Decarboxylation and carboxylation of pyruvate in the living mice

BIOCHIMIB, 1979, 61, 101-107.

Decarboxylation and carboxylation of pyruvate in the living mice. Pierre FAVARGER ~, Simonne ROUS and Sylvette BAS (*).

D~partement de Biochimie m~dicale, 20, r. Ecole de M~decine, 1211 Gendve 4, Suisse.

(10-10-1978).

R~sum~.

Summary.

Diff6rents lots de souris reqoivent des injections intraveineuses d ' a c 6 t a t e [1- ou 2-14C], de p y r u v a t e [1-, 2- o u 3-14C] et sont ex6cut6es 1, 3, 5 ou 15 rain plus tard. La radioactivit6 du CO.> ou du HCO3- du foie et de la c a r c a s s e ainsi que la radioactivit6 du g l u c o s e circulant sont mesur6es. Les rapports des radioactivit6s trouv6es d a n s ces compos6s apr~s injection de p y r u v a t e [3-~4C] et de p y r u v a t e [2-14C] permettent de sugg6rer :

Mice received i n t r a v e n o u s l y [1- or 2-14C]acetate, [1-, 2- or 3-1+C]pyruvate a n d w e r e killed 1, 3, 5 or 15 rain later. The radioactivity of CO2 or H C O 3 of liver or c a r c a s s as well as the radioactivity of blood glucose w e r e m e a s u r e d . The ratio of the radioactivity found in these comp o u n d s after [3-1+C] or [2-~4C]pyruvate injection s u g g e s t s that in the fed a n i m a l s :

1. que chez l ' a n i m a l aliment6 la d6carboxylation du p y r u v a t e est plus r a p i d e clue s a carboxylation, 2. q u ' u n e proportion tr~s notable du g l u c o s e n6oform6 d6rive de mol&cules de p y r u v a t e qui ont subi p r 6 a l a b l e m e n t une d 6 c a r b o x y l a tion et u n e c o n d e n s a t i o n en citrate, 3. que chez les a n i m a u x nourris a v e c une alimentation p a u v r e en qraisse le 1/3 ou le 1/4 environ des mol6cules de p y r u v a t e sont carboxyl~es, les 2/3 ou les 3/4 d6carboxyl6es.

2. most of the n e o s y n t h e s i z e d glucose w a s derived from p y r u v a t e molecules w h i c h h a d u n d e r g o n e a d e c a r b o x y l a t i o n followed b y a c o n d e n s a t i o n to citrate,

Introduction.

from [14C]pyruvate labelled in the 2 or 3 position.

There is a good deal of available informations about the utilization and transformation of the endogenous pyruvate by the two competitive pathways involving pyruvate carboxylase (P'CX, EC. 6.4.1.2) and pyruvate dehydrogenase (PDH, EC. 1.2.4.1). Such stu,dies have been carried out mainly in the perfused liver systems [1-3]. The present study was undertaken in the living animals in order to work under the most appropriate physiological conditions. On the other hand, we used a new approach for estimating the amount of pyruvate which was carboxylated or decarboxylated, i.e., the formation of tissular 14CO2

Previous work in our laboratory bad shown that the amount of 14COz found in the tissues was different when acetate was labelled at different positions. The activity of 1~C0~ obtained 3 minutes after administration of [2-14C]acetate was one-half of that obtained from [1-14C]acetate in the liver and adipose tissues of the mice [4]

(') With technical Ry.chner.

a~sistance

of M~'s Monique

<>To whom all correspondence should be addressed.

1. the d e c a r b o x y l a t i o n of the p y r u v a t e w a s more rapid t h a n its carboxTlation,

3. 1/4 to 1/3 of the p y r u v a t e w a s c a r b o x y l a t e d a n d 2/3 to 3/4 w a s d e c a r b o x y l a t e d in anim a l s receivinq a diet poor in fats.

These results suggested ,that during the degradation of acetyl CoA through the Krebs cycle, oxaloacetate was only regenerated 3 to 4 times before following alternate metabolic pathways. If the same difference could be found between the amount of 14C02 emerging from [3-~C]pyruvate or of [2-1+C]pyruvale, the conclusion could be that most of the injected pyruvate was totally de_ carboxylated and then converted to citrate.

P. Favarger and coll.

102

I n d e e d , t h e p r e l i m i n a r y c a r b o x y l a t i o n of p y r u v ~ t e to o x a l o a c e ~ a t e follow.ed b y its p a r t i c i p a t i o n i n t h e K r e b s c y c l e "~vonld yi.eld m o r e 14CO 2 w i t h [3J+C]pyruvate as a p r e c u r s o r t h a n w i t h t h e E2-~C] p y r u v a t e . H e n c e , i n t h e p r e s e n t w o r k [1-, 2- o r 3 - 1 4 C ] p y r u v a t e w a s i n j e c t e d to m i c e a n d t h e I+CO 2 of t h e t i s s u e s as ~ve.ll as t h e 14C of b l o o d glucose were measured a short time later. The s p e e d of g l u c o s e [51 a n d p y r u v a t e [6] u t i l i z a t i o n in mice would render indispensable this condition. This last measurement was performed to g i v e i n f o r m a t i o n o n t h e p r o p o r t i o n of p y r u v a t e which gives glucose ~fter being converted into t h e a c e t y l CoA p a r t o f c i t r a t e .

[3-14C] p y r u v a t e was checked by h i g h voltage electrophoresis. The first c o n t a i n e d 2.6 per cent of [~4C] acetate and th.e second 3.3 per cent. After blood collection, the liver a n d the carcass were i m m e d i a t e l y fixed in boiling alcoholic p o t a s s i u m hydroxide.

The radioactivity of blood glucose was m e a s u r e d after precipitation of the p r o t e i n according to Somogyi I71 and e l i m i n a t i o n of ionized m e t a b o l i t e s w i t h ion-exchanging resins [8]. The radioactivity of tissue C02 and respiratory CO~ was d e t e r m i n e d as. reported e a r l i e r [4]. The ~4C0_0 exhaled b y some mice d u r i n g the first m.inutes after [1-14C] or [2-14C] acetate i n j e c t i o n was also measured.

Results. T h e q u a n t i t i e s o f 1~CO~ e x h a l e d b y m i c e a f t e r a d m i n i s t r a t i o n of [1- o r 2 - 1 + C l a c e l a t e a r e g i v e n i n f i g u r e 1. T h e e v o l u t i o n of t h e d e g r a d a t i o n c u r v e s c l e a r l y shonvs t h a t t h e p r e d o m i n a n c e of 14C0~ f r o m [ 1 - t + C ~ a c e t a t e o n l y e x i s t s d u r i n g t h e first 5 rain after injection.

Experimental part. Ten vceeks old albin,u mice (Swiss) weighing 25 to 40 g were fed ad l i b i t u m ¢ Nafag >> food from 4 a.m. to 6 p.m. They were killed a t 8 a.m. 1, 5 or 15 m l n a f t e r a n i n t r a v e n o u s i n j e c t i o n of [1-14C], [2-14C] or [3-14C] p y r u v a t e (1,0 mCA/mmol) (Radiochemical Center, A m e r s h a m , England). The p u r i t y of [2-14C] a n d

T h e f i g u r e s of t a b l e s I a n d II c o r r e s p o n d to 1+C0 2 + 14CO3H- f o u n d i n l i v e r a n d c a r c a s s of

so,~o'

e~c]

*¢.l.l+

~.,o'

3o,~6

Fro. 1. - - Radioactivity of respiratory CO+ after intravenous administration of [1- or 2-~C] acetate to mice.

2o.lo

1010 +

5, m +a

'

t

1

, 2

3

*

s

6

7

I I

9

Io

;. m

2 mice received 5 ~Ei of ei.ther [1-14C] acetate or [2-14C] acetate a n d were killed, a t different t i m e s a f t e r t h i s injection. ~ h e r e s u l t represents t h e total t4C02 expired.

TABLE I.

Total radioactivity of [1+C]0 e in liver and carcass after injection to mice of 5 ~Ci of [1-] or [2-1+C] acetate. [1-1+C] acetate

[2-1+C] acetate

Time Fasted (t)

Fed {2)

Fasted 13)

Fed (4)

[2-t4C] / acetate

Fasted

[t-uC] acetate Fed

n=9 n~9 34,615-~- 1 , 6 7 0 46,760-t-3,060 n --~ 9 n ~- 9 0.63 0.47 (1)and(3) (2) and(4) 21,860 ~ 1,860 22,100 -q- 3,530 p = 0.005 p ~ 0.001 u~5 n~5 845,550-t-28,000 771,720-t-65,490 n -~ 5 n = 5 0.53 0.44 [l+C]0~carcass 1 mn (1) and (3) (2) and (4) 450,370~__16,500 338,500-+38,600 p ~ 0.00l p ~ 0.00l The significanees b e t w e e n the m e a n s are given o n l y for the results w h i c h can be i u t e r p r e t a t e d , i.e. w h i c h concern the c o m p a r i s o n of t h e same p r e c u r s o r differently labelled, p w a s calculated according to S t u d e n t <
[14C]0~ liver

1 mn

BIOCHIMIE, 19'79, 61, n ° 1.

Decarboxylalion and carboxylalion of pyruvale.

103

TABLE II.

Total radioactivity of [l~C]Oz in liver and carcass a[ter injection to mice of 5 ~Ci of [1-l~C], [2-t~C] or [3-~C~pgruvate. [I-14C] pyruvate Time Fasted {t)

n =

[t4C]O~ liver

n = 13 1 2 1 , 8 3 0 _+ 8 , 3 0 0

13

1 4 6 , 2 5 0 -4- 6 , 3 2 0

1 mn

(1) and (3)

(2) and (4)

p <

p <

n = [14C]0~ c a r c a s s

Fed (2)

0.001 13

13

n =

1,228,000 ~ 105,000

1 mn

0.001

1,350,0O0-4- 93,000

(1) a n d (3) p ~ 0.001

(2) a n d (4) p ~ 0.001

[2-t4C] pyruvate

[3-1~C] pyruvate

[3-uC] / pyruvate

[2-t~C] pyruvate

Time Fasted (3)

Fed (~}

Fasted (5)

Fed (6)

Fasted

Fed

n=13 33,505-4-3,265 (~)and(6) p < 0.001

n = 13 4 0 , 9 3 0 _____5 , 1 4 0

n = 13 17,730-{-1,670

1.27

0.53

n= 6 91,265-{-5,660 (4) a n d (6) 0.05 > p>O.02

n = 6 6 0 , 0 4 0 _--+- 1 , 8 3 0

n = 6 7 6 , 3 0 0 -+- 2 , 1 7 0

1.0

0.84

n=13 32,060-{-2,530

1 mn

(3) a n d (5) n.s. [14C]0~ liver 5 mn

n =6 60,600+2,850 --

(3) a n d (5) n.s.

1 mn

n = 13 n = 13 180,345!16,955:324,130+33,930 (3) a n d (5) (4)and(6) 0.01>p>0.005 p < O_001

n = 13 126,500 -}- 6,470

n = 13 138,400 ! - 1 3 , 6 2 5

0.70

0.43

5 mn

n=6 653,900+30,492 (3) a n d (5) p < 0.001

n = 6 398,160 -~- 14,920

n = 6 741,000 ~___23,745

0 61

0 76

[~C]0~ c a r c a s s n=6 975,030+68,076 (4) a n d (6) p < 0.001

T h e significan.ces b e t w e e n t h e m e a n s a r e g i v e n o n l y f o r t h e r e s u l t s w h i c h c a n be i n t e r p r e t a t e d , i.e. w h i c h c o n c e r n t h e c o m p a r i s o n of t h e s a m e p r e c u r s o r d i f f e r e n t l y l a b e l l e d , p w a s c a l c u l a t e d a c c o r d i n g to S t u d e n t <~t >> t e s t . n.s. = n o t s i g n i f i c a n t , n = n u m b e r of a n i m a l s .

TABLE III.

Total radioactivity of blood glucose after injection of 5 ~Ci of [1-1',C] or [2-l',Ciacetate. [l-'~'C] acetate

[2-~4C] acetate

[2-'~C] acetate

/

[t-"C]

acetate

Time Fasted~(t)

n = 5

R a d i o a c t i v i t y of blood glucose

I mn

1 8 , 6 6 0 ___ 915 (1) a n d (3) p < 0. 001

The results are expressed in dpm/ml n = n u m b e r of a n i m a l s .

BIOCHIMIE, 1979,

61, n ° 1.

Fed~4~)

n : 5

1 5 , 6 6 5 ± 315 (2) a n d (4) p < 0.00l

Fasted (3)

n----5

32,540 A- 1 , 8 7 0

Fed (4)

Fasted

Fed

n:5 27,300 -4=_ 1 , 0 0 0

1.74

1.74

of b l o o d -4- S.E.M. p w a s c a l c u l a t e d a c c o r d i n g to S t u d e n t < t e s t .

P. Favarger a n d coll.

104

after i n j e c t i o n the difference became m u c h smaller. These ratios were not very m u c h different for 14C02 of the carcass.

mice 1 rain after an i n t r a v e n o u s i n j e c t i o n of either [1- or 2-14C]acetate or [1-, 2- or 3-14C] pyruvate. The ratio ~CO 2 from I2-~4C]acetate

I n fed mice, the i n c o r p o r a t i o n of [2J4C] acetate i n t o blood glucose was double than that obtained w i t h [1J4C]acetate (table III).

14CO2 from [1-14C]acetate f o u n d i n this e x p e r i m e n t was slightly lower t h a n the values r e p o r t e d by us else~vhere [4]. In this case the delay b e t w e e n a~etate i n j e c t i o n a n d sacrifi~ce (3 m i n ) was p r o b a b l y too long to rule out the i n f l u e n c e of some i n t e r c o n v c r s i o n reactions.

Discussion.

The q u a n t i t y of 14.C0u f o u n d in tiss.ues 1 m i n after [1-14C]pyruvate injec.tion was six to ten times h i g h e r t h a n after C2- or 3-14C]pyruvate injection. F a s t i n g c o n s i d e r a b l y changed the ratio of 14C0 2 issued from [3- a n d 2-1*C]pyruvate and the observed c h a n g e was more m a r k e d t h a n that o b t a i n e d after [2- or 1-14C]acetate injection. So, the ratio 14C0 2 from [3-14C]pyruvate 14CO2 from [2-14C]pyruvate f o u n d i n Jiver was 0.53 ~vith fed mice and 1.27 with fasted mice (1 m i n after injection). Five rain

1. - - 1 ~ C 0 e formation. - - The p r e d o m i n a n c e of 14C02 issued from [1-14C]acetate w a s only visible d u r i n g the five first m i n u t e s of the e x p e r i m e n t (fig. 1),. Therefore it was essential to c a r r y out only short-time experimen.ts. I n this w a y a significant p r o d u c t i o n of 14CO2from [14CJpyruvate or [14C]acetate o r i g i n a t i n g from less direct pathways t h a n the i m m e d i a t e degradation of the citrate was avoided. We m e a s u r e d the hepatic 1~C02 r a t h e r t h a n the r e s p i r a t o r y I~CO 2 i n o r d e r to obtain d i r e c t i n f o r m a t i o n on the decarboxylations o c c u r i n g i n l i v e r w h i c h is direct'ly implicated i n gluconeogenesis.

TABLE I V .

Total radioactivity of blood glucose after injection of 5 ~Ci of [1-l',C], [2-I',C~ or E3-1~C]pyruvate. [t-1'-C] pyruvale Time Fasted (t)

Fed {2)

n=9 Radioactivity n ~ 9 8,400-t- 8 1 5 of blood 1 mn 30,355 ~___2,040 (2) and(4) glucose (l)and ( 3 ) p : 0.01 0.05 ~ r~ ~ 0 . 0 2

[2-14C] pyruvate

[3-t~C] / [2-~4C] pyruvate pyruvate

[3-14C] pyruvate

Time Fasted (3)

Radioactivity of b l o o d

n =

13

Fed (~) n =

13

1 mn

85,620 ~ 7,200 17,090 ~_ 1,300 (3) a n d (5) n.s. t4) and (6) n.s.

5 mn

n : 6 274,870-+- 29,850 (3) and (5) n.s.

glucose 15 mn

n=6 47,080~-2,100 (4) a n d (6) p : 0.02

n~-~-8 41,440~ 5,020 (4) and (6) 0,01 ~ p > 0.P,05

Fasted (5) n =

13

76,900-+- 6,600

n : 6 229,175-1- 18,650

Fed {6) n ~

13

20,520 + 2,400

n : 72,900 ~

6 8,980

n = 8 61,860 ~___2,780

Fasted

Fed

0.89

1.20

0.83

1.55

1.50

The results are expressed in dpm/ml of blood 4- S.E.M. p was calculated according to Student ¢ t >> test. n •s.--. -- not significant, n. ---- number of animals.

BIOCHIMIE, 19'79, 61, n ° 1.

Decarboxylation and carboxylation of pyruvate. If the p y r u v a t e w a s e n t i r e l y d e c a r b o x y l a t e d , its 'C 2 a n d 3 w o u l d r e s p e c t i v e l y c o r r e s p o n d to the C 1 a n d 2 of acetate. The r a t i o 14CO2 from [3-1¢C]:pyruvate 14C02 f r o m [2-1¢C]pyruvate w o u l d be the s a m e t h a n that f r o m [2-14C] acetate [1-1~C] acetate

RP

(RA)

E v e n u n d e r the p r e s e n t experimen,tal c o n d i t i o n s w h e r e the d e l a y b e t w e e n the i n j e c t i o n of r a d i o a c t i v e p r e c u r s o r s a n d the execution of animals w a s v e r y s h o r t (1 rain) a great n u m b e r of K r e b s c y c l e r e v o l u t i o n s h a d t i m e to take p l a c e [9]. C o n s e q u e n t l y the r a d i o a c t i v i t y of ~4GO~ s h o u l d be g r e a t e r after i n j e c t i o n of [1-14C]acetate or [2-14C]pyruvate only if the oxaloacetate escap e d the K r e b s cycle after a small n u m b e r of revolutions [4].

105

The first r a t i o of 0.5 f o u n d for RP w i t h fed m i c e k i l l e d 1 m i n after the i n j e c t i o n w a s almost the same after i n j e c t i o n of [2-14C a n d 1-14G]ace rate. The most l i k e l y sequence of r e a c t i o n s explain i n g t h i s result is : d e c a r b o x y l a t i o n into acetyl CoA, c i t r a t e s y n t h e s i s f o l l o w e d b y 3 successive cycles. In this .manner all the o r i g i n a l [2-14C] of p y r u v a t e a n d half of the [3-1¢C]pyruvate w i l l a p p e a r in the CO 2. I n o r d e r to o b t a i n m o r e 14C0 2 f o r m e d from [3-14C],pyruvate, it is n e c e s s a r y t h a t pyruvate undergoes a preliminary carboxylation into o x a l o a c e t a t e f o l l o w e d b y at least 2 K r e b s cycles. This w a s the case 5 m i n later (table II) w h e n the r a t i o ~ w a s [2- C]O 2

significantly higher.

We can t h e r e f o r e suggest that in fed mice, the p y r u v a t e p e n e t r a t e s m o r e r a p i d l y into the acetate p a r t of c i t r a t e t h a n into its oxaloacetate part. This w a s e v i d e n t l y not true in 14 h fasted m i c e

H o w e v e r , p a r t of the p y r u v a t e is also c a r b o x y lated, y i e l d i n g oxaloacetate. 3:laree p o s s i b i l i t i e s exist if w e assume that the w h o l e o.f p y r u v a t e is carboxylated.

for ~vhich the r a t i o [3-14C] 02 i n c r e a s e d conside[ 2-14C] 02 r a b l y , m a i n l y in the liver a n d even in the mice k i l l e d 1 m i n after the p y r u v a f e injection.

a) The before its citrate. In after one w o u l d be

2. - - [ltC] glucose formation. - - T h e r a t i o s 3/2 of the radioactiviti.es f o u n d in b l o o d glucose after i n j e c t i o n of [3-14C~ p y r u v a l e and [2-~C] p y r u v a t e w e r e significantly h i g h e r t h a n one in fed m i c e a n d s o m e w h a t l o w e r in fasted mice. Conseq u e n t l y it can be suggested that in fed m i c e m a n y of the p y r u v a t e m o l e c u l e s d o not follow the classical p a t h w a y l e a d i n g to the glucose (mitoc h o n d r i a l oxaloacetate, malale, extra~mitochon.drial o x a t o a c e t a t e a n d P E P ) . The most l i k e l y p a t h w a y e x p l a i n i n g the excess of r a d i o a c t i v i t y in the glucose a f t e r injectioal of [3-14C] p y r u v a t e is a d e c a r b o x y l a t i o n f o l l o w e d b y the c o n d e n s a t i o n to yiel:d c i t r a t e a n d a d e g r a d a t i o n to malate. Malate in t u r n w o u l d y i e l d in the e x t r a - m i t o c h o n d r i a l c o m p a r t m e n t t w i c e m o r e r a d i o a c t i v e glucose w h e n it is issued f r o m [3-14C] p y r u v a t e rat h e r [2-14C] p y r u v a t e . In t h e 5 and 15 m i n experime,nts t h e excess of 14C f r o m [3-14C] p y r u v a t e in the glucose of fed m i c e c o r r e s p o n d e d to the p r e l i m i n a r y d e c a r b o x y l a t i o n of h a l f of the p y r u v a t e m o l e c u l e s finaly t r a n s f o r m e d in,to glucose (cf. a p pendix).

t o t a l i t y .of o x a l o a c e t a l e is r a n d o m i z e d c o n d e n s a t i o n w i t h acetyl CoA to give this case the 1~C02 w h i c h w o u l d a p p e a r or several r e v o l u t i o n s of Krebs c y c l e i d e n t i c a l f r o m [2- or 3-1~C]pTruvate.

b) Only a p a r t of o x a l o a c e t a t e is r a n d o m i z e d a n d in t h i s case the r a d i o a c t i v i t y f o u n d into ~4C02 f r o m [3-14C]pyruvate w i l l be h i g h e r t h a n that of [2-14C] p y r u v a t e . c) No r a n d o m i z a t i o n of oxaloacetate occurs. In this case the r a t i o 14C02 f r o m [3-14C]pyruvate 1~C02 f r o m [2-14C]pyruvate will be i d e n t i c a l to 14C0z f r o m [ 1 J ~ C ] a c e t a t e 14C02 f r o m [2-14C]acetate The i m p o r t a n c e of r a n d o m i z a t i o n could v a r y c o n s i d e r a b l y ~ i t h different p h y s i o l o g i c a l or exper i m e n t a l ,conditions. It is p o s s i b l e that t h e r e is co,mpart'mentation of the o x a l o a c e t a t e pool, the one p a r t i c i p a t i n g d i r e c t l y in gluconeogenesis being d i f f e r e n t f r o m t h a t ~vhi.ch forms citrate. A c c o r d i n g to Miillhofer [10, 11] this r a n d o m i z a tion is c o m p l e t e in l i v e r p e r f u s e d ~vith [2-I4C] lactate as a p r e c u r s o r , but not w i t h [2-14C]pyruvate. Since the i n f r a m i t o c h o n d r i a l m e t a b o l i s m of o x a l o a c e t a t e is d i f f e r e n t in rats and mice [12], it is difficult to say w h e t h e r u n d e r the p r e s e n t conditions t h e r e is r a n d o m i z a t i o n or c o m p a r t m e n t a [ion.

BIOCHIMIE, 19q9, 61, u ° I.

I n c o n c l u s i o n the results of the p r e s e n t s t u d y suggest that : 1. T h e d e c a r b o x y l a t i o n of p y r u v a t e into acetyl CoA is m o r e r a p i d t h a n its c a r b o x y l a t i o n into oxaloacetate. 2. Even in fed a n i m a l s the gluconeogenesis is imp o r t a n t and about half of the oxaloacet:~te

106

P. Faoarger and coll. APPENDIX.

3CH3-2CO-1COOH 3CH3-2COSCoA

3CH3-2COSCoA

>

COOH-CO-CH2-COOH

+

3CH2-2COOH HO-C~ COOH

31H2-2COOH ......

~

l

3C0 J

CH2--COOH

2COOH

.....

3CHO ! 3CHOH 2t

CHOH CHOH

2! ! 3CHOH

3~H20H

3CH3-2CO-1COOH CH2COOH~I

~

+

CO2 ~

'J COOH-3CH2-2CO-1COOH (I)COOH (2)3CH2-(3)2CHOH-1COOH

-

HO-~2C~IcooH

(2)3~H~_I~OOH (3)2C~

0 OH

(1)COOH-~2)3CH!--~3) 2C0-JCOOH (2)3CHo I

(3)2CHOH I

1CHOH p

(~)2CHo

1

t

(3)2CHOH (2)3CHOH I (2)3CHOH I (3)2CHOH (3)2~H20H ( ) : randomization. BIOCHIMII¢, 19'79, 61, n ° 1.

11CHOH I (3)2CHOH I (2)3CH20H

D e c a r b o x y l a t i o n a n d c a r b o x y l a t i o n o[ p y r u v a t e . w h i c h i s c o n v e r t e d ,into g l u c o s e d,er.ives f r o m t h e d e c a r b o x y l a t e d p a r t of p y r u v a t e w h i c h p a r t i c i pates in the synthesis of citrate. It w a s s h o w n b y u s e a r l i e r [4] t h a t o x a l o a c e t a t e was recycled twice or thrice before leaving the K r e b s c y c l e , it c a n no'w b e s u g g e s t e d t h a t u n d e r o u r c x p e r i m e n t a l c o n d i l i o n s (i.e. a f a t p o o r d i e t ) t h e c a r b o x y l a t i o n of p y r u v a t e i n t o o x a l o a c e t a t e n e e d s n o t e x c e e d 1 / 3 o r 1 / 4 of t h e d e c a r b o x y l a t e d part.

Acknowledgements. This work was supported by a grant of the Swiss National Science Foundation, Bern, No. 3.66#.075.

BIOCHIMIE, 1979, 61, n ° 1.

107

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