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Carbohydrate metabolism in local and generalized tetanus
I84
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 1 0
(I953)
CARBOHYDRATE METABOLISM IN LOCAL AND GENERALIZED TETANUS by F. \ V E N S I N C K AND J. A. C O H E N
Medical Biological Institute o[ the National Detence Research Council T.N.O., Leyden (Netherlands)
T h e m a j o r i t y of e x p e r i m e n t a l d a t a s u p p o r t s t h e h y p o t h e s i s t h a t m u s c u l a r rigidity r e s u l t i n g f r o m i n t r a m u s c u l a r injection of t e t a n u s t o x i n is n o t of c e n t r a l origin b u t i n d u c e d b y f u n c t i o n a l d i s t u r b a n c e s residing a t t h e m y o n e u r a l j u n c t i o n or in m u s c l e m e t a b o l i s m . T h e s y m p t o m s of a generalized t e t a n u s are r e a d i l y e x p l a i n e d on t h e basis of a n i m p a i r e d f u n c t i o n of t h e muscle, t h e c e n t r a l n e r v o u s s y s t e m a n d possibly t h e liver. No s y s t e m a t i c i n v e s t i g a t i o n into t h e c h a r a c t e r of m e t a b o l i c d i s t u r b a n c e s in local t e t a n u s h a s b e e n carried out. I n f o r m a t i o n h a s so far been l i m i t e d to i n c i d e n t a l d a t a on glycogen a n d p h o s p h a t e c o n t e n t a n d t h e a c t i v i t y of a few e n z y m e s like A T P - a s e a n d cholinesterase in t e t a n i z e d muscles. I n t h e p r e s e n t p a p e r e x p e r i m e n t s are described w h i c h f o r m p a r t of a n i n v e s t i g a t i o n into t h e m e t a b o l i s m of a n i m a l s t r e a t e d w i t h t e t a n u s toxin, w h i c h involves a s t u d y of t h e g l y c o g e n s y n t h e s i s a n d glycolysis in t h a t condition.
Glycogen synthesis Male albino rats, w e i g h i n g lOO-15o g a n d s t a r v e d for 4 8 hours, were used. A f t e r t h e s t a r v a t i o n period, glucose w a s a d m i n i s t e r e d b y s t o m a c h t u b e or i n t r a p e r i t o n e a l injection, while in o t h e r experim e n t s l a b o r a t o r y s t o c k diet w a s fed ad libitum. L o c a l t e t a n u s w a s i n d u c e d b y i n t r a m u s c u l a r injection, generalized t e t a n u s b y i n t r a p e r i t o n e a l injection of toxin. Glycogen w a s isolated b y t h e m e t h o d of GooD et al.t; r e s u l t s are e x p r e s s e d as glucose (NELSON'S methodZ). Glycogen s y n t h e s i s in vitro w a s e v a l u a t e d a c c o r d i n g to WALAAS AND WALAAS3. I n c o n t r a s t to t h e m a r k e d increase in g l y c o g e n c o n t e n t of n o r m a l m u s c l e following t h e i n g e s t i o n of food b y s t a r v e d a n i m a l s , g l y c o g e n in t e t a n i z e d m u s c l e r e m a i n s practically u n c h a n g e d (Table I). T h e a d m i n i s t r a t i o n of glucose b y s t o m a c h t u b e to a n i m a l s in t h e p r e t e t a n i c period r e s u l t s in glycogen figures in m u s c l e (pretetanic period of local t e t a n u s ) a n d liver (pretetanic period of generalized t e t a n u s ) , w h i c h are significantly lower t h a n t h o s e o b s e r v e d in controls. I m p a i r m e n t of g l y c o g e n s y n t h e s i s , therefore, s e e m s to precede s y m p t o m s of t e t a n u s i n t o x i c a t i o n (Table II). F u r t h e r investig a t i o n s h o w e d t h a t e v e n in a n i m a l s w i t h a severe general i n t o x i c a t i o n s y n t h e s i s of liver glycogen TABLE I GLYCOGEN CONTENT OF NORMAL AND TETANIZED GASTROCNEMII OF PREVIOUSLY STARVED RATS DURING A 96-HOUR FEEDING PERIOD
Feeding period (hours)
Glycogen content (%) Normal
Tetanus
0.31 I.I 7 0.57 0.52
o. I8 0.32 °-14 0.24
T A B L E 12 GLYCOGEN CONTENT OF NORMAL AND TETANIZED MUSCLE (PRETETANIC P~RIOD OF LOCAL TETANUS) A N D LIVER (PRETETANIC P E R I O D O F G E N E R A L I Z E D TETANUS) 3 HOURS AFTER ADMINISTRATION OF G L U C O S E (I.5 g) BY S T O M A C H T U B E TO 4 8 - H O U R STARVED ANIMALS
Glycogen (%) o 24 48 96
(3)* (8) (5) (2)
liver
TABLE ANAEROBIC
GLYCOLYSIS O F D I A P H R A G M S QCO2 =
micro i C O z / m g
n o r m a l (15)*: t e t a n u s (15):
muscle
FROM NORMAL
{ n o r m a l (12): t e t a n u s (12) :
0.70 _E 0.o33 o.51 4- o.o31 2.4o 4- o.194 0.69 i o.12o
III AND TETANIZED RATS (GENERALIZED
dryweight/6o rain. Glucose concentration o.2 % .
Qc02 n o r m a l (6)*: t e t a n u s (6) :
3.65 4- 0.38 2.03 + o.12
* Figures b e t w e e n b r a c k e t s give n u m b e r of e x p e r i m e n t s .
TETANUS)
VOL.
10 11953)
SHORT COMMUNICATIONS, PRELIMINARY" NOTES
I85
occurs at a normal rate during the first hours after intraperitoneal glucose administration; after 4 hours, however, nearly all glycogen has disappeared. In vitro experiments showed t h a t glycogen synthesis in diaphragms from rats with a generalized tetanus proceeds normally during a 35 rain period; the insulin stimulation of glycogen synthesis and the breakdown of glycogen b y adrenalin a r e not altered.
Glycolysis Glycolysis in diaphragms was measured by the conventional WARBURG technique. Glucose was added in a final concentration of o.2 %. Muscle extracts were prepared by extraction of minced muscle with M/I 5 phosphate buffer p H 7.4. Glycogen, glucose-l-phosphate, and hexose diphosphate or 3-phosphoglyceric acid were used as alternate substrates. Samples were deproteinized with trichloroacetic acid and analysed for lactic acid 4, pyruvic acid 5, fructoses and phosphate~ before and after incubation (6o min, 37 ° C). A separation of intermediates was accomplished by Ba-fractionations. Anaerobic glycolysis in intact diaphragm (generalized tetanus) and in muscle extracts (local tetanus) is inhibited (Tables III and IV). It is concluded from the results of Table IV t h a t differences in lactic acid formation are due to the accumulation of hexosediphosphate in tetanus extracts, which could be isolated in the Ba-insoluble fraction. At fiist sight this seems suggestive of a decreased aldolase activity in tetanized muscle. We found, however, t h a t the addition of coenzyme I to extracts of such muscles completely restored glycolytic activity. This activation by coenzyme suggests that a blockade at the triosephosphatedehydrogenase stage should also be kept in mind. TABLE IV GLYCOLYTIC ACTIVITY OF I~XTRACTS FROM NORMAL AND TETANIZED MUSCLES (LOCAL TI~TANUS) Substrates: glucose-l-phosphate, hexosediphosphate, 3-phosphoglyceric acid. Figures between brackets give initial amounts of substrates. Results are expressed as mg found after incubation (6o rain, 37 o C).
Normal
Tetanus
Glucose-i-phosphate (8.5 mg) Glucose-l-phosphate Hexosediphosphate (as fructose) Pyrnvie acid Lactic acid
< i.o < i.o < o.io 1.39 + 0.062 o.36 3=_ o.o72 < o . I o 3.18 ± o.o77 1.64 4- o.148
Hexosediphosphate (3.8 mg as fructose) Hexosediphosphate (as fructose)
0.4o ± 0.080
1.9o 4- 0.220
1.47 i 0.025
1.42 ± o.o23
3-Phosphoglyceric acid (5 rag) Pyruvic acid
DISCUSSION I t is found t h a t the glycogen synthesis by skeletal muscles in vivo (local tetanus) and the glycolysis in diaphragms from rats with a generalized tetanus are severely impaired; the glycolytic activity of muscle extracts, also, is diminished. The site of the blockade can be located at the aldolase or the triosephosphatedehydrogenase stage. As it can be established, moreover, t h a t the synthesis of fiver-glycogen is considerably diminished in generalized tetanus, the conclusion seems justified t h a t tetanus intoxication is accompanied or caused by disturbances in carbohydrate metabolism. In contrast to rigor morris, the amount of energy-rich phosphate compounds is only slightly diminished in local tetanus. No evidence was obtained for the occurrence of gross changes in aerobic metabolism (cyclophorase activity, respiration in diaphragm and liver slices). Full details and discussion of results will be published in due time. REFERENCES 1 C. A. GooD, H. KRAMER AND M. SOMOGYI,J. Biol. Chem., ioo (1933) 485 • N. NELSON, J. Biol. Chem., 153 (1944) 375. 3 0 . WALAASAND E. WALAAS,J. Biol. Chem., 187 (195o) 77 o. 4 C. LonG, Biochem, f., 4° (1946) 27. T. E. FRIEDEMANI~rAND G. E. HAUGEN, J. Biol. Chem., I44 (1942) 67. s j . H. RoE, f . Biol. Chem., lO7 (1934) 15. C. H. FISKE AND Y. SUBBAROW,f . Biol. Chem., 66 (1925) 375. 8 V~r. W. [J'MBREIT, R. H. BURRIS AND J. F. ~TAUFFER, Manometric Techniques, Burgess, Minn., 1951. Received November i3th, 1952
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 1 0
(I953)
CARBOHYDRATE METABOLISM IN LOCAL AND GENERALIZED TETANUS by F. \ V E N S I N C K AND J. A. C O H E N
Medical Biological Institute o[ the National Detence Research Council T.N.O., Leyden (Netherlands)
T h e m a j o r i t y of e x p e r i m e n t a l d a t a s u p p o r t s t h e h y p o t h e s i s t h a t m u s c u l a r rigidity r e s u l t i n g f r o m i n t r a m u s c u l a r injection of t e t a n u s t o x i n is n o t of c e n t r a l origin b u t i n d u c e d b y f u n c t i o n a l d i s t u r b a n c e s residing a t t h e m y o n e u r a l j u n c t i o n or in m u s c l e m e t a b o l i s m . T h e s y m p t o m s of a generalized t e t a n u s are r e a d i l y e x p l a i n e d on t h e basis of a n i m p a i r e d f u n c t i o n of t h e muscle, t h e c e n t r a l n e r v o u s s y s t e m a n d possibly t h e liver. No s y s t e m a t i c i n v e s t i g a t i o n into t h e c h a r a c t e r of m e t a b o l i c d i s t u r b a n c e s in local t e t a n u s h a s b e e n carried out. I n f o r m a t i o n h a s so far been l i m i t e d to i n c i d e n t a l d a t a on glycogen a n d p h o s p h a t e c o n t e n t a n d t h e a c t i v i t y of a few e n z y m e s like A T P - a s e a n d cholinesterase in t e t a n i z e d muscles. I n t h e p r e s e n t p a p e r e x p e r i m e n t s are described w h i c h f o r m p a r t of a n i n v e s t i g a t i o n into t h e m e t a b o l i s m of a n i m a l s t r e a t e d w i t h t e t a n u s toxin, w h i c h involves a s t u d y of t h e g l y c o g e n s y n t h e s i s a n d glycolysis in t h a t condition.
Glycogen synthesis Male albino rats, w e i g h i n g lOO-15o g a n d s t a r v e d for 4 8 hours, were used. A f t e r t h e s t a r v a t i o n period, glucose w a s a d m i n i s t e r e d b y s t o m a c h t u b e or i n t r a p e r i t o n e a l injection, while in o t h e r experim e n t s l a b o r a t o r y s t o c k diet w a s fed ad libitum. L o c a l t e t a n u s w a s i n d u c e d b y i n t r a m u s c u l a r injection, generalized t e t a n u s b y i n t r a p e r i t o n e a l injection of toxin. Glycogen w a s isolated b y t h e m e t h o d of GooD et al.t; r e s u l t s are e x p r e s s e d as glucose (NELSON'S methodZ). Glycogen s y n t h e s i s in vitro w a s e v a l u a t e d a c c o r d i n g to WALAAS AND WALAAS3. I n c o n t r a s t to t h e m a r k e d increase in g l y c o g e n c o n t e n t of n o r m a l m u s c l e following t h e i n g e s t i o n of food b y s t a r v e d a n i m a l s , g l y c o g e n in t e t a n i z e d m u s c l e r e m a i n s practically u n c h a n g e d (Table I). T h e a d m i n i s t r a t i o n of glucose b y s t o m a c h t u b e to a n i m a l s in t h e p r e t e t a n i c period r e s u l t s in glycogen figures in m u s c l e (pretetanic period of local t e t a n u s ) a n d liver (pretetanic period of generalized t e t a n u s ) , w h i c h are significantly lower t h a n t h o s e o b s e r v e d in controls. I m p a i r m e n t of g l y c o g e n s y n t h e s i s , therefore, s e e m s to precede s y m p t o m s of t e t a n u s i n t o x i c a t i o n (Table II). F u r t h e r investig a t i o n s h o w e d t h a t e v e n in a n i m a l s w i t h a severe general i n t o x i c a t i o n s y n t h e s i s of liver glycogen TABLE I GLYCOGEN CONTENT OF NORMAL AND TETANIZED GASTROCNEMII OF PREVIOUSLY STARVED RATS DURING A 96-HOUR FEEDING PERIOD
Feeding period (hours)
Glycogen content (%) Normal
Tetanus
0.31 I.I 7 0.57 0.52
o. I8 0.32 °-14 0.24
T A B L E 12 GLYCOGEN CONTENT OF NORMAL AND TETANIZED MUSCLE (PRETETANIC P~RIOD OF LOCAL TETANUS) A N D LIVER (PRETETANIC P E R I O D O F G E N E R A L I Z E D TETANUS) 3 HOURS AFTER ADMINISTRATION OF G L U C O S E (I.5 g) BY S T O M A C H T U B E TO 4 8 - H O U R STARVED ANIMALS
Glycogen (%) o 24 48 96
(3)* (8) (5) (2)
liver
TABLE ANAEROBIC
GLYCOLYSIS O F D I A P H R A G M S QCO2 =
micro i C O z / m g
n o r m a l (15)*: t e t a n u s (15):
muscle
FROM NORMAL
{ n o r m a l (12): t e t a n u s (12) :
0.70 _E 0.o33 o.51 4- o.o31 2.4o 4- o.194 0.69 i o.12o
III AND TETANIZED RATS (GENERALIZED
dryweight/6o rain. Glucose concentration o.2 % .
Qc02 n o r m a l (6)*: t e t a n u s (6) :
3.65 4- 0.38 2.03 + o.12
* Figures b e t w e e n b r a c k e t s give n u m b e r of e x p e r i m e n t s .
TETANUS)
VOL.
10 11953)
SHORT COMMUNICATIONS, PRELIMINARY" NOTES
I85
occurs at a normal rate during the first hours after intraperitoneal glucose administration; after 4 hours, however, nearly all glycogen has disappeared. In vitro experiments showed t h a t glycogen synthesis in diaphragms from rats with a generalized tetanus proceeds normally during a 35 rain period; the insulin stimulation of glycogen synthesis and the breakdown of glycogen b y adrenalin a r e not altered.
Glycolysis Glycolysis in diaphragms was measured by the conventional WARBURG technique. Glucose was added in a final concentration of o.2 %. Muscle extracts were prepared by extraction of minced muscle with M/I 5 phosphate buffer p H 7.4. Glycogen, glucose-l-phosphate, and hexose diphosphate or 3-phosphoglyceric acid were used as alternate substrates. Samples were deproteinized with trichloroacetic acid and analysed for lactic acid 4, pyruvic acid 5, fructoses and phosphate~ before and after incubation (6o min, 37 ° C). A separation of intermediates was accomplished by Ba-fractionations. Anaerobic glycolysis in intact diaphragm (generalized tetanus) and in muscle extracts (local tetanus) is inhibited (Tables III and IV). It is concluded from the results of Table IV t h a t differences in lactic acid formation are due to the accumulation of hexosediphosphate in tetanus extracts, which could be isolated in the Ba-insoluble fraction. At fiist sight this seems suggestive of a decreased aldolase activity in tetanized muscle. We found, however, t h a t the addition of coenzyme I to extracts of such muscles completely restored glycolytic activity. This activation by coenzyme suggests that a blockade at the triosephosphatedehydrogenase stage should also be kept in mind. TABLE IV GLYCOLYTIC ACTIVITY OF I~XTRACTS FROM NORMAL AND TETANIZED MUSCLES (LOCAL TI~TANUS) Substrates: glucose-l-phosphate, hexosediphosphate, 3-phosphoglyceric acid. Figures between brackets give initial amounts of substrates. Results are expressed as mg found after incubation (6o rain, 37 o C).
Normal
Tetanus
Glucose-i-phosphate (8.5 mg) Glucose-l-phosphate Hexosediphosphate (as fructose) Pyrnvie acid Lactic acid
< i.o < i.o < o.io 1.39 + 0.062 o.36 3=_ o.o72 < o . I o 3.18 ± o.o77 1.64 4- o.148
Hexosediphosphate (3.8 mg as fructose) Hexosediphosphate (as fructose)
0.4o ± 0.080
1.9o 4- 0.220
1.47 i 0.025
1.42 ± o.o23
3-Phosphoglyceric acid (5 rag) Pyruvic acid
DISCUSSION I t is found t h a t the glycogen synthesis by skeletal muscles in vivo (local tetanus) and the glycolysis in diaphragms from rats with a generalized tetanus are severely impaired; the glycolytic activity of muscle extracts, also, is diminished. The site of the blockade can be located at the aldolase or the triosephosphatedehydrogenase stage. As it can be established, moreover, t h a t the synthesis of fiver-glycogen is considerably diminished in generalized tetanus, the conclusion seems justified t h a t tetanus intoxication is accompanied or caused by disturbances in carbohydrate metabolism. In contrast to rigor morris, the amount of energy-rich phosphate compounds is only slightly diminished in local tetanus. No evidence was obtained for the occurrence of gross changes in aerobic metabolism (cyclophorase activity, respiration in diaphragm and liver slices). Full details and discussion of results will be published in due time. REFERENCES 1 C. A. GooD, H. KRAMER AND M. SOMOGYI,J. Biol. Chem., ioo (1933) 485 • N. NELSON, J. Biol. Chem., 153 (1944) 375. 3 0 . WALAASAND E. WALAAS,J. Biol. Chem., 187 (195o) 77 o. 4 C. LonG, Biochem, f., 4° (1946) 27. T. E. FRIEDEMANI~rAND G. E. HAUGEN, J. Biol. Chem., I44 (1942) 67. s j . H. RoE, f . Biol. Chem., lO7 (1934) 15. C. H. FISKE AND Y. SUBBAROW,f . Biol. Chem., 66 (1925) 375. 8 V~r. W. [J'MBREIT, R. H. BURRIS AND J. F. ~TAUFFER, Manometric Techniques, Burgess, Minn., 1951. Received November i3th, 1952