- Email: [email protected]
Octanoic acid-induced coma and reticular formation energy metabolism
Brain Research, 335 (1985) 131-137 Elsevier
131
BRE 10759
Octanoic Acid-Induced Coma and Reticular Formation Energy Metabolism* DAVID W. McCANDLESS
Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, TX 77025 ( U.S. A.) (Accepted August 21st, 1984)
Key words: energy metabolism - - coma - - octanoic acid - - ATP - - reticular formation - - metabolic encephalopathy
The medium chain fatty acid octanoic acid was injected i.p. into 20-22 g Swiss-Albino mice at a dose of 15 ~mol/g. This dose produced a reproducible response consisting of a 3-4 min period of drowsiness, followed by coma. These mice as well as suitable controls were sacrificed by rapid submersion in liquid N2, or by microwave irradiation in a 7.3 kW microwave oven. Tissue from the reticular formation and the inferior colliculus was prepared for microanalysis of the energy metabolites glucose, glycogen, ATP and phosphocreatine. Results from this study showed a selective effect on energy metabolism in cells of the reticular formation. Both glucose and glycogen were elevated in the coma and precoma state. In addition, ATP and phosphocreatine were decreased in the reticular formation during coma. These results show a selective effect of octanoic acid on energy metabolism in the reticular formation both in the precoma stage, and during overt coma. The selective vulnerability of the reticular formation to metabolic insult may act in a beneficial manner to the animal by inducing coma. This lowers the overall demand for energy, thereby placing the animal in a milieu in which there is an increased chance for correction of the perturbation.
INTRODUCTION
octanoic acid on cerebral energetics, and have found no change in either the net levels of metabolites such
E n c e p h a l o p a t h y associated-with liver failure has been
studied
extensively,
and
the
concept
as A T P and phosphocreatine, or on their t u r n o v e r 26.
has
The adenylate energy charge has been r e p o r t e d to be
evolved that a 'toxin' or a combination of toxins is responsible for the neurological symptoms6,22, 32. A m -
normal in octanoic acid induced coma 2. R e c e n t stud-
monia 4, mercaptans 31, phenols 28 and short and medi-
reticular formation
um chain fatty acids 20 have all b e e n implicated in he-
have shown a selective effect 15. Since previous stud-
patic encephalopathy. The evidence for an encepha-
ies on octanoic acid have used large pieces of tissue~
lopathic role for the m e d i u m chain fatty acid octanoic
the present study was u n d e r t a k e n to examine the ef-
acid is somewhat circumstantial. Octanoic acid is ele-
fects of octanoic acid on energy metabolism in micro-
vated in the serum of patients with cirrhosis and he-
sections from the reticular formation, uncontami-
patic encephalopathy 18, and has also b e e n r e p o r t e d
nated by other perhaps less affected tissue, in this
ies on energy metabolism in discrete regions of the in metabolic encephalopathy
to be elevated in R e y e ' s SyndromelO, 24. In vitro, oc-
study we have used octanoic acid to induce coma in
tanoic acid has been shown to uncouple oxidative phosphorylationS, 29 and inhibit Na, K A T P a s e 1.25.
small mice, and have used both liquid N 2 and micro-
When octanoic acid is injected into rats, hypoglyce-
lites glucose, glycogen, A T P , and phosphocreatine
mia results 21, h o w e v e r the extent of the hypoglyce-
were measured in 100-500 ng samples from the reticular formation and the inferior colliculus, and the re-
mia is not severe enough in itself to cause c o m a 14. Previous investigators have e x a m i n e d the effect of
wave irradiation for sacrifice. The energy metabo-
suits are the basis for this report.
* Presented in part at the Eleventh International Symposium on Cerebral Blood Flow and Metabolism, Paris, 1983. Correspondence: D. W. McCandless, Department of Neurobiology and Anatomy, University of Texas Medical School at Houston. P.O. Box 20708, Houston, TX 77025, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division )
MATERIALS AND METHODS Female Swiss-Albino mice weighing 2 0 - 2 2 g were injected i.p. with octanoic acid at a dose of 15 ktmol/g. Control mice received an e q u i m o l a r dose of saline, or no injection. Mice were sacrificed at 3 rain following injection (precoma), or 1 min after the onset of coma. Animals were sacrificed by rapid submersion in liquid N 2. In order to eliminate the possibility of spurious results due to a delay in freezing of the deeply located reticular formation, a separate series of mice was sacrificed using a 7.3 k W microwave oven at 2450 M H z t6. Following sacrifice, the brains were r e m o v e d in a W e d e e n cryostat at - 2 0 °C, transferred to an I E C cryostat and sectioned into sections 20 u m thick. These sections at the level of the inferior coUiculus (an easily identifiable l a n d m a r k ) were transferred to aluminum holders and freeze-dried at - 4 0 °C for 24 h. Following this t r e a t m e n t , the sections can be brought to r o o m t e m p e r a t u r e u n d e r vacuum and manipulated without loss of labile metabolites. Small 100-500 ng samples from the inferior colliculus and reticular formation were freehand dissected and weighed on a quartz fiber fishpole balance (sensitivity -+ 2 rig). Samples were subsequently l o a d e d into oil well racks for m e t a b o l i t e analysis using enzymatic cyclingS.12. D a t a were analyzed using the non-parametric statistic, the M a n n - W h i t n e y U test for statistical significance 23. In another series of mice, turnover (utilization) was assessed by converting the brain to a closed system by decapitation 7. The head was m a i n t a i n e d at 37 °C for 15 s after which it was frozen in liquid N 2 and processed the same as intact animals.
GLUCOSE
.:~i RAG
~O.C
• tO T
80
>,
E ©
CONTROL
PRECOMA
COMA
Fig. 1. Effects of octanoic acid induced coma on regional cerebral glucose. Animals sacrificed by submersion in liquid N2. R.A.S., reticular activating system; I.C., inferior coUiculus. 3-5 animals per group (Figs. 1-7). Data expressed as mean + S.E. *, P < 0.00l.
acid injection on glucose in the inferior colliculus and the reticular formation is shown in Fig. 1. Glucose was elevated in the reticular formation only. The increase was present in both the p r e c o m a and coma stage. Fig. 2 shows the response of cerebral glycogen to octanoic acid. In this case there was a significant increase in glycogen in the inferior colliculus in coma
RESULTS The i.p. injection of octanoic acid at a dose of 15 ~mol/g results in a reproducible response characterized by a 3 - 4 rain p e r i o d of drowsiness which is followed by coma. Seizure activity was not o b s e r v e d in octanoic acid treated mice. Figs. 1 - 4 , and Figs. 8 and 9 represent the effects of octanoic acid on metabolites from mice sacrificed by submersion in liquid N:, whereas Figs. 5 - 7 show metabolites m e a s u r e d in brains from mice sacrificed by microwave irradiation. The effects of octanoic
"O
PRE~
Fig. 2. Effects of octanoic acid induced coma on regional cerebral glycogen. Animals sacrificed by submersion in liquid N2. Data expressed as mean _4-S.E. *, P < 0.001.
133 iii R.A.S.
•
ooo
• "- R.A.S.
ATP
elo
I.C.
•
10.0
GLYCOGEN
I.C.
16£
~72
:>,
°°°,,
,°,,, ,,°,, ,,°°,
05
'o
12.0
o)
E
°,°°° °°°°° °°°°°
O
°°,., °,°°, .°,°, °°,,, °°,,° .°°° .°.°
~ E c--
8.O
°,°° ,°°°° ,°°°° °°°°.
°°°,° ° . . ° .
, , ° . . °,,°° °,°,,
4.0
,.,°° , , , ° ° °,°°°
CONTROL PRECOMA COMA Fig. 3. Effects of octanoic acid induced coma on regional cerebral ATP. Animals sacrificed by submersion in liquid N 2. Data expressed as mean + S.E. *, P < 0.05; **, P < 0.002.
only whereas glycogen was elevated in the reticular formation in both the precoma and coma stages. Fig. 3 shows the effects of octanoic acid on ATP in the brain. Note that ATP is reduced in both the precoma and coma stage in cells of the reticular formation only. Interestingly, phosphocreatine was decreased only in the coma stage, and again, only in the reticular formation (Fig. 4). In order to validate the data obtained from mice
CONTROL
COMA
Fig. 5. Effects of octanoic acid induced coma on regional glycogen. Animals sacrificed by microwave irradiation. Data expressed as mean _+ S.E. *, P < 0.01.
frozen in liquid N2, another series of mice was treated with the same dose of octanoic acid, then sacrificed at 7.3 kW in a 2450 MHz microwave oven. Fig. 5 shows "•." " RA S o o l
.
•
•
I ,.c ATP
~ R.AS.
• [C.
Per
8.0
6.0
E O9
4£
O
E c-
2£
CONTROL
PRECOMA
COMA
Fig. 4. Effects of octanoic acid induced coma on regional cerebral phosphocreatine. Animals sacrificed by submersion in liquid N 2. Data expressed as mean _+ S.E. *, P < 0.02.
CONTROL
COMA
Fig. 6. Effects of octanoic acid induced coma on regional cerebral ATP. Animals sacrificed by microwave irradiation. Data expressed as mean + S.E. *, P < 0.05.
!34 oeo
• *" eee
RA.S
"2o
I I tc
,,
oF.-L_-7_
6O
POt
16.0
IC
ATP /
-
.
....... .-.,,
-
' O,
120~
ATP
RAS
PCr
;:~A
>,
o~ E
15
CO
C
as
"
Fig. 9. Effects of octanoic acid induced coma on regional turnover rates of ATP and phosphocreatine. C. control mice frozen intact in liquid N2; 15, decapitated heads maintained at 15 s at 37 °C, then frozen in liquid N 2. Solid lines represent control mice, dash lines represent precoma mice and dots represent comatose mice.
o E t"
CONTROL
ic decrease in phosphocreatine localized to the reticular formation in the coma stage.
COMA
Fig. 7. Effects of octanoic acid induced coma on regional cerebral phosphocreatine. Animals sacrificed by microwave irradiation. Data expressed as mean _+S.E. *, P < 0.05.
the effects of octanoic acid on glycogen, and as in the case of mice sacrificed in liquid N2, glycogen was elevated in the reticular formation only. Fig. 6 shows a selective decrease in A T P in the reticular formation in the coma stage, and Fig. 7 shows a similar dramat-
Fig. 8 shows the utilization rate at 15 s for glycogen and glucose in control, precoma, and comatose mice. Glycogen utilization rates are similar in both regions, whereas glucose utilization rates in coma induced by octanoic acid are increased in both the inferior colliculus and the reticular formation. Fig. 9 shows that in the case of A T P , the utilization rate over 15 s is lower in the coma stage than in controls or the precoma stage, and phosphocreatine showed a similar response in that the rate of utilization in comatose mice was lower than that in controls or precomatose mice.
12C
It must be r e m e m b e r e d that these data show trends
GLYCOGEN-LC
only; since they compare 15 s decapitation levels to '0' second non-decapitation metabolite levels, they
To 8
6[ 4(
are derived data, and as such are not a m e n a b l e to statistical analysis.
..... ~.~:..LLLLLL..~C..:.:
2(
~12c
GLYCOGEN-R.A.S
GLUCOSE-RA
S
DISCUSSION
~1oc
"~~tL........... ..;.:.:.~.:~ Fig. 8. Effects of octanoic acid induced coma on regional turnover rates of glucose and glycogen. C, control mice frozen intact in liquid N2; 15, decapitated heads maintained at 15 s at 37 °C, then frozen in liquid N2. Solid lines represent control mice, dash lines represent precoma mice and dots represent comatose mice.
Evidence for an encephalopathic role for octanoic acid has been increasing since the classic study of Samson et al. in 1956 describing the narcotic action of short and medium chain fatty acids from butyrate to caprate 20. Octanoic acid was shown to produce unconsciousness in the relatively short time of about 5 min in rats at a dose of 3.8 mmol/kg. Later studies showed that at a concentration of 5 - 1 0 mM, octanoic acid in vitro acts to uncouple oxidative phosphoryla-
135 tion, and associated with this is loss of respiratory control, and a decrease in oxygen consumption 5,29. In later studies it was shown that octanoic acid was capable of inhibiting rat brain Na, K, ATPase in vitro, and at a concentration of 15.4 mM, the inhibition amounted to 50 percent 1.25. Whether or not this is a direct cause-effect relation is uncertain; it has been shown that octanoic acid is capable of inhibiting a number of other enzyme systems in vitro, including hexokinase, phosphofructokinase, and glucose-6phosphate dehydrogenase, among others 2v. These early data on the effects of octanoic acid on energy related enzyme systems provided the rationale for subsequent in vivo experiments designed to examine effects of fatty acids on energy metabolism. In one study of the in vivo effect of the short chain fatty acids butyrate, valerate, and octanoate, energy metabolism was assessed in the cerebral cortex and brainstem in rats which were precomatose and comatose. Normal concentrations of both ATP and phosphocreatine were found in both brain regions studied in animals made encephalopathic with the aforementioned fatty acids 26. Furthermore, turnover of high energy phosphates was assessed in these animals using the Lowry 'closed system', and found to be unchanged as compared to suitable controls. In another study of the effect of octanoic acid on energy metabolism in rats, it was found that the adenylate charge was similar in encephalopathic animals as compared to controls 2. Brain mitochondrial oxidative phosphorylation in these animals was normal. A problem with all these previous studies was that they were performed on large pieces of brain. Neurophysiological studies have clearly shown a direct effect of fatty acids on the midbrain ascending reticular activating system 11,17 and, therefore, chemical studies using large samples would by necessity include contiguous non-affected tissue. Such non-affected tissue acts to dilute the sample rendering detection of more localized changes impossible. We have recently been investigating the effects of various encephalopathic agents on the energy metabolism of cells in the reticular formation. In these studies, we have found that both ammonia induced coma 15 and insulin induced hypoglycemiO4 coma are associated with statistically significant changes in energy metabolites, and these changes are selectively localized to the reticular formation. Cells in adjacent
non-reticular formation areas are not affected. These results have prompted the present study, in which the reticular formation in octanoic acid induced comatose mice was assessed in terms of its energy status. In the present study, microwave irradiation was used as an additional sacrifice technique in order to be certain that changes observed in mice sacrificed by submersion in liquid N 2 represent actual in vivo alterations, and were not enhanced by anoxia during the time necessary for the freezing front to reach the brainstem. This is an important consideration in any study in which labile metabolites are to be measured in brain structures deeper than the level of the cerebral cortex. It has been shown that as the freezing front advances in animals sacrificed by submersion in liquid N 2, anoxic changes may occur in labile metabolites 3,9. The values were higher in animals sacrificed by microwave irradiation because enzyme activity is more rapidly inactivated than by submersion in liquid nitrogen. In the present study, the inferior colliculus was chosen for comparison purposes because it represents an easily identifiable landmark. Results from this study using 7.3 kW of microwave irradiation confirmed the changes observed in the brains of mice made comatose with octanoic acid, and sacrificed by rapid submersion in liquid N 2. These changes consisted of a decrease in ATP and phosphocreatine selectively localized to the reticular formation in the coma stage. A decrease in ATP was observed in the reticular formation in the precoma stage as well. Glucose and glycogen were both elevated in the reticular formation in both the precoma and coma stages. The only statistically significant change observed in the inferior colliculus consisted of an increase in glycogen in the comatose animals. The changes localized to the cells of the reticular formation represent a highly selective effect, and furthermore, are present in the precoma animals before the actual onset of coma. Results from the turnover studies, in which the brain is converted to a closed system during which time theoretically only utilization occurs 7, show a similar response between inferior colliculus and reticular formation, which consists of a gradual decrease in the utilization rate of all metabolites studied. This is not unexpected since as the animal becomes first lethargic and stuporous, then comatose, energy demands might be expected to de-
136 crease throughout the brain, and in fact, throughout the animal. The results from this study are quite similar to those o b t a i n e d in earlier studies on the effects of ammonia induced coma 15, and insulin induced hypoglycemia coma 14. In each instance there was a selective effect on the high energy phosphates A T P a n d phosphocreatine in cells of the reticular formation in that they were decreased as c o m p a r e d to controls. The neurotoxic mechanism of each of these metabolic encephalopathies is thought to be different, and yet each has a c o m m o n biochemical change which is localized to cells of the reticular formation. The reason for the a p p a r e n t difference in response of reticular formation cells as c o m p a r e d to cells of the inferior colliculus is at present uncertain. It should be pointed out, however, that such biochemical h e t e r o g e n e i t y is not u n c o m m o n in the brain. The cerebellum for example has been shown to possess responses to metabolic p e r t u r b a t i o n which differs from that of o t h e r brain regions13,19. The results of this study show a selective effect of octanoic acid on energy metabolism in the reticular formation in the p r e c o m a as well as the c o m a stage. These changes are consistent with etectrophysiological studies which show a decrease in electrical output from the reticular formation during coma. These data therefore, represent a biochemical correlate to the neurophysiological changes in coma. It is interesting to speculate that these energy m e t a b o l i s m changes in the reticular formation, and the concomitant coma serve in a beneficial m a n n e r to the animal. This m a y represent a c o m p e n s a t o r y mechanism
REFERENCES 1 Dahl, D. R., Short chain fatty acid inhibition of rat brain Na, K, adenosine triphosphatase, J. Neurochem., 15 (1968) 815-820. 2 Derr, R. F. and Zieve, L., Decreased cerebral uptake of oxygen in coma - - A consequence of decreased utilization of ATP, J. Neurochem., 21 (1973) 1555-1557. 3 Ferrendelli, J. A., Gay, M. H., Sedgwick, W. G. and Chang, M. M., Quick freezing of the murine CNS: comparison of regional cooling rates and metabolite levels when using liquid N 2 or freon12, J. Neurochem., 19 (1972) 979-984. 4 Gabuzda, G. J., Hepatic coma: clinical considerations, pathogenesis, and management, Adv. Int. Med., 11 (1962) 11-73. 5 Hird, F. J. R. and Weiderman, M. J., Oxidative phosphory-
which acts to place the animal in a miliet~ of lowered energy demands, which, in the case of metabolic eno cephalopathy, provides an opportunity ~;c,r the organism to correct its threatened energy status. Two brief comments are pertinent t,; this discussion. First, the metabolite analyses in this study were p e r f o r m e d on samples weighing from 100-500 ng. Single neurons in the reticular formation weigh from 2 - 4 ng, and so clearly, these relatively large samples contain other cell types which may not respond the same to metabolic insult. Studies on the effects of octanoic acid induced coma on energy metabolism of single cells are in progress. Secondly, the present study has examined the effects of octanoic acid alone on cerebral energy metabolism. In patients with hepatic encephalopathy, not only short and m e d i u m chain fatty acids are elevated, but also ammonia, mercaptans, and phenols are elevated. Substantial data exist showing that there is a considerable synergistic effect of these cerebrotoxins ~0.33. It would be of interest to examine the effects of combinations of these c o m p o u n d s on energy metabolism in the reticular formation. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. William Stavinoha who allowed us to use his microwave oven for this study. The expert secretarial assistance of Ms. D i a n a P a r k e r is gratefully acknowledged. S u p p o r t e d in part by U . S . P . H . S . G r a n t N.S. 17130.
lation accompanying oxidation of short chain fatty acids by rat liver mitochondria, Biochem. J., 98 (1966) 378-388. 6 Hoyumpa, A. M., Desmond, P. V., Avant, G. R~, Roberts, R. K. and Schenker, S., Clinical Conference. Hepatic Encephalopathy, Gastroenterology, 76 (1979) 84-195. 7 Lowry, O. H., Passonneau, J. V., Hasselberger, F. X. and Schulz, D. W., Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. Biol. Chem., 239 (1964) 18-40. 8 Lowry, O. H. and Passonneau, J. V., A Flexible System of Enzymatic Analysis, Academic Press, N.Y., 1972. 9 Lust, W. D., Murakami, N., deAzeredo, F. and Passonneau, J. V., A comparison of methods forebrain fixation. In J. V. Passonneau, R. A. Hawkins, W. D. Lust and F. A. Welsh (Eds.), Cerebral Metabolism and Neural Function. Williams and Wilkins, Baltimore, 1980. 10 Manunes, P., DeVries, C. H., Miller, C. D. and David, R.
137 B., Fatty acid quantitation in Reye's Syndrome. In J. D. Pollack (Ed.), Reye's Syndrome, Grune and Stratton Inc., N.Y., 1975, pp. 245-254. 11 Matsuzaki, M. and Takagi, H., Para-sleep induction by sodium butyrate in acute brainstem preparations, Brain Research, 4 (1967) 223-240. 12 McCandless, D. W., Feussner, G. K., Lust, W. D. and Passonneau, J. V., Metabolite levels in brain following experimental seizures: the effects of electroshock and phenytoin in cerebellar layers, J. Neurochem., 32 (1979) 743-753. 13 McCandless, D. W., Feussner, G. K., Lust, W. D. and Passonneau, J. V., Sparing of metabolic stress in Purkinje cells after maximal electroshock, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 1482-1484. 14 McCandless, D. W., Insulin induced hypoglycemic coma and regional cerebral energy metabolism, Brain Research, 215 (1981) 225-233. 15 McCandless, D. W. and Schenker, S., Effects of acute ammonia intoxication on energy stores in the cerebral reticular activating systems, Exp. Brain Res., 44 (1981) 325-330. 16 Medina, M. A., Deam, A. P. and Stavinoha, W. B., Inactivation of brain tissue by microwave irradiation. In J. V. Passonneau, R. A. Hawkins, W. D. Lust and F. A. Welsh (Eds.), Cerebral Metabolism and Neural Function, Williams and Wilkins, Baltimore, 1980, pp. 56-69. 17 Muto, Y. I., Takahashi, Y. and Kawamura, H., Effects of short chain fatty acids on the electrical activity of the neopaleo- and archicortical system, Brain and Nerve (Tokyo), 16 (1964) 601-608. 18 Rabinowitz, J. L., Staeffen, J., Blanquet, P., Vincent, J. D., Terme, R., Series, C. and Myerson, R. M., Sources of serum [14C]octanoate in cirrhosis of the liver and hepatic encephalopathy, J. Lab. clin. Med., 91 (1978) 223-227. 19 Ratcheson, R. A., Blank, A. C. and Ferrendelli, J. A., Regionally selective metabolic effects of hypoglycemia in brain, J. Neurochem., 36 (1981) 1952-1958. 20 Samson, F. E., Dahl, N. and Dahl, D. R., A study on the narcotic study of the short chain fatty acids, J. clin. Invest., 35 (1956) 1291-1298. 21 Sanbar, S. S., Hetenyi, G., Forbath, N. and Evans, J. K., Effects of infusion of octanoate on glucose concentration in plasma and the rates of glucose production and utilization in
dogs, Metabolism, 14 (1965) 1311-1323. 22 Schenker, S., Henderson, G. I., Hoyumpa, A. M. and McCandless, D. W., Hepatic and Wernicke's encephalopathies: current concepts of pathogenesis, Amer. J. clin. Nutr., 33 (1980) 2719-2726. 23 Seigel, S., Non-Parametric Statistics for the Behavioral Sciences, McGraw-Hill, N.Y., 1956. 24 Trauner, D. A. and HutterLocher, P. R., Short chain fatty acid induced central hyperventillation in rabbits, Neurology, 28 (1978) 940-947. 25 Trauner, D. A., Regional cerebral Na, K, ATPase activity following octanoate administration, Pediat. Res., 14 (1980) 844. 26 Walker, C. O., McCandless, D. W., McGarry, J. D. and Schenker, S., Cerebral energy metabolism in short chain fatty acid induced coma, J. Lab. clin. Med., 76 (1970) 569-583. 27 Weber, G., Convery, H. J. H., Lea, M. A. and Stature, N. B., Feedback inhibition of key glycolytic enzymes in liver: action of free fatty acids, Science, 154 (1966) 1357-1360. 28 Windus-Podehl, G., Lyftogt, C., Zieve, L. and Brunner, G., Encephalopathic effect of phenol in rats, J. Lab. clin. Med., 101 (1983) 589-592. 29 Wojtczak, L. and Wojtczak, A. B., Uncoupling of oxidative phosphorylation and inhibition of ATP-Pi exchange by a substance from insect mitochondria, Biochim. Biophys. Acta, 39 (1960) 277-283. 30 Zeneroli, M. L., Ventura, E., Baraldi, M., Penne, A., Messori, E. and Zieve, L., Visual evoked potentials in encephalopathy induced by galactoseamine, ammonia, dimethyldisulfide and octanoic acid, Hepathology, 2 (1982) 532-538. 31 Zieve, L., Doizaki, W. M. and Zieve, F. J., Synergism between mercaptans and ammonia or fatty acids in the production of coma: a possible role for mercaptans in the pathogenesis of hepatic coma, J. Lab. clin. Med., 83 (1974) 16-28. 32 Zieve, L., Pathogenesis of hepatic coma, Arch. Int. Med., 118 (1966) 211-223. 33 Zieve, L., Encephalopathy due to short and medium chain fatty acids. In D. W. McCandless (Ed.), Cerebral Energy Metabolism and Metabolic Encephalopathy, Plenum, New York, 1985, pp. 163-177.
131
BRE 10759
Octanoic Acid-Induced Coma and Reticular Formation Energy Metabolism* DAVID W. McCANDLESS
Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, TX 77025 ( U.S. A.) (Accepted August 21st, 1984)
Key words: energy metabolism - - coma - - octanoic acid - - ATP - - reticular formation - - metabolic encephalopathy
The medium chain fatty acid octanoic acid was injected i.p. into 20-22 g Swiss-Albino mice at a dose of 15 ~mol/g. This dose produced a reproducible response consisting of a 3-4 min period of drowsiness, followed by coma. These mice as well as suitable controls were sacrificed by rapid submersion in liquid N2, or by microwave irradiation in a 7.3 kW microwave oven. Tissue from the reticular formation and the inferior colliculus was prepared for microanalysis of the energy metabolites glucose, glycogen, ATP and phosphocreatine. Results from this study showed a selective effect on energy metabolism in cells of the reticular formation. Both glucose and glycogen were elevated in the coma and precoma state. In addition, ATP and phosphocreatine were decreased in the reticular formation during coma. These results show a selective effect of octanoic acid on energy metabolism in the reticular formation both in the precoma stage, and during overt coma. The selective vulnerability of the reticular formation to metabolic insult may act in a beneficial manner to the animal by inducing coma. This lowers the overall demand for energy, thereby placing the animal in a milieu in which there is an increased chance for correction of the perturbation.
INTRODUCTION
octanoic acid on cerebral energetics, and have found no change in either the net levels of metabolites such
E n c e p h a l o p a t h y associated-with liver failure has been
studied
extensively,
and
the
concept
as A T P and phosphocreatine, or on their t u r n o v e r 26.
has
The adenylate energy charge has been r e p o r t e d to be
evolved that a 'toxin' or a combination of toxins is responsible for the neurological symptoms6,22, 32. A m -
normal in octanoic acid induced coma 2. R e c e n t stud-
monia 4, mercaptans 31, phenols 28 and short and medi-
reticular formation
um chain fatty acids 20 have all b e e n implicated in he-
have shown a selective effect 15. Since previous stud-
patic encephalopathy. The evidence for an encepha-
ies on octanoic acid have used large pieces of tissue~
lopathic role for the m e d i u m chain fatty acid octanoic
the present study was u n d e r t a k e n to examine the ef-
acid is somewhat circumstantial. Octanoic acid is ele-
fects of octanoic acid on energy metabolism in micro-
vated in the serum of patients with cirrhosis and he-
sections from the reticular formation, uncontami-
patic encephalopathy 18, and has also b e e n r e p o r t e d
nated by other perhaps less affected tissue, in this
ies on energy metabolism in discrete regions of the in metabolic encephalopathy
to be elevated in R e y e ' s SyndromelO, 24. In vitro, oc-
study we have used octanoic acid to induce coma in
tanoic acid has been shown to uncouple oxidative phosphorylationS, 29 and inhibit Na, K A T P a s e 1.25.
small mice, and have used both liquid N 2 and micro-
When octanoic acid is injected into rats, hypoglyce-
lites glucose, glycogen, A T P , and phosphocreatine
mia results 21, h o w e v e r the extent of the hypoglyce-
were measured in 100-500 ng samples from the reticular formation and the inferior colliculus, and the re-
mia is not severe enough in itself to cause c o m a 14. Previous investigators have e x a m i n e d the effect of
wave irradiation for sacrifice. The energy metabo-
suits are the basis for this report.
* Presented in part at the Eleventh International Symposium on Cerebral Blood Flow and Metabolism, Paris, 1983. Correspondence: D. W. McCandless, Department of Neurobiology and Anatomy, University of Texas Medical School at Houston. P.O. Box 20708, Houston, TX 77025, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division )
MATERIALS AND METHODS Female Swiss-Albino mice weighing 2 0 - 2 2 g were injected i.p. with octanoic acid at a dose of 15 ktmol/g. Control mice received an e q u i m o l a r dose of saline, or no injection. Mice were sacrificed at 3 rain following injection (precoma), or 1 min after the onset of coma. Animals were sacrificed by rapid submersion in liquid N 2. In order to eliminate the possibility of spurious results due to a delay in freezing of the deeply located reticular formation, a separate series of mice was sacrificed using a 7.3 k W microwave oven at 2450 M H z t6. Following sacrifice, the brains were r e m o v e d in a W e d e e n cryostat at - 2 0 °C, transferred to an I E C cryostat and sectioned into sections 20 u m thick. These sections at the level of the inferior coUiculus (an easily identifiable l a n d m a r k ) were transferred to aluminum holders and freeze-dried at - 4 0 °C for 24 h. Following this t r e a t m e n t , the sections can be brought to r o o m t e m p e r a t u r e u n d e r vacuum and manipulated without loss of labile metabolites. Small 100-500 ng samples from the inferior colliculus and reticular formation were freehand dissected and weighed on a quartz fiber fishpole balance (sensitivity -+ 2 rig). Samples were subsequently l o a d e d into oil well racks for m e t a b o l i t e analysis using enzymatic cyclingS.12. D a t a were analyzed using the non-parametric statistic, the M a n n - W h i t n e y U test for statistical significance 23. In another series of mice, turnover (utilization) was assessed by converting the brain to a closed system by decapitation 7. The head was m a i n t a i n e d at 37 °C for 15 s after which it was frozen in liquid N 2 and processed the same as intact animals.
GLUCOSE
.:~i RAG
~O.C
• tO T
80
>,
E ©
CONTROL
PRECOMA
COMA
Fig. 1. Effects of octanoic acid induced coma on regional cerebral glucose. Animals sacrificed by submersion in liquid N2. R.A.S., reticular activating system; I.C., inferior coUiculus. 3-5 animals per group (Figs. 1-7). Data expressed as mean + S.E. *, P < 0.00l.
acid injection on glucose in the inferior colliculus and the reticular formation is shown in Fig. 1. Glucose was elevated in the reticular formation only. The increase was present in both the p r e c o m a and coma stage. Fig. 2 shows the response of cerebral glycogen to octanoic acid. In this case there was a significant increase in glycogen in the inferior colliculus in coma
RESULTS The i.p. injection of octanoic acid at a dose of 15 ~mol/g results in a reproducible response characterized by a 3 - 4 rain p e r i o d of drowsiness which is followed by coma. Seizure activity was not o b s e r v e d in octanoic acid treated mice. Figs. 1 - 4 , and Figs. 8 and 9 represent the effects of octanoic acid on metabolites from mice sacrificed by submersion in liquid N:, whereas Figs. 5 - 7 show metabolites m e a s u r e d in brains from mice sacrificed by microwave irradiation. The effects of octanoic
"O
PRE~
Fig. 2. Effects of octanoic acid induced coma on regional cerebral glycogen. Animals sacrificed by submersion in liquid N2. Data expressed as mean _4-S.E. *, P < 0.001.
133 iii R.A.S.
•
ooo
• "- R.A.S.
ATP
elo
I.C.
•
10.0
GLYCOGEN
I.C.
16£
~72
:>,
°°°,,
,°,,, ,,°,, ,,°°,
05
'o
12.0
o)
E
°,°°° °°°°° °°°°°
O
°°,., °,°°, .°,°, °°,,, °°,,° .°°° .°.°
~ E c--
8.O
°,°° ,°°°° ,°°°° °°°°.
°°°,° ° . . ° .
, , ° . . °,,°° °,°,,
4.0
,.,°° , , , ° ° °,°°°
CONTROL PRECOMA COMA Fig. 3. Effects of octanoic acid induced coma on regional cerebral ATP. Animals sacrificed by submersion in liquid N 2. Data expressed as mean + S.E. *, P < 0.05; **, P < 0.002.
only whereas glycogen was elevated in the reticular formation in both the precoma and coma stages. Fig. 3 shows the effects of octanoic acid on ATP in the brain. Note that ATP is reduced in both the precoma and coma stage in cells of the reticular formation only. Interestingly, phosphocreatine was decreased only in the coma stage, and again, only in the reticular formation (Fig. 4). In order to validate the data obtained from mice
CONTROL
COMA
Fig. 5. Effects of octanoic acid induced coma on regional glycogen. Animals sacrificed by microwave irradiation. Data expressed as mean _+ S.E. *, P < 0.01.
frozen in liquid N2, another series of mice was treated with the same dose of octanoic acid, then sacrificed at 7.3 kW in a 2450 MHz microwave oven. Fig. 5 shows "•." " RA S o o l
.
•
•
I ,.c ATP
~ R.AS.
• [C.
Per
8.0
6.0
E O9
4£
O
E c-
2£
CONTROL
PRECOMA
COMA
Fig. 4. Effects of octanoic acid induced coma on regional cerebral phosphocreatine. Animals sacrificed by submersion in liquid N 2. Data expressed as mean _+ S.E. *, P < 0.02.
CONTROL
COMA
Fig. 6. Effects of octanoic acid induced coma on regional cerebral ATP. Animals sacrificed by microwave irradiation. Data expressed as mean + S.E. *, P < 0.05.
!34 oeo
• *" eee
RA.S
"2o
I I tc
,,
oF.-L_-7_
6O
POt
16.0
IC
ATP /
-
.
....... .-.,,
-
' O,
120~
ATP
RAS
PCr
;:~A
>,
o~ E
15
CO
C
as
"
Fig. 9. Effects of octanoic acid induced coma on regional turnover rates of ATP and phosphocreatine. C. control mice frozen intact in liquid N2; 15, decapitated heads maintained at 15 s at 37 °C, then frozen in liquid N 2. Solid lines represent control mice, dash lines represent precoma mice and dots represent comatose mice.
o E t"
CONTROL
ic decrease in phosphocreatine localized to the reticular formation in the coma stage.
COMA
Fig. 7. Effects of octanoic acid induced coma on regional cerebral phosphocreatine. Animals sacrificed by microwave irradiation. Data expressed as mean _+S.E. *, P < 0.05.
the effects of octanoic acid on glycogen, and as in the case of mice sacrificed in liquid N2, glycogen was elevated in the reticular formation only. Fig. 6 shows a selective decrease in A T P in the reticular formation in the coma stage, and Fig. 7 shows a similar dramat-
Fig. 8 shows the utilization rate at 15 s for glycogen and glucose in control, precoma, and comatose mice. Glycogen utilization rates are similar in both regions, whereas glucose utilization rates in coma induced by octanoic acid are increased in both the inferior colliculus and the reticular formation. Fig. 9 shows that in the case of A T P , the utilization rate over 15 s is lower in the coma stage than in controls or the precoma stage, and phosphocreatine showed a similar response in that the rate of utilization in comatose mice was lower than that in controls or precomatose mice.
12C
It must be r e m e m b e r e d that these data show trends
GLYCOGEN-LC
only; since they compare 15 s decapitation levels to '0' second non-decapitation metabolite levels, they
To 8
6[ 4(
are derived data, and as such are not a m e n a b l e to statistical analysis.
..... ~.~:..LLLLLL..~C..:.:
2(
~12c
GLYCOGEN-R.A.S
GLUCOSE-RA
S
DISCUSSION
~1oc
"~~tL........... ..;.:.:.~.:~ Fig. 8. Effects of octanoic acid induced coma on regional turnover rates of glucose and glycogen. C, control mice frozen intact in liquid N2; 15, decapitated heads maintained at 15 s at 37 °C, then frozen in liquid N2. Solid lines represent control mice, dash lines represent precoma mice and dots represent comatose mice.
Evidence for an encephalopathic role for octanoic acid has been increasing since the classic study of Samson et al. in 1956 describing the narcotic action of short and medium chain fatty acids from butyrate to caprate 20. Octanoic acid was shown to produce unconsciousness in the relatively short time of about 5 min in rats at a dose of 3.8 mmol/kg. Later studies showed that at a concentration of 5 - 1 0 mM, octanoic acid in vitro acts to uncouple oxidative phosphoryla-
135 tion, and associated with this is loss of respiratory control, and a decrease in oxygen consumption 5,29. In later studies it was shown that octanoic acid was capable of inhibiting rat brain Na, K, ATPase in vitro, and at a concentration of 15.4 mM, the inhibition amounted to 50 percent 1.25. Whether or not this is a direct cause-effect relation is uncertain; it has been shown that octanoic acid is capable of inhibiting a number of other enzyme systems in vitro, including hexokinase, phosphofructokinase, and glucose-6phosphate dehydrogenase, among others 2v. These early data on the effects of octanoic acid on energy related enzyme systems provided the rationale for subsequent in vivo experiments designed to examine effects of fatty acids on energy metabolism. In one study of the in vivo effect of the short chain fatty acids butyrate, valerate, and octanoate, energy metabolism was assessed in the cerebral cortex and brainstem in rats which were precomatose and comatose. Normal concentrations of both ATP and phosphocreatine were found in both brain regions studied in animals made encephalopathic with the aforementioned fatty acids 26. Furthermore, turnover of high energy phosphates was assessed in these animals using the Lowry 'closed system', and found to be unchanged as compared to suitable controls. In another study of the effect of octanoic acid on energy metabolism in rats, it was found that the adenylate charge was similar in encephalopathic animals as compared to controls 2. Brain mitochondrial oxidative phosphorylation in these animals was normal. A problem with all these previous studies was that they were performed on large pieces of brain. Neurophysiological studies have clearly shown a direct effect of fatty acids on the midbrain ascending reticular activating system 11,17 and, therefore, chemical studies using large samples would by necessity include contiguous non-affected tissue. Such non-affected tissue acts to dilute the sample rendering detection of more localized changes impossible. We have recently been investigating the effects of various encephalopathic agents on the energy metabolism of cells in the reticular formation. In these studies, we have found that both ammonia induced coma 15 and insulin induced hypoglycemiO4 coma are associated with statistically significant changes in energy metabolites, and these changes are selectively localized to the reticular formation. Cells in adjacent
non-reticular formation areas are not affected. These results have prompted the present study, in which the reticular formation in octanoic acid induced comatose mice was assessed in terms of its energy status. In the present study, microwave irradiation was used as an additional sacrifice technique in order to be certain that changes observed in mice sacrificed by submersion in liquid N 2 represent actual in vivo alterations, and were not enhanced by anoxia during the time necessary for the freezing front to reach the brainstem. This is an important consideration in any study in which labile metabolites are to be measured in brain structures deeper than the level of the cerebral cortex. It has been shown that as the freezing front advances in animals sacrificed by submersion in liquid N 2, anoxic changes may occur in labile metabolites 3,9. The values were higher in animals sacrificed by microwave irradiation because enzyme activity is more rapidly inactivated than by submersion in liquid nitrogen. In the present study, the inferior colliculus was chosen for comparison purposes because it represents an easily identifiable landmark. Results from this study using 7.3 kW of microwave irradiation confirmed the changes observed in the brains of mice made comatose with octanoic acid, and sacrificed by rapid submersion in liquid N 2. These changes consisted of a decrease in ATP and phosphocreatine selectively localized to the reticular formation in the coma stage. A decrease in ATP was observed in the reticular formation in the precoma stage as well. Glucose and glycogen were both elevated in the reticular formation in both the precoma and coma stages. The only statistically significant change observed in the inferior colliculus consisted of an increase in glycogen in the comatose animals. The changes localized to the cells of the reticular formation represent a highly selective effect, and furthermore, are present in the precoma animals before the actual onset of coma. Results from the turnover studies, in which the brain is converted to a closed system during which time theoretically only utilization occurs 7, show a similar response between inferior colliculus and reticular formation, which consists of a gradual decrease in the utilization rate of all metabolites studied. This is not unexpected since as the animal becomes first lethargic and stuporous, then comatose, energy demands might be expected to de-
136 crease throughout the brain, and in fact, throughout the animal. The results from this study are quite similar to those o b t a i n e d in earlier studies on the effects of ammonia induced coma 15, and insulin induced hypoglycemia coma 14. In each instance there was a selective effect on the high energy phosphates A T P a n d phosphocreatine in cells of the reticular formation in that they were decreased as c o m p a r e d to controls. The neurotoxic mechanism of each of these metabolic encephalopathies is thought to be different, and yet each has a c o m m o n biochemical change which is localized to cells of the reticular formation. The reason for the a p p a r e n t difference in response of reticular formation cells as c o m p a r e d to cells of the inferior colliculus is at present uncertain. It should be pointed out, however, that such biochemical h e t e r o g e n e i t y is not u n c o m m o n in the brain. The cerebellum for example has been shown to possess responses to metabolic p e r t u r b a t i o n which differs from that of o t h e r brain regions13,19. The results of this study show a selective effect of octanoic acid on energy metabolism in the reticular formation in the p r e c o m a as well as the c o m a stage. These changes are consistent with etectrophysiological studies which show a decrease in electrical output from the reticular formation during coma. These data therefore, represent a biochemical correlate to the neurophysiological changes in coma. It is interesting to speculate that these energy m e t a b o l i s m changes in the reticular formation, and the concomitant coma serve in a beneficial m a n n e r to the animal. This m a y represent a c o m p e n s a t o r y mechanism
REFERENCES 1 Dahl, D. R., Short chain fatty acid inhibition of rat brain Na, K, adenosine triphosphatase, J. Neurochem., 15 (1968) 815-820. 2 Derr, R. F. and Zieve, L., Decreased cerebral uptake of oxygen in coma - - A consequence of decreased utilization of ATP, J. Neurochem., 21 (1973) 1555-1557. 3 Ferrendelli, J. A., Gay, M. H., Sedgwick, W. G. and Chang, M. M., Quick freezing of the murine CNS: comparison of regional cooling rates and metabolite levels when using liquid N 2 or freon12, J. Neurochem., 19 (1972) 979-984. 4 Gabuzda, G. J., Hepatic coma: clinical considerations, pathogenesis, and management, Adv. Int. Med., 11 (1962) 11-73. 5 Hird, F. J. R. and Weiderman, M. J., Oxidative phosphory-
which acts to place the animal in a miliet~ of lowered energy demands, which, in the case of metabolic eno cephalopathy, provides an opportunity ~;c,r the organism to correct its threatened energy status. Two brief comments are pertinent t,; this discussion. First, the metabolite analyses in this study were p e r f o r m e d on samples weighing from 100-500 ng. Single neurons in the reticular formation weigh from 2 - 4 ng, and so clearly, these relatively large samples contain other cell types which may not respond the same to metabolic insult. Studies on the effects of octanoic acid induced coma on energy metabolism of single cells are in progress. Secondly, the present study has examined the effects of octanoic acid alone on cerebral energy metabolism. In patients with hepatic encephalopathy, not only short and m e d i u m chain fatty acids are elevated, but also ammonia, mercaptans, and phenols are elevated. Substantial data exist showing that there is a considerable synergistic effect of these cerebrotoxins ~0.33. It would be of interest to examine the effects of combinations of these c o m p o u n d s on energy metabolism in the reticular formation. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. William Stavinoha who allowed us to use his microwave oven for this study. The expert secretarial assistance of Ms. D i a n a P a r k e r is gratefully acknowledged. S u p p o r t e d in part by U . S . P . H . S . G r a n t N.S. 17130.
lation accompanying oxidation of short chain fatty acids by rat liver mitochondria, Biochem. J., 98 (1966) 378-388. 6 Hoyumpa, A. M., Desmond, P. V., Avant, G. R~, Roberts, R. K. and Schenker, S., Clinical Conference. Hepatic Encephalopathy, Gastroenterology, 76 (1979) 84-195. 7 Lowry, O. H., Passonneau, J. V., Hasselberger, F. X. and Schulz, D. W., Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. Biol. Chem., 239 (1964) 18-40. 8 Lowry, O. H. and Passonneau, J. V., A Flexible System of Enzymatic Analysis, Academic Press, N.Y., 1972. 9 Lust, W. D., Murakami, N., deAzeredo, F. and Passonneau, J. V., A comparison of methods forebrain fixation. In J. V. Passonneau, R. A. Hawkins, W. D. Lust and F. A. Welsh (Eds.), Cerebral Metabolism and Neural Function. Williams and Wilkins, Baltimore, 1980. 10 Manunes, P., DeVries, C. H., Miller, C. D. and David, R.
137 B., Fatty acid quantitation in Reye's Syndrome. In J. D. Pollack (Ed.), Reye's Syndrome, Grune and Stratton Inc., N.Y., 1975, pp. 245-254. 11 Matsuzaki, M. and Takagi, H., Para-sleep induction by sodium butyrate in acute brainstem preparations, Brain Research, 4 (1967) 223-240. 12 McCandless, D. W., Feussner, G. K., Lust, W. D. and Passonneau, J. V., Metabolite levels in brain following experimental seizures: the effects of electroshock and phenytoin in cerebellar layers, J. Neurochem., 32 (1979) 743-753. 13 McCandless, D. W., Feussner, G. K., Lust, W. D. and Passonneau, J. V., Sparing of metabolic stress in Purkinje cells after maximal electroshock, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 1482-1484. 14 McCandless, D. W., Insulin induced hypoglycemic coma and regional cerebral energy metabolism, Brain Research, 215 (1981) 225-233. 15 McCandless, D. W. and Schenker, S., Effects of acute ammonia intoxication on energy stores in the cerebral reticular activating systems, Exp. Brain Res., 44 (1981) 325-330. 16 Medina, M. A., Deam, A. P. and Stavinoha, W. B., Inactivation of brain tissue by microwave irradiation. In J. V. Passonneau, R. A. Hawkins, W. D. Lust and F. A. Welsh (Eds.), Cerebral Metabolism and Neural Function, Williams and Wilkins, Baltimore, 1980, pp. 56-69. 17 Muto, Y. I., Takahashi, Y. and Kawamura, H., Effects of short chain fatty acids on the electrical activity of the neopaleo- and archicortical system, Brain and Nerve (Tokyo), 16 (1964) 601-608. 18 Rabinowitz, J. L., Staeffen, J., Blanquet, P., Vincent, J. D., Terme, R., Series, C. and Myerson, R. M., Sources of serum [14C]octanoate in cirrhosis of the liver and hepatic encephalopathy, J. Lab. clin. Med., 91 (1978) 223-227. 19 Ratcheson, R. A., Blank, A. C. and Ferrendelli, J. A., Regionally selective metabolic effects of hypoglycemia in brain, J. Neurochem., 36 (1981) 1952-1958. 20 Samson, F. E., Dahl, N. and Dahl, D. R., A study on the narcotic study of the short chain fatty acids, J. clin. Invest., 35 (1956) 1291-1298. 21 Sanbar, S. S., Hetenyi, G., Forbath, N. and Evans, J. K., Effects of infusion of octanoate on glucose concentration in plasma and the rates of glucose production and utilization in
dogs, Metabolism, 14 (1965) 1311-1323. 22 Schenker, S., Henderson, G. I., Hoyumpa, A. M. and McCandless, D. W., Hepatic and Wernicke's encephalopathies: current concepts of pathogenesis, Amer. J. clin. Nutr., 33 (1980) 2719-2726. 23 Seigel, S., Non-Parametric Statistics for the Behavioral Sciences, McGraw-Hill, N.Y., 1956. 24 Trauner, D. A. and HutterLocher, P. R., Short chain fatty acid induced central hyperventillation in rabbits, Neurology, 28 (1978) 940-947. 25 Trauner, D. A., Regional cerebral Na, K, ATPase activity following octanoate administration, Pediat. Res., 14 (1980) 844. 26 Walker, C. O., McCandless, D. W., McGarry, J. D. and Schenker, S., Cerebral energy metabolism in short chain fatty acid induced coma, J. Lab. clin. Med., 76 (1970) 569-583. 27 Weber, G., Convery, H. J. H., Lea, M. A. and Stature, N. B., Feedback inhibition of key glycolytic enzymes in liver: action of free fatty acids, Science, 154 (1966) 1357-1360. 28 Windus-Podehl, G., Lyftogt, C., Zieve, L. and Brunner, G., Encephalopathic effect of phenol in rats, J. Lab. clin. Med., 101 (1983) 589-592. 29 Wojtczak, L. and Wojtczak, A. B., Uncoupling of oxidative phosphorylation and inhibition of ATP-Pi exchange by a substance from insect mitochondria, Biochim. Biophys. Acta, 39 (1960) 277-283. 30 Zeneroli, M. L., Ventura, E., Baraldi, M., Penne, A., Messori, E. and Zieve, L., Visual evoked potentials in encephalopathy induced by galactoseamine, ammonia, dimethyldisulfide and octanoic acid, Hepathology, 2 (1982) 532-538. 31 Zieve, L., Doizaki, W. M. and Zieve, F. J., Synergism between mercaptans and ammonia or fatty acids in the production of coma: a possible role for mercaptans in the pathogenesis of hepatic coma, J. Lab. clin. Med., 83 (1974) 16-28. 32 Zieve, L., Pathogenesis of hepatic coma, Arch. Int. Med., 118 (1966) 211-223. 33 Zieve, L., Encephalopathy due to short and medium chain fatty acids. In D. W. McCandless (Ed.), Cerebral Energy Metabolism and Metabolic Encephalopathy, Plenum, New York, 1985, pp. 163-177.