Gut, 1991,32, 1261-1263
1261
Gut Leading article Neurochemistry of hepatic encephalopathy Since the first biochemical descriptions of hepatic encephalopathy several unifying aetiological hypotheses have been developed that could account for the main clinical and biochemical features of the condition. Initially the concept of portosystemic encephalopathy was developed. This postulated the origin of a nitrogenous product in the gut that reached the cerebral circulation because of defective hepatic clearance by the diseased liver. Ammonia was the prime candidate neurotoxin and several studies were undertaken to determine the value of amino acid infusions, particularly glutamic acid and arginine, to stimulate urea cycle activity and hepatic urea synthesis.' Catharsis, gut sterilisation, and later lactulose, designed to diminish ammonia production by the colon, were the therapeutic consequences that were widely promoted and used. The mercaptan hypothesis followed, later modified to include synergism between ammonia short chain fatty acids and mercaptans,2 but subsequent studies3'4 using elegant trapping techniques' were negative. The next phase was characterised by the false neurotransmitter hypothesis.6 High plasma concentrations of aromatic amino acids were recognised in liver failure and thought to be associated with the synthesis of derivatives such as octopamine which interfered with neurotransmission. Because of the competition between aromatic and branch chain amino acids for transport into the brain, amino acid infusions rich in branch chain varieties were introduced,7 designed to impede cerebral uptake of precursor amino acids, tyrosine and phenylalanine, and thus decrease false transmitter formation in the brain. The concept of modulation of neurotransmission was extended by the finding of arousal after the administration of the dopamine agonist L dopa8 and various studies showed decreased brain catecholamine concentrations in animal models of encephalopathy, although these were not subsequently confirmed in man.910 The leucine degradation product, isovaleraldehyde, was increased in hepatic encephalopathy but no important role could be attributed to this product in man. " 5 Hydroxytryptamine (5HT) acts as a central neurotransmitter, so excess 5HT activity could account for the neuroinhibition of hepatic encephalopathy. Increased concentrations of the degradation product 5 hydroxy-indoleacetic acid (5HIAA) has been a consistent finding in the brain of animal models'2 and in the cerebrospinal fluid'3 and frontal cortex'4 of patients with fulminant hepatic failure, although in the latter brain 5HT concentrations were normal.'4 The findings were similar in prefrontal cortex and caudate nucleus of cirrhotic patients dying of hepatic coma.'° In the portacaval shunted rat, however, when compared with controls, there was increased 5HT turnover as shown by the 5HIAA:5HT
ratio but no further alteration in the ratio was observed when deep coma was precipitated by ammonia administration.'5 Thus, the changes in 5HT metabolism are probably the result of increased tryptophan availability in the brain'416 owing to the high plasma tryptophan concentrations observed in acute'4 and chronic liver disease7 rather than representing a primary abnormality. The gamma amino butyric acid hypothesis When the gamma amino butyric acid (GABA) hypothesis was first formulated it was suggested that excess activity of this neuroinhibitory transmitter in the brain caused the development of somnolence. GABA was thought to arise from bacterial action in the gut and reach the brain because of defective hepatic clearance and increased permeability of the blood brain barrier.'" Increased concentrations of GABA in the blood of patients with hepatic encephalopathy were described, perhaps associated with abnormalities of brain GABA receptors, which were increased in a rabbit model of acute encephalopathy induced by galactosamine. However, it was not possible to show penetration of the brain after intravenous administration of GABA.'8 More recent studies have found that brain GABA receptors, particularly in man, are normal in number and affinity'9 20 while in the portacaval shunted rat, quantitative autoradiographic studies of GABAA receptors are also normal.2' Furthermore brain GABA concentrations at necropsy in cirrhotic patients with encephalopathy were not significantly different from control values.22 Although brain GABA rises within one hour of death,23 when samples from patients dying of acute liver failure were collected within this period concentrations were still identical to those found in control subjects.'4 More recently, cerebrospinal fluid GABA values in man24 and in encephalopathic dogs with congenital portacaval shunts were normal,25 while brain GABA concentrations in dogs'8 and the thioacetamide rat model were normal or significantly decreased rather than increased, as would have been expected if GABAergic transmission were enhanced.26 The hypothesis has now been modified to include modulation of GABA transmission by a
benzodiazepine agonist. The benzodiazepine hypothesis27 Interest in the role of benzodiazepines in hepatic encephalopathy first developed because of the close association of the benzodiazepine and GABA receptors in the brain and the finding that binding sites were increased in galactosamine induced acute liver failure.28 29 Much greater interest followed early reports of a striking arousal effect ofthe benzodiazepine
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antagonist flumazenil in hepatic encephalopathy in man.30 Subsequent reports showed beneficial results in animal models,2931 while cerebellar Purkinje neurones from the rabbit galactosamine model were hypersensitive to GABA and benzodiazepine receptor agonists.32 On the other hand, alterations in GABA benzodiazepine receptors were not found in the brains of rats with chronic encephalopathy as a result of portal vein and bile duct ligation33 or in cirrhotic patients dying of encephalopathy.'9 Furthermore, quantitative autoradiography has shown that benzodiazepine receptors in the galactosamine rabbit model are within normal limits.34 The benzodiazepine hypothesis of hepatic encephalopathy is only tenable if the presence and source of endogenous benzodiazepine like substances can be shown. The presence of such ligands has been reported in the cerebrospinal fluid35 and brain extracts36 of animal models and also in the cerebrospinal fluid37 38 and plasma38 of patients with hepatic encephalopathy. The difficulty with these studies in man is the reliable exclusion of benzodiazepine drugs, which are readily available and have a prolonged half life. Naturally occurring benzodiazepines may be of dietary origin.35 38 The most compelling evidence for the benzodiazepine hypothesis comes from the use of the specific antagonist flumazenil both in man and experimental animals. Unfortunately, the early enthusiastic reports for the drug have not been sustained. In the thioacetamide model of acute liver failure flumazenil may reverse the deficits in spontaneous motor function and visually evoked response abnormalities but the motor deficit usually persists after drug administration.39 No effect could be shown in the rat hepatic ischaemia model (portacaval shunt plus hepatic artery ligation) despite administration of a dose sufficient to reverse benzodiazepine induced coma.' In man, the results are even more controversial - some authors claiming beneficial results4' in 60% of patients42 while others,43 including a placebo controlled trial, found only a 27% response rate." The ammonia hypothesis revisited The modern theories for the pathogenesis of hepatic encephalopathy do not account for the traditional and well documented role of the gut in the development of the condition. The precipitation of encephalopathy by protein ingestion in the form of variceal bleeding is familiar to all in clinical practice and any neurochemical hypothesis must take account of such clinical observations, including the efficacy of gut sterilisation and lactulose. Any mechanism encompassing the role of the gut must also be explicable in terms of current concepts of dissordered neurotransmission. For obvious physiological reasons any neurotransmitter substance arising from the gut will not cross the blood brain barrier, and while blood brain barrier permeability may be disturbed in the latter stages of encephalopathy there is little evidence to support any change during its initial development.58 45-I8 Ammonia arising from the gut is, however, freely permeable to the brain, while cerebrospinal fluid values49 and arterial concentrations30 bear a reasonable correlation with coma grade. Ammonia taken up by the brain is incorporated into glutamine which may deplete brain glutamate. Glutamate is the principal excitatory transmitter in the brain and a disturbance in glutamatergic transmission could account for the neuroinhibition of hepatic encephalopathy. Early studies of human brain amino acid concentrations in hepatic encephalopathy showed that glutamine concentrations were increased two to threefold in the frontal cortex of patients dying from fulminant hepatic failure.'4 Increased concentrations were then found in cirrhotic patients22 while cerebrospinal fluid values showed a close correlation with coma grade.495' Brain glutamate concentrations in patients with cirrhosis and encephalopathy were decreased in several
areas22 but were normal in fulminant hepatic failure in unventilated patients or increased in those dying after a prolonged period of ventilation. 14 The latter finding probably reflects brain death and equilibration with the high concentrations of glutamate found in the blood of the patients studied. Glutamate is thought to be present in the brain in several distinct pools including metabolic, GABA precurser, astrocytic, and finally a releasable pool available for glutamatergic transmission. Depletion of the latter alone may be sufficient to depress excitatory neurotransmission. Several studies have attempted to evaluate the concentration of glutamate in vivo in animal models of hepatic encephalopathy. The results using nuclear magnetic resonance52 have suggested decreased concentrations, while studies with brain dialysis have shown normal53 or increased glutamate output in the perfusate.5 156 The latter observation could be the result of decreased uptake of glutamate from the synaptic cleft into astrocytes, and indeed no further glutamate could be released into the brain perfusate in acute hepatic failure when potassium was included in the perfusion medium54 suggesting an absolute decrease in transmitter pool. In the rat thioacetamide model brain glutamate is significantly decreased in frontal cortex and striatum when plasma concentrations are increased more than fourfold.26 An ammonia induced decrease in cerebral glutamate has been described by Hindfelt.5 Interest in the glutamate receptor has increased since the finding that the anaesthetic agent ketamine leads to depression of the reticuloendothelial system by changing glutamatergic transmission.58 Glutamate receptors can be devided into Ca++/Cl- dependent (presynaptic) and independent (postsynaptic) classes and can be recognised by their ability to bind n methyl D aspartate (NMDA) and quisquolate/ kainate (nonNMDA). In chronically encephalopathic dogs with congenital portacaval shunts, NMDA binding sites in the brain were normal or increased while kainate binding was decreased.59 In the thioacetamide rat model Ca++/Cl[ dependent and independent glutamate binding was unchanged but this may have been because of the short duration of encephalopathy in this model (about seven hours.)26 In portacaval shunted rats (3H) glutamate binding was increased consistent with decreased values of synaptic glutamate and up regulation of the postsynaptic receptor.60 Glutamate receptor binding in hepatic encephalopathy in man has not yet been determined. More direct evidence for the role of disturbed glutamatergie transmission comes from studies of the effect of ammonia on evoked glutamate release and excitatory post synaptic potentials. Several investigations have shown diminished responses.6' The attraction of the glutamate depletion hypothesis is that it accounts for many of the original observations concerning the role of ammonia and reestablishes the role of the gastrointestinal tract. Much work remains to be done but ammonia is currently returning to the forefront of hepatic neurochemistry. C O RECORD
Gastroenterology Unit, Royal Victoria Infirmary and University of Newcastle upon Tyne, Newcastle NEI 4LP 1 McDermott WV. Metabolism and toxicity of ammonia. N Engl J Med 1957; 257: 1076-81. 2 Zieve L, Doizaki WM, Zieve FJ. 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 1974- 83: 16-27. 3 Al Mardini H, Bartlett K, Record GO. Blood and brain concentrations of mercaptans in hepatic and methanethiol induced coma. Gut 1984; 25: 284-90. 4 Al Mardini H, Leonard J, Bartlett K, Lloyd S, Record CO. Effect of methionine loading and endogenous hypermethionin-aemia on blood mercaptans in man. Glin Chim Acta 1988; 176: 83-90. 5 Blom HJ, Chamuleau RAFM, Rothuizen J, Deutz NEP, Tangerman A. Methanethiol metabolism and its role in the pathogenesis of hepatic encephalopathy in rats and dogs. Hepatology 1990; 11: 682-9. 6 Fisher JE, Baldessarini RJ. Pathogenesis and therapy of hepatic coma. In: Popper H, Schaffner F, eds. Progress in liver disease. Vol V. New York: Grune and Stratton, 1976: 363-97.
Neurochemistry ofhepatic encephalopathy 7 Erickson LS. Branched chain amino acids in the management of hepatic encephalopathy. In: Conn HO, Bircher MD, eds. Hepatic encephalopathy. E Lancing. Medi-Ed Press, 1988: 129-44. 8 Parks JD, Sharpstone P, Williams R. Levodopa in hepatic coma. Lancet 1970; ii: 1341-3. 9 Cuilleret G, Pomier-Layrargues G, Pons F, Cadilhac J, Michel H. Changes in brain catecholamine levels in human cirrhotic hepatic encephalopathy. Gut 1980; 21: 565-9. 10 Bergeron M, Reader TA, Pomier Layrargues G, Butterworth RF. Monoamines and metabolites in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Neurochem Res 1989; 14: 853-9. 11 Al Mardini H, O'Brien CJ, Bartlett K, Wiliams R, Record CO. Thermal desorption-gas chromatography of plasma isovaler-aldehyde in hepatic encephalopathy in man. Clin Chim Acta 1987; 165: 61-7 1. 12 Yurdaydin C, Hortnagl H, Steinol P et al. Increased serotinergic and noradnergic activity in hepatic encephalopathy in rats with thioacetamideinduced acute liver failure. Hepatology 1990; 12: 695-700. 13 Knell AJ, Davidson AR, Williams R, Kantamaneni BD, Curzon G. Dopamine and serotoxin metabolism in hepatic encephalopathy. BMJ 1974; i: 549-51. 14 Record CO, Buxton B, Chase RA, Curzon G, Murray Lyon I, Williams R, Plasma and brain amino acids in fulminant hepatic failure and their relationship to hepatic encephalopathy. EurJ Clin Invest 1976; 6: 387-94. 15 Bergeron M, Swain MS, Reader TA, Grondin L, Butterworth RF. Effect of ammonia on brain serotonin metabolism in relation to function in the portacaval shunted rat. J Neurochem 1990; 55: 222-9. 16 Mans AM, Saunders SJ, Kirsch RE, Biebuyck JF. Correlation of plasma and brain amino acid and putative neurotransmitter alterations during acute hepatic coma in the rat. J Neurochem 1979; 32: 285-92. 17 Schafer DF, Jones EA. Hepatic encephalopathy and the y-aminobutyric acid neurotransmitter system. Lancet 1982; ii: 18-20. 18 Roy S, Pomier-Layrargues G, Butterworth RF, Michel Huet P. Hepatic encephalopathy in cirrhotic and portacaval shunted dogs: lack of changes in brain GABA uptake, brain GABA levels, brain glutamic acid decarboxylase activity and brain postsynaptic GABA receptors. Hepatology 1988; 8: 845-9. 19 Butterworth RF, Lavoie J, Giguere JF, Pomier-Layrargues G. Affinities and densities of high affinity ('H) Muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Hepatology 1988; 8: 1084-8. 20 Al Mardini H, Bartlett K, Wiliams R, Record CO. Plasma levels and brain receptors of gamma amino butyric acid in human hepatic encephalopathy. [Abstract]. Gut 1988; 29: A1430. 21 Mans A, Kukulka K, McAvoy K, Hawkins R. Quantitative autoradiography of binding to the GABAA receptor after portacaval shunting. J Hepatol 1990; 10: S15. 22 Lavoie Joel, Giguere JF, Pomier-Layrargues G, Butterworth RF. Amino acid changes in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. J Neurochem 1987; 49: 692-7. 23 Perry TL, Hansen S, Gandham SS. Postmortem changes of amino compounds in human and rat brainj Neurochem 1981; 36: 406-12. 24 Moroni F, Raiggio 0, Carla V, et al. Hepatic encephalopathy: lack of changes of -aminobutyric acid content in plasma and cerebrospinal fluid. Hepatology 1987; 7:816-20. 25 MaddisonjE, Yau D, Stewart P, Farrell GC. Cerebrospinalfluidy-aminobutyric acid levels in dogs with chronic portosystemic encephalopathy. Clin Sci 1986; 71: 749-53. 26 Zimmermann C, Ferenci P, Pifl C, et al. Hepatic encephalopathy in thioacetamide-induced acute liver failure in rats: characterisation of an improved model and study of amino acid-ergic neurotransmission. Hepatology 1989; 9: 594-601. 27 Mullen KD, Martin JV, Mendelson WB, Bassett ML, Jones EA. Could an endogenous benzodiazepine ligand contribute to hepatic encephalopathy. Lancet 1988; i: 457-9. 28 Schafer DF, Fowler JM, Munson PJ, Thakur AK, Waggoner JG, Jones EA. Gamma-aminobutyric acid and benzodiazepine receptors in an animal model of fulminant hepatic failure.J Lab Clin Med 1983; 102: 870-80. 29 Baraldi M, Zeneroli ML, Ventura E, et al. Supersensitivity of benzodiazepine receptors in hepatic encephalopathy due to fulminant hepatic failure in the rat: reversal by a benzodiazepine antagonist. Clin Sci 1984; 67: 167-75. 30 Scollo-Levizzaire G, Steinmann E. Reversal of hepatic coma by benzodiazepine antagonist (RO 15-1788). Lancet 1985; i: 1324. 31 Bassett ML, Mullen KD, Skolnick P, Jones EA. Amelioration of hepatic encephalopathy by pharmacologic antagonism of the GABAP>A-benzodiazepine receptor complex in a rabbit model of fulminant hepatic failure. Gastroenterology 1987; 93: 1069-77. 32 Basile AS, Gammal SH, Mullen KD, Jones EA, Skolnick P. Differential responsiveness or cerebellar Purkinje neurons to GABA and benzodiazepine receptor ligands in an animal model of hepatic encephalopathy. J Neurosci 1988; 8: 2414-21. 33 Maddison JE, Dodd PR, Morrison M, Johnston GAR, Farrell GC. Plasma GABA, GABA-like activity and the brain GABA-benzodiazepine receptor
1263 complex in rats with chronic hepatic encephalopathy. Hepatology 1987; 7: 621-8. 34 Rossle M, Deckert J, Jones EA. Autoradiographic analysis of GABAbenzodiazepine receptors in an animal model of acute hepatic encephalopathy. Hepatology 1989; 10: 143-7. 35 Mullen KD, Szauter KM, Kaminsky K, Tolentino PD, McCullough AJ. Detection and characterisation of endogenous benzodiazepine activity in both animal models and humans with hepatic encephalopathy. In: Butterworth RF, Pomier Langrarques G, eds. Hepatic encephalopathy: pathophysiology and treatment. Clifton NJ: Humana Press, 1989: 287-94. 36 Basile AS, Gammal SH, Jones EA, Skolnick P. GABAA receptor complex in an experimental model of encephalopathy: evidence for elevated levels of an endogenous benzodiazepine receptor ligand. JNeurochem 1989; 53: 1057-63. 37 Olasmaa M, Guidotti A, Rothstein JD, Paul SM, Costa E. Naturally occurring benzodiazepines in CSF of patients with hepatic encephalopathy. [Abstract]. Soc NeurosciAbs 1989; 15: 199.8. 38 Mullen KD, Szauter KM, Kaminsky-Russ K. Endogenous benzodiazepine activity in body fluids of patients with hepatic encephalopathy. Lancet 1990; 336: 81-3. 39 Gammal SH, Basile AS, Geller D, Skolnick P, Jones EA. Reversal of the behavioural and electrophysiological abnormalities of an animal model of hepatic encephalopathy by benzodiazepine receptor ligands. Hepatology 1990; 11: 371-8. 40 Zieve L, Ferenci P, Rzepczynski D, Ebner J, Zimmermann C. A benzodiazepine antagonist does not alter the course of hepatic encephalopathy or neural y-amino butyric acid (GABA) binding. Metab Brain Dis 1987; 2: 201-5. 41 Bansky G, Meier PJ, Riederer E, Walser H, Ziegler WH, Schmid M. Effects of benzodiazepine receptor antagonist flumazenil in hepatic encephalopathy in humans. Gastroenterology 1989; 97: 744-50. 42 Grimm G, Ferenci P, Katzenschlager R, et al. Improvement of hepatic encephalopathy treated with flumazenil. Lancet 1988; ii: 1392-4. 43 Van der Riit CCD, Schaln SW, Meulstee J, Stiinen Th. Flumazenil therapy for hepatic encephalopathy: a double-blind crossover study. [Abstract]. Hepatology 1989; 10: 590. 44 Pomier Layrargues G, Giguere JF, Butterworth RF. Clinical trials of the efficacy of flumazenil in hepatic coma. J Hepatol 1990; 10: S 13. 45 Lo WD, Ennis SR, Goldstein GW, McNeely DL, Betz Al. The effects of galactosamine-induced hepatic failure upon blood-brain barrier permeability. Hepatology 1987; 7: 452-6. 46 Knudsen GM, Poulsen HE, Paulson OB. Blood-brain barrier permeability in galactosamine-induced hepatic encephalopathy. J Hepatol 1988; 6:187-92. 47 Knudsen GM, Schmidt J, Vilstrup H. Blood-brain barrier transport of amino acids in patients with hepatic encephalopathy. J Hepatol 1990; 10: S17. 48 Bassett ML, Mullen KD, Scholz B, Fenstermacher JD, Jones EA. Increased brain uptake of y-amino butyric acid in a rabbit model of hepatic encephalopathy. Gastroenterology 1990; 98: 747-57. 49 Plum F, Hindfelt B. Neurological complications ot liver disease. In: Vinken PJ, Bruyn GW, eds. Handbook of clinical neurology. Vol 27. Amsterdam: North Holland, 1976: 349-77. 50 Stahl S. Studies of blood ammonia in liver disease, its diagnostic, prognostic and therapeutic significance. Ann Intern Med 1963; 58: 1-24. 51 Hourani BT, Hamblin EM, Reynolds TB. Cerebrospinal fluid glutamine as a measure of hepatic encephalopathy. Arch Intern Med 1971; 127: 1033-6. 52 Bosman DK, Deutz N, De Graaf A, et al. Changes in brain metabolism during hyperamonemia and acute liver failure: results of a comparative 'H-NMR specroscopy and biochemical investigation. Hepatology 1990; 12: 281-90. 53 Tossmann U, Eriksson S, Delin A, Hagenfeldt L, Law D, Ungerstedt U. Brain amino acids measured by intracerebral dialysis in portacaval shunted rats. J3Neurochem 1983; 41: 1046-51. 54 Bosman DK, Deutz NEP, Maas MAW, Van Eyk HMH, Chamuleau RAFM. Brain cortex amino acids (AA) during acute hepatic encephalopathy (AHE) in the rat studied by brain dialysis (BD). J Hepatol 1990; 10: S3. 55 de Knegt RJ, vd Riut CCD, Schalm SW, Fekkes D, Dalm E, Voogd J. Brain glutamate and glutamine in rabbits with acute liver failure and hyperammonemia using in vivo brain dialysis. J Hepatol 1990; 10: S12. 56 Tossmann U, Delin A, Eriksson S, Ungerstedt U. Brain cortical amino acids measured by intracerebral dialysis in portacaval shunted rats. Neurochem Res 1987; 12: 265-9. 57 Hindfelt B, Plum F, Duffy TF. Effect of acute ammonia intoxication on central metabolism in rats with portacaval shunts. J Clin Invest 1977; 59: 386-96. 58 Kemp JA, Foster AC, Wong EH. Non competitive antagonists of excitatory amino acids. Trends Neurosci 1988; 11: 294-8. 59 Maddison JE, Watson WEJ, Dodd PR, Johnston GAR. The glutamate receptor complex in canine congenital portasystemic encephalopathy (PSE). J Hepatol 1990; 10: S 14. 60 Butterworth RF, Lavoie J, Giguere JF, Pomier-Layrargues G, Bergeron M. Cerebral GABA-ergic and glutamatergic function in hepatic encephalopathy. Neurochem Pathol 1987; 6: 131-44. 61 Raabe W. Synaptic transmission in ammonia intoxication. Neurochem Pathol 1987; 6:145-66.
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Gut Leading article Neurochemistry of hepatic encephalopathy Since the first biochemical descriptions of hepatic encephalopathy several unifying aetiological hypotheses have been developed that could account for the main clinical and biochemical features of the condition. Initially the concept of portosystemic encephalopathy was developed. This postulated the origin of a nitrogenous product in the gut that reached the cerebral circulation because of defective hepatic clearance by the diseased liver. Ammonia was the prime candidate neurotoxin and several studies were undertaken to determine the value of amino acid infusions, particularly glutamic acid and arginine, to stimulate urea cycle activity and hepatic urea synthesis.' Catharsis, gut sterilisation, and later lactulose, designed to diminish ammonia production by the colon, were the therapeutic consequences that were widely promoted and used. The mercaptan hypothesis followed, later modified to include synergism between ammonia short chain fatty acids and mercaptans,2 but subsequent studies3'4 using elegant trapping techniques' were negative. The next phase was characterised by the false neurotransmitter hypothesis.6 High plasma concentrations of aromatic amino acids were recognised in liver failure and thought to be associated with the synthesis of derivatives such as octopamine which interfered with neurotransmission. Because of the competition between aromatic and branch chain amino acids for transport into the brain, amino acid infusions rich in branch chain varieties were introduced,7 designed to impede cerebral uptake of precursor amino acids, tyrosine and phenylalanine, and thus decrease false transmitter formation in the brain. The concept of modulation of neurotransmission was extended by the finding of arousal after the administration of the dopamine agonist L dopa8 and various studies showed decreased brain catecholamine concentrations in animal models of encephalopathy, although these were not subsequently confirmed in man.910 The leucine degradation product, isovaleraldehyde, was increased in hepatic encephalopathy but no important role could be attributed to this product in man. " 5 Hydroxytryptamine (5HT) acts as a central neurotransmitter, so excess 5HT activity could account for the neuroinhibition of hepatic encephalopathy. Increased concentrations of the degradation product 5 hydroxy-indoleacetic acid (5HIAA) has been a consistent finding in the brain of animal models'2 and in the cerebrospinal fluid'3 and frontal cortex'4 of patients with fulminant hepatic failure, although in the latter brain 5HT concentrations were normal.'4 The findings were similar in prefrontal cortex and caudate nucleus of cirrhotic patients dying of hepatic coma.'° In the portacaval shunted rat, however, when compared with controls, there was increased 5HT turnover as shown by the 5HIAA:5HT
ratio but no further alteration in the ratio was observed when deep coma was precipitated by ammonia administration.'5 Thus, the changes in 5HT metabolism are probably the result of increased tryptophan availability in the brain'416 owing to the high plasma tryptophan concentrations observed in acute'4 and chronic liver disease7 rather than representing a primary abnormality. The gamma amino butyric acid hypothesis When the gamma amino butyric acid (GABA) hypothesis was first formulated it was suggested that excess activity of this neuroinhibitory transmitter in the brain caused the development of somnolence. GABA was thought to arise from bacterial action in the gut and reach the brain because of defective hepatic clearance and increased permeability of the blood brain barrier.'" Increased concentrations of GABA in the blood of patients with hepatic encephalopathy were described, perhaps associated with abnormalities of brain GABA receptors, which were increased in a rabbit model of acute encephalopathy induced by galactosamine. However, it was not possible to show penetration of the brain after intravenous administration of GABA.'8 More recent studies have found that brain GABA receptors, particularly in man, are normal in number and affinity'9 20 while in the portacaval shunted rat, quantitative autoradiographic studies of GABAA receptors are also normal.2' Furthermore brain GABA concentrations at necropsy in cirrhotic patients with encephalopathy were not significantly different from control values.22 Although brain GABA rises within one hour of death,23 when samples from patients dying of acute liver failure were collected within this period concentrations were still identical to those found in control subjects.'4 More recently, cerebrospinal fluid GABA values in man24 and in encephalopathic dogs with congenital portacaval shunts were normal,25 while brain GABA concentrations in dogs'8 and the thioacetamide rat model were normal or significantly decreased rather than increased, as would have been expected if GABAergic transmission were enhanced.26 The hypothesis has now been modified to include modulation of GABA transmission by a
benzodiazepine agonist. The benzodiazepine hypothesis27 Interest in the role of benzodiazepines in hepatic encephalopathy first developed because of the close association of the benzodiazepine and GABA receptors in the brain and the finding that binding sites were increased in galactosamine induced acute liver failure.28 29 Much greater interest followed early reports of a striking arousal effect ofthe benzodiazepine
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antagonist flumazenil in hepatic encephalopathy in man.30 Subsequent reports showed beneficial results in animal models,2931 while cerebellar Purkinje neurones from the rabbit galactosamine model were hypersensitive to GABA and benzodiazepine receptor agonists.32 On the other hand, alterations in GABA benzodiazepine receptors were not found in the brains of rats with chronic encephalopathy as a result of portal vein and bile duct ligation33 or in cirrhotic patients dying of encephalopathy.'9 Furthermore, quantitative autoradiography has shown that benzodiazepine receptors in the galactosamine rabbit model are within normal limits.34 The benzodiazepine hypothesis of hepatic encephalopathy is only tenable if the presence and source of endogenous benzodiazepine like substances can be shown. The presence of such ligands has been reported in the cerebrospinal fluid35 and brain extracts36 of animal models and also in the cerebrospinal fluid37 38 and plasma38 of patients with hepatic encephalopathy. The difficulty with these studies in man is the reliable exclusion of benzodiazepine drugs, which are readily available and have a prolonged half life. Naturally occurring benzodiazepines may be of dietary origin.35 38 The most compelling evidence for the benzodiazepine hypothesis comes from the use of the specific antagonist flumazenil both in man and experimental animals. Unfortunately, the early enthusiastic reports for the drug have not been sustained. In the thioacetamide model of acute liver failure flumazenil may reverse the deficits in spontaneous motor function and visually evoked response abnormalities but the motor deficit usually persists after drug administration.39 No effect could be shown in the rat hepatic ischaemia model (portacaval shunt plus hepatic artery ligation) despite administration of a dose sufficient to reverse benzodiazepine induced coma.' In man, the results are even more controversial - some authors claiming beneficial results4' in 60% of patients42 while others,43 including a placebo controlled trial, found only a 27% response rate." The ammonia hypothesis revisited The modern theories for the pathogenesis of hepatic encephalopathy do not account for the traditional and well documented role of the gut in the development of the condition. The precipitation of encephalopathy by protein ingestion in the form of variceal bleeding is familiar to all in clinical practice and any neurochemical hypothesis must take account of such clinical observations, including the efficacy of gut sterilisation and lactulose. Any mechanism encompassing the role of the gut must also be explicable in terms of current concepts of dissordered neurotransmission. For obvious physiological reasons any neurotransmitter substance arising from the gut will not cross the blood brain barrier, and while blood brain barrier permeability may be disturbed in the latter stages of encephalopathy there is little evidence to support any change during its initial development.58 45-I8 Ammonia arising from the gut is, however, freely permeable to the brain, while cerebrospinal fluid values49 and arterial concentrations30 bear a reasonable correlation with coma grade. Ammonia taken up by the brain is incorporated into glutamine which may deplete brain glutamate. Glutamate is the principal excitatory transmitter in the brain and a disturbance in glutamatergic transmission could account for the neuroinhibition of hepatic encephalopathy. Early studies of human brain amino acid concentrations in hepatic encephalopathy showed that glutamine concentrations were increased two to threefold in the frontal cortex of patients dying from fulminant hepatic failure.'4 Increased concentrations were then found in cirrhotic patients22 while cerebrospinal fluid values showed a close correlation with coma grade.495' Brain glutamate concentrations in patients with cirrhosis and encephalopathy were decreased in several
areas22 but were normal in fulminant hepatic failure in unventilated patients or increased in those dying after a prolonged period of ventilation. 14 The latter finding probably reflects brain death and equilibration with the high concentrations of glutamate found in the blood of the patients studied. Glutamate is thought to be present in the brain in several distinct pools including metabolic, GABA precurser, astrocytic, and finally a releasable pool available for glutamatergic transmission. Depletion of the latter alone may be sufficient to depress excitatory neurotransmission. Several studies have attempted to evaluate the concentration of glutamate in vivo in animal models of hepatic encephalopathy. The results using nuclear magnetic resonance52 have suggested decreased concentrations, while studies with brain dialysis have shown normal53 or increased glutamate output in the perfusate.5 156 The latter observation could be the result of decreased uptake of glutamate from the synaptic cleft into astrocytes, and indeed no further glutamate could be released into the brain perfusate in acute hepatic failure when potassium was included in the perfusion medium54 suggesting an absolute decrease in transmitter pool. In the rat thioacetamide model brain glutamate is significantly decreased in frontal cortex and striatum when plasma concentrations are increased more than fourfold.26 An ammonia induced decrease in cerebral glutamate has been described by Hindfelt.5 Interest in the glutamate receptor has increased since the finding that the anaesthetic agent ketamine leads to depression of the reticuloendothelial system by changing glutamatergic transmission.58 Glutamate receptors can be devided into Ca++/Cl- dependent (presynaptic) and independent (postsynaptic) classes and can be recognised by their ability to bind n methyl D aspartate (NMDA) and quisquolate/ kainate (nonNMDA). In chronically encephalopathic dogs with congenital portacaval shunts, NMDA binding sites in the brain were normal or increased while kainate binding was decreased.59 In the thioacetamide rat model Ca++/Cl[ dependent and independent glutamate binding was unchanged but this may have been because of the short duration of encephalopathy in this model (about seven hours.)26 In portacaval shunted rats (3H) glutamate binding was increased consistent with decreased values of synaptic glutamate and up regulation of the postsynaptic receptor.60 Glutamate receptor binding in hepatic encephalopathy in man has not yet been determined. More direct evidence for the role of disturbed glutamatergie transmission comes from studies of the effect of ammonia on evoked glutamate release and excitatory post synaptic potentials. Several investigations have shown diminished responses.6' The attraction of the glutamate depletion hypothesis is that it accounts for many of the original observations concerning the role of ammonia and reestablishes the role of the gastrointestinal tract. Much work remains to be done but ammonia is currently returning to the forefront of hepatic neurochemistry. C O RECORD
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