nd, 195 4
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