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The relationship between plasma and brain quinolinic acid levels and the severity of hepatic encephalopathy
GASTROENTEROLOGY1995;108:818-823
The Relationship Between Plasma and Brain Quinolinic Acid Levels and the Severity of Hepatic Encephalopathy ANTHONY S. BASILE,*
KUNIAKI SAITO, t HANAAN A L - M A R D I N I , § CHRISTOPHER O. RECORD, §
ROBIN D. HUGHES, II PHIL HARRISON, H ROGER WILLIAMS, II YONG L I , * and MELVYN P. HEYES* *Laboratory of Neuroscience, National Institute of Diabetes and Digestiveand Kidney Diseases, National Institutes of Health, Bethesda, Maryland; tSection on Analytical Biochemistry, Laboratoryof Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland; ~GastroenterologyUnit, The Royal Victoria Infirmary, Newcastle-Upon-Tyne,England; and illnstitute of Liver Studies, King's College School of Medicine and Dentistry, London, England
Background/Aims: Quinolinic acid is an endogenous
neuroexcitant derived from tryptophan. Brain quinolinic acid concentrations are reportedly elevated in chronic liver failure. The aim of this study was to determine if brain quinolinic acid levels correlate with the severity of hepatic encephalopathy. Methods: Postmortem samples of selected brain regions and plasma samples taken at several stages of encephalopathy were obtained from patients with acute and chronic liver failure. Quinolinic acid levels were measured by mass spectroscopy using [180]quinolinic acid. Results: Plasma quinolinic acid levels were significantly increased by stage I encephalopathy in patients with acute liver failure and by stages II and III in patients with chronic liver failure. Brain quinolinic acid levels were elevated only in patients with acute liver failure and were uniformly distributed at concentrations below those observed in plasma. Conclusions: The uniform distribution of quinolinic acid at subplasma concentrations in the brains of patients with acute liver failure suggests that it is synthesized peripherally and enters the brain across a permeabilized blood-brain barrier. Whereas the elevation of brain quinolinic acid levels in patients who died of acute but not chronic liver failure suggests that the involvement of quinolinic acid in the pathogenesis of hepatic encephalopathy is minimal, it could predispose these patients to seizures.
he tryptophan metabolite quinolinic acid is an endogenous neuronal excitant acting at the N-methylD-aspartate receptor that can cause convulsions and neuronal degeneration. 1 Because the plasma and brain concentrations of tryptophan are elevated in acute and chronic liver failure, 2'~ increased rates of quinolinic acid synthesis in the central nervous system (CNS) and periphery might result from the increased availability of tryptophan in these regions. Indeed, increased quinolinic acid levels have been reported in the brains of hyperammonemic and portacaval shunted rats 4 and in the cerebrospinal fluid and frontal cortex of humans who died of
chronic liver failure) This led to the proposal that quinolinic acid may play a role in the pathogenesis of hepatic encephalopathy (HE), a neuropsychiatric disorder that often accompanies acute or chronic hepatic failure. A role for quinolinic acid in the pathogenesis of HE is particularly intriguing because the neuroexcitatory, convulsant, and neurotoxic characteristics of this agent contrast with the general manifestations of HE. Specifically, patients with HE show evidence of depressed neuronal activity manifested as lethargy, decreased cognitive function, and electroencephalographic slowing. 6.s Seizures are relatively rare, and neuronal degeneration is not a significant characteristic of this reversible, metabolic encephalopathy. 8'9 Moreover, because the mechanisms underlying the pathogenesis of HE caused by acute or chronic liver failure are not entirely the same, the involvement of quinolinic acid in patients with HE associated with chronic liver failure may not be relevant to patients with HE caused by acute or fulminant hepatic failure. In view of these incongruities, we have measured quinolinic acid levels in the plasma and selected brain regions of patients with either acute or chronic liver failure at several stages of HE. This may provide an insight into the role that quinolinic acid may play in the pathogenesis of acute episodes of HE associated with either acute or chronic liver failure.
Materials and Methods
T
Patient Characteristics The clinical characteristics of the 28 patients with acute liver failure, 30 patients with chronic liver failure, and 18 normal subjects are presented in Table 1. The manifestations of HE appeared acutely in the patients with chronic liver failure as well as the acute liver failure group. One of the patients with an acute presentation of Wilson's disease was included Abbreviations used in this paper: HE, hepatic encephalopathy. This is a U.S. governmentwork, There are no restrictionson its
use, 0016-5085/95/$0,00
March 1995
QUINOLINIC ACID AND HEPATIC ENCEPHALOPATHY 819
Table 1. Clinical Data on Patients With HE and Control Subjects Sex (M/F)
Age (yr)
Acute liver failure
16/12
29 _+ 2
Chronic liver failure
20/12
53 + 2
9/9
46 + 4
Group
Control subjects
Cause of death Acetaminophen overdose (19) Other drug overdose (2) Hepatitis (5) Wilson's disease (1) Alcoholic cirrhosis (11) Primary biliary cirrhosis (10) Cryptogenic cirrhosis (4) Hepatic carcinomatosis (2) Hepatitis (3) Wilson's disease (1) Ischemic heart failure (14) Motoneuron disease (1) Rheumatic heart disease (1) Lung cancer (2)
Prothrombin time
(seconds)
85 _+ 9
41 + 7
NOTE. The data represent the mean _+ SE.
in the acute liver failure group because the clinical course of the condition was identical to that of patients with acute liver failure rather than that of a patient with chronic liver failure. The primary cause of death for patients in the acute liver failure category was acetaminophen overdose (68%), whereas most patients with chronic liver failure had alcoholic cirrhosis (37%) or primary biliary cirrhosis (33%). The principal cause of death among the controls was ischemic heart failure (78%). Of the patients with hepatitis, 3 of 5 patients with acute liver failure had non-A, non-B hepatitis; 1 of 5 had ischemic hepatitis; and 1 of 5 had drug-induced hepatitis, whereas all 3 of the patients with chronic liver failure had hepatitis B. Some of the patients received ranitidine, cefuroxime, vitamin K, and parentrovite (containing 500 mg ascorbic acid, 900 mg glucose, 160 mg nicotinamide, 50 mg pyridoxine, 4 mg riboflavine, and 250 mg thiamine HC1 per 10 mL) as routine therapy. Furthermore, 9 patients received noradrenaline or adrenaline for hypotension. The severity of HE in each of these patients was graded according to the general criteria previously described, lo Patient plasma samples from only one stage of HE were included in subsequent analyses. There was no significant difference between the groups in the numbers of men and women studied. The age of the patients in the acute liver failure group was significantly less than either the control or chronic liver failure groups (P < 0.01; analysis of variance). However, the ages of the patients in the control and chronic liver failure groups were not significantly different from each other. Not all patients were sampled for both brain and plasma.
Quantification of Quinolinic Acid Plasma samples ( 3 - 1 5 mL) were taken from patients at various times during their hospital care in the United Kingdom with the first sample taken within 24 hours after admission. Brain samples were taken an average of 20 _+ 2 hours after death according to previously described techniques. 11 The samples were stored at - 8 0 ° C for 3 - 4 months and then shipped on dry ice for analysis in the United States, where they were analyzed within 1 month.
Aliquots of the plasma samples (20 btL) were diluted with 1 mL of 1 mol/L HC1, whereas brain samples (50 mg) were homogenized by sonication with 1 mL of 1 mol/L HC1. Both preparations were then centrifuged at 12,000g for 20 minutes at 4°C, and aliquots of the supernatant (300 }.tL of plasma and 800 btL of brain) were retained. Quinolinic acid standards were dissolved in equivalent volumes of deionized water. [iSO]Quinolinic acid (2 ng) was added as an internal standard to each tube. All tissue extracts and corresponding quinolinic acid standards were washed with 3 mL of chloroform, and equal volumes of the aqueous layer were collected. The samples and standards were then freeze-dried overnight. Quinolinic acid and [lSO]quinolinic acid were derivatized to their dihexafluoroisopropanol esters with complete retention of the isotopes, washed with 250 [.tL of water, extracted into 3 0 0 - 1 0 0 0 btL of heptane, and then quantified using a Hewlett-Packard 5988 mass spectrometer (San Fernando, CA) operated in the electron capture negative chemical ionization mode using methane as the reagent gas. Aliquots of each sample were injected into a 1 m X 0.53 m m (inside diameter) fused silica precolumn (on-column injection at 80°C) that had been sealed to a 15 m X 0.25 m m (inner diameter) DB5 analytical column (J & W Scientific, Folsum, CA). Column temperature was 112°C, and helium was the carrier gas. The molecular anions of quinolinic acid (m/z 467) and the [180]quinolinic acid internal standard (mass/charge = 471) were monitored and each peak area quantified at the appropriate retention time. All quantification was performed at a signal/noise ratio of >--20:1. The minimum detectability was 300 fg at a signal/noise ratio of -----5:1.
Statistics Intergroup comparisons were made using analysis of variance followed by post hoc multiple comparison analysis with Fisher's least significant difference test (Systat, Evanston, IL). Correlations were determined using the Pearson correlation matrix with Bonferroni-adjusted probabilities.
820
0 4J
BASILE ET AL.
GASTROENTEROLOGY Vol. 108, No. 3
tients with chronic liver failure were not significantly increased above control levels until stages II and III of HE, where the maximum concentrations reached approximately 5 btmol/L (Figure 1 and Table 1). Overall, plasma samples from 13 of 23 patients with chronic liver failure had quinolinic acid levels within the range of normal values. Analyzed by themselves, a strong correlation was found between the plasma concentration of quinolinic acid and the stage of HE (Pearson correlation coefficient = 0.75; P < 0.01) in patients with chronic liver failure. When the plasma quinolinic acid concentrations from both patients with acute and chronic liver failure were pooled, there was a moderate but significant correlation between the stage of HE and the quinolinic acid concentration (Pearson correlation coefficient = 0.33; P
18-
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16 14
12lo-
8 6-
o
o A
0
O
AA
:
2
B
Ct:rl
O
oo , , , t a ~
o
i Stage
O
~o
o
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0
AA
O
4
o_
CY
8
A
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ziz
ooo
i~,
HE
Figure 1. Distribution of plasma quinolinic acid levels in controls (F1)
and patients with acute (©) or chronic (A) liver failure. Patients were sampled only once at any stage of encephalopathy. Approximately 35% of the patients with acute liver failure and 57% of the patients with chronic liver failure had quinolinic acid levels that fell within the control range, regardless of the stage of HE.
=
0.01).
The quinolinic acid concentrations in samples of cerebral cortex, caudate nucleus, or cerebellum from patients who died from chronic liver failure in stage IV HE were not significantly different from controls (Figure 2 and Table 3). However, mean quinolinic acid concentrations in patients who died from acute liver failure were elevated by approximately 1 0 0 % - 4 0 0 % above control levels in the cerebral cortex, caudate nucleus, and cerebellum (F(8,795 = 3.736; P < 0.01, P < 0.05, and P < 0.05, respectively). There was no significant difference in quinolinic acid concentrations between the brain regions from patients with acute liver failure (P = 0.06, caudate nucleus vs. cortex; P = 0.19 caudate nucleus vs. cerebellum). Approximately 35% of the patients with acute liver failure had brain quinolinic acid concentrations that fell within normal levels.
Results
Approximately 35% of the patients with acute liver failure had plasma quinolinic acid levels within the control range (<1.2 btmol/L). Overall, patients with acute liver failure had mean plasma quinolinic acid concentrations 11.3 times greater than control by stage I HE (F(8,51) = 2.29; P < 0.05), which remained significantly elevated throughout stages I I - I V of HE (Figure 1 and Table 2). The mean plasma concentration of quinolinic acid in patients with acute liver failure was stable at approximately 5 btmol/L through stages I - I V . There was no significant difference between the plasma quinolinic acid concentrations in patients with acute hepatic failure at various stages of HE, and there was no correlation with the severity of HE (Pearson correlation coefficient = 0.25; P = 0.15). Contrasting with the observations in patients with acute liver failure, plasma quinolinic acid levels in pa-
Discussion
Increased brain and plasma tryptophan levels are a hallmark of liver failure2'3 and may enhance the synthesis of metabolites such as serotonin and quinolinic acid. Elevated concentrations of these metabolites may then alter neuronal activity and contribute to the manifesta-
Table 2. Ouinolinic Acid Levels in Plasma From Patients With Acute and Chronic Liver Failure Stage of HE Group Control Acute liver failure Chronic liver failure
0
I
II
III
IV
0.57 (1)
5.4 _+ 3.0 a (4) 1.4 + 0.3 c (18)
5.0 + 1.4 a (5) 4.7 (2)
4.6 + 2.2 b (4) 5.2 + 0.7 a (3)
5.1 + 1.6 a (12)
0.44 + 0.11 (10)
NOTE. All values are mean _+ SE in micromolar. Values in parentheses represent the number of observations. "P < 0.05; bp < 0.01; significantly different from control values, analysis of variance followed by Fisher's least significant difference multiple comparison test. cSignificantly different from stage I acute liver failure, P < 0.05, analysis of variance followed by Fisher's least significant difference multiple comparison test.
March 1 9 9 5
QUINOLINIC ACID AND HEPATIC ENCEPHALOPATHY
Table 3. Quinolinic Acid Levels in Brain Regions From
16-
E o
Humans With Acute and Chronic Liver Failure
A
Brain region
6o
@
o
og
L~ c
-6 v
5-
4: 3: 2:
0
OoO
n°
o~o Q"D
r-
o
~oo~on ~
nBo°~On
Control
Chronic
°1
0 0
5o
4:
O v
0
2:
1:
0
O0 Control
0~0 Chronic
BB OoO Acute
c
o
0 0 o
0 l__
3o
o
C= 0
cv
2"
0
%%
(EY
moO0O Control
000
Chronic
Group
Cerebral cortex
Caudate nucleus
Cerebellum
Control Acute liver failure Chronic liver failure
0.52 _+ 0.25 (12)
0.65 _+ 0.50 (11)
0.63 _ 0.42 (6)
1.9 + 0.592 (13)
3.4 _+ 1.3 b (11)
2.3 _+ 0.65 b (9)
0.48 _+ 0.12 (13)
0.54 _+ 0.13 (13)
0.53 ± 0.14 (3)
NOTE. The data represent the mean _+ SE of quinolinic acid concentrations in nanomole per gram wet weight. Values in parentheses represent the number of observations. ap < 0.01, bp < 0.05; significantly different from control values, analysis of variance followed by Fisher's least significant difference multiple comparison test.
Acute
' TB
.2
1
821
0
Acute
Figure 2. Quinolinic acid levels in (A) samples of cerebral cortex, (B) caudate nucleus, and (C) cerebellum from controls and patients who died from either acute or chronic liver failure in stage IV HE. The mean quinolinic acid levels in patients with chronic liver failure were not significantly different from control levels in any brain region. Although quinolinic acid levels in approximately 35% of the patients who died of acute liver failure in stage IV HE were within the control range, the mean quinolinic acid levels were significantly higher than controls in all three brain regions•
tions of HE. Indeed, increased turnover of serotonin 11'12 as well as elevated concentrations of quinolinic acid 5 are observed in patients with liver failure. However, the relationship of either brain or plasma quinolinic acid concentrations to the severity of HE are not known. In patients with either acute or chronic liver failure, plasma quinolinic acid levels were significantly increased above control levels to an average maximum of ~ 5 btmol/ L. However, elevated plasma quinolinic acid levels were not a reliable feature of liver failure because approximately 3 0 % - 5 0 % of the patients at all stages of HE had quinolinic acid levels in the normal range. This factor and the relatively stable elevation of quinolinic acid in the plasma of patients with acute liver failure regardless of the stage of HE may explain why the correlation between the plasma concentration of quinolinic acid and the severity of encephalopathy was only moderate. More important are the differences between the brain levels ofquinolinic acid in patients with acute vs. chronic liver failure. No significant changes in quinolinic acid levels were observed in the cortex, caudate nucleus, or cerebellum of patients who died in stage IV HE caused by chronic liver failure despite evidence of elevated plasma quinolinic acid concentrations. This observation contrasts with that of a previous report 5 indicating that quinolinic acid levels in the frontal cortex of patients with chronic liver failure (caused by hepatic cirrhosis or metastatic liver cancer) were increased approximately threefold, implying that CNS synthesis of quinolinic acid is not significantly enhanced in patients with chronic liver failure despite the presence of elevated concentrations of its precursor, tryptophan) This strongly suggests that quinolinic acid has a limited role in the pathogenesis of HE. In contrast, quinolinic acid levels were significantly increased in the cortex, caudate nucleus, and cerebellum of patients with acute liver failure arising primarily from drug overdose. The level of quinolinic acid in these re-
822
BASILE ET AL.
gions was lower than the plasma level and was evenly distributed throughout the brain regions sampled, providing further evidence that the quinolinic acid measured in the CNS may be of peripheral origin. The origin of the quinolinic acid in the CNS of patients with acute liver failure is unclear. Normal brain does not convert tryptophan to quinolinic acid, 13 and quinolinic acid does not penetrate a normal blood-brain barrier. 14'15 Whereas quinolinic acid can be synthesized from tryptophan by activated macrophages, 16'1v it is not known whether macrophages invade the CNS of patients with acute liver failure. Thus, the plasma is the most probable source of the quinolinic acid we measured in the brain, entering through a permeabilized blood-brain barrier. ~8'~9 The dependence of the entry of quinolinic acid into the CNS on the permeability of the blood-brain barrier may explain the differences in CNS quinolinic acid concentrations between patients with acute vs. chronic liver failure because increased blood-brain barrier permeability is believed to play a role in the pathogenesis of HE complicating acute liver failure,9'~8'~9 whereas its role in chronic liver failure is unclear. 9'2° Plasma quinolinic acid levels may increase as a result of enhanced quinolinic acid synthesis in the liver resulting from glucocorticoid stimulation of hepatocytes2~ or by cytokine activation of sinusoidal endothelial cells as a result of inflammatory liver disease processes associated with both acute and chronic liver failure. 22-25 Increases in plasma and brain quinolinic acid levels similar to those observed in patients with acute liver failure are also observed in the galactosamine-treated rabbit and thioacetamidetreated rat models of HE due to fulminant hepatic failure. 26 The elevation in quinolinic acid concentrations is accompanied by increased activity of hepatic tryptophan2,3-dioxygenase (the rate-limiting enzyme for the hepatic synthesis of quinolinic acid from L-tryptophan) but not brain indoleamine-2,3-dioxygenase (the rate-limiting enzyme for extrahepatic conversion of tryptophan to quinolinic acid), 21 supporting the proposal that the primary source of quinolinic acid in hepatic failure is the liver. Thus, the evidence implicating quinolinic acid in the pathogenesis of HE should be reassessed. Not only are the outward manifestations of HE one of depressed CNS function and neuronal activity, but the correlation between plasma quinolinic acid concentrations and the stage of HE is only moderate. Indeed, patients with chronic liver failure show no evidence of increased CNS quinolinic acid levels during an acute episode of HE. Furthermore, the CNS concentrations of quinolinic acid observed in patients with acute liver failure are ~20% of those observed in patients with infectious or inflam-
GASTROENTEROLOGY Vol. 108, No. 3
matory diseases 2v'28 and are probably insufficient to cause a permanent neuronal lesion. Nonetheless, quinolinic acid may play a differential role in CNS function in patients with acute rather than chronic liver failure, and that role may be to facilitate seizure development. While seizures are not considered a principal manifestation of HE per se, they occur more commonly in patients with acute or fulminant hepatic failure29 than in those with chronic hepatic failure. The lack of increase in CNS quinolinic acid concentrations, the possible development of "tolerance" to ammonia, 3° and the generalized increase in ]'-aminobutyricergic neurotransmission in patients with chronic liver failure7 would act to reduce the frequency of seizures in this population. Given the ability of quinolinic acid to cause neuronal hyperexcitability and seizures, 1 it is possible that the generalized increase in CNS quinolinic acid levels observed in patients with acute liver failure may predispose them to convulsions, a condition that would be exacerbated by the rapid increase in CNS ammonia levels 31'32 and cerebral edema33 observed in fulminant hepatic failure.
References 1. Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev 1993;45:309-379. 2. James JH, Hodgman JM, Funovics JM, Yoshimura N, Fischer JE. Brain tryptophan, plasma free tryptophan and the distribution of plasma neutral aminoacids. Metabolism 1976;25:471-476. 3. Record CA, Buxton B, Chase RA, Curzon G, Murray-Lyon IM, Williams R. Plasma and brain amino acids in fulminant hepatic failure and their relationship to hepatic encephalopathy. Eur J Clin Invest 1976;6:387-394. 4. Moroni F, Lombardi G, Carla V, Pellegrini D, Carassale GL, Cortesini C. Content of quinolinic acid and of other tryptophan metabolites increases in brain regions of rats used as experimental models of hepatic encephalopathy. J Neurochem 1986; 46:869874. 5. Moroni F, Lombardi G, Carla V, Lal S, Etienne P, Nair NPV. Increase in the content of quinolinic acid in cerebrospinal fluid and frontal cortex of patients with hepatic failure. J Neurochem 1986; 47:1667-1771. 6. van der Rijt C, Schalm SW, deGroot GH, de Vlieger M. Objective measurement of hepatic encephalopathy by means of automated EEG analysis. Electroencephalogr Clin Neurophysiol 1984;57: 423-426. 7. Basile AS, Jones EA, Skolnick P. The pathogenesis and treatment of hepatic encephalopathy: evidence for the involvement of benzodiazepine receptor iigands. Pharmacol Rev 1991;43:27-71. 8. Conn HO. Hepatic encephalopathy. In: Schiff L, Schiff ER, eds. Diseases of the liver. Volume 2. Philadelphia: Lippincott, 1993; 1036-1056. 9. Jones EA, Gammal SH. Hepatic encephalopathy. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven, 1988;985-1005. 10. Basile AS, Hughes RD, Harrison PM, Gu Z-Q, Pannell L, McKinnon A, Jones EA, Williams R. Correlation between plasma benzodiazepine receptor ligand concentrations and the severity of hepatic encephalopathy in patients with fulminant hepatic failure. Hepatology 1994; 19:112-121. 11. AI Mardini H, Harrison EJ, Ince PG, Bartlett K, Record CO. Brain
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indoles in human hepatic encephalopathy. Hepatology 1993; 17: 1033-1040. Bergeron M, Reader TA, Layrargues GP, Butterworth RF. Monoamines and metabolites in autopsiad brain tissue from cirrhotic patients with hepatic encephalopathy. Neurochem Res 1989; 14:853-859. Speciale C, Ungerstedt U, Schwarcz R. Production of extracellular quinolinic acid in the striatum studied by microdialysis in unanesthetized rats. Neurosci Lett 1989; 104:345-350. During M J, Freese A, Heyes MP, Swartz KJ, Markey SP, Roth RH, Martin JB. Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid. Effect of tryptophan loading. FEBS Lett 1989;247:438-444. Fukui S, Schwarcz R, Rapoport SI, Takada Y, Smith QR. Bloodbrain barrier transport of kynurenines: implications for brain synthesis and metabolism. J Neurochem 1991;56:2007-2015. Saito K, Chen CY, Masana M, Crowley JS, Markey SP, Heyes MP. 4-Chloro-3-hydroxyanthranilate, 6-chlorotryptophan and norharmane attenuate quinolinic acid formation by interferon-~'-stimulated monocytes (THP-1 cells). Biochem J 1993;291:11-14. Saito K, Nowak TS, Markey SP, Heyes MP. Mechanism of delayed increases in kynurenine pathway metabolism in damaged brain regions following transient cerebral ischemia. J Neurochem 1993; 60:180-192. Zaki AEO, Ede RJ, Davis M, Williams R. Experimental studies of blood brain barrier permeability in acute hepatic failure. Hepatology 1983; 4 : 3 5 9 - 3 6 3 . Horowitz ME, Schafer DF, Molnar P, Jones EA, Blasberg RG, Patlak CS, Waggoner J, Fenstermacher JD. Increased blood-brain transfer in a rabbit model of acute liver failure. Gastroenterology 1983; 84:1003-1011. Huet PM, Rocheleau B, Pomier-Layrargues G, Williams B. Blood brain barrier in dogs with and without hepatic encephalopathy. In: Kleinberger G, Ferenci P, Riederer P, Thaler H, eds. Advances in hepatic encephalopathy and urea cycle diseases. Basel: Karger, 1984; 261-271. Saito K, Markey SP, Heyes MP. Effects of immune activation on quinolinic acid and neuroactive kynurenines in the mouse. Neuroscience 1992; 51:25-39. Tilg H. The role of cytokines in the pathophysiology of chronic liver diseases. Int J Clin Lab Res 1993;23:179-185. Shiratori Y, Moriwaki H, Kawashima Y, Ando K, Asano F, Shimazaki M, Ohnishi H, Mute Y, Okuno M. Elevated interleukin-6 levels in sera of patients with fulminant hepatitis. Gastroenterol Jpn 1991; 26:233.
QUINOLINIC ACID AND HEPATIC ENCEPHALOPATHY 823
24. Sheron N, Goka J, Wend J, Keays R, Keane H, Alexander G, Williams R. Highly elevated plasma cytokines in fulminant hepatic failure: correlation with multiorgan failure and death (abstr). Hepatology 1990; 12:939. 25. Mute Y, Nouri-Aria KT, Meager A, Alexander GJM, Eddleston ALWF, Williams R. Enhanced tumor necrosis factor and intedeukin-1 in fulminant hepatic failure. Lancet 1988;2:72-74. 26. Basile AS, Saito K, Li Y, Heyes MP. The relationship between plasma and brain quinolinic acid levels and the severity of hepatic encephalopathy. J Neurochem (in press). 27. Heyes MP, Brew BJ, Martin A, Price RW, Salazar AM, Sidtis JJ, Yergey JA, Mouradian MM, Sadler AE, Keilp J, Rubinow D, Markey SP. Quinolinic acid in cerebrospinal fluid and serum in HIV-1 infection: relationship to clinical and neurological status. Ann Neurol 1991; 29:202-209. 28. Heyes MP, Saito K, Crowley JS, Davis LE, Demitrack MA, Der M, Dilling LA, Ella J, Kruesi MJP, Lackner A, Larsen SA, Lee K, Leonard HL, Markey SP, Martin A, Milstein S, Mouradian MM, Pranzatelli MR, Quearry BJ, Salazar A, Smith M, Strauss SE, Sunderland T, Swede SW, Tourtellotte WW. Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease. Brain 1991; 115:1249-1273. 29. Schafer DF, Jones EA. Hepatic encephalopathy. In: Zakim D, Boyer TD, eds. Hepatology: a textbook of liver disease. Philadelphia: Saunders, 1990:447-459. 30. Raabe W, Onstad G. Perta-caval shunting changes neuronal sensitivity to ammonia. J Neurol Sci 1985;71:307-314. 31. lies JF, Jack JJB. Ammonia: assessment of its action on postsynaptic inhibition as a cause of convulsions. Brain 1980; 103:555578. 32. Raabe W. Neurophysiology of ammonia intoxication. In: Butterworth R, Pomier-Layrargues G, eds. Hepatic encephalopathy: pathophysiology and treatment. Clifton, NJ: Humana, 1 9 8 9 ; 4 9 78. 33. Gazzard BG, Portmann B, Murray-Lyon IM, Williams R. Causes of death in fulminant hepatic failure and relationship to quantitative histological assessment of parenchymal damage. Q J Med 1975;44:615-626.
Received June 21, 1994. Accepted October 17, 1994. Address requests for reprints to: Anthony S. Basile, Ph.D., Laboratory of Neuroscience, Building 8, Room 111, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. Fax: (301) 402-2872.
The Relationship Between Plasma and Brain Quinolinic Acid Levels and the Severity of Hepatic Encephalopathy ANTHONY S. BASILE,*
KUNIAKI SAITO, t HANAAN A L - M A R D I N I , § CHRISTOPHER O. RECORD, §
ROBIN D. HUGHES, II PHIL HARRISON, H ROGER WILLIAMS, II YONG L I , * and MELVYN P. HEYES* *Laboratory of Neuroscience, National Institute of Diabetes and Digestiveand Kidney Diseases, National Institutes of Health, Bethesda, Maryland; tSection on Analytical Biochemistry, Laboratoryof Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland; ~GastroenterologyUnit, The Royal Victoria Infirmary, Newcastle-Upon-Tyne,England; and illnstitute of Liver Studies, King's College School of Medicine and Dentistry, London, England
Background/Aims: Quinolinic acid is an endogenous
neuroexcitant derived from tryptophan. Brain quinolinic acid concentrations are reportedly elevated in chronic liver failure. The aim of this study was to determine if brain quinolinic acid levels correlate with the severity of hepatic encephalopathy. Methods: Postmortem samples of selected brain regions and plasma samples taken at several stages of encephalopathy were obtained from patients with acute and chronic liver failure. Quinolinic acid levels were measured by mass spectroscopy using [180]quinolinic acid. Results: Plasma quinolinic acid levels were significantly increased by stage I encephalopathy in patients with acute liver failure and by stages II and III in patients with chronic liver failure. Brain quinolinic acid levels were elevated only in patients with acute liver failure and were uniformly distributed at concentrations below those observed in plasma. Conclusions: The uniform distribution of quinolinic acid at subplasma concentrations in the brains of patients with acute liver failure suggests that it is synthesized peripherally and enters the brain across a permeabilized blood-brain barrier. Whereas the elevation of brain quinolinic acid levels in patients who died of acute but not chronic liver failure suggests that the involvement of quinolinic acid in the pathogenesis of hepatic encephalopathy is minimal, it could predispose these patients to seizures.
he tryptophan metabolite quinolinic acid is an endogenous neuronal excitant acting at the N-methylD-aspartate receptor that can cause convulsions and neuronal degeneration. 1 Because the plasma and brain concentrations of tryptophan are elevated in acute and chronic liver failure, 2'~ increased rates of quinolinic acid synthesis in the central nervous system (CNS) and periphery might result from the increased availability of tryptophan in these regions. Indeed, increased quinolinic acid levels have been reported in the brains of hyperammonemic and portacaval shunted rats 4 and in the cerebrospinal fluid and frontal cortex of humans who died of
chronic liver failure) This led to the proposal that quinolinic acid may play a role in the pathogenesis of hepatic encephalopathy (HE), a neuropsychiatric disorder that often accompanies acute or chronic hepatic failure. A role for quinolinic acid in the pathogenesis of HE is particularly intriguing because the neuroexcitatory, convulsant, and neurotoxic characteristics of this agent contrast with the general manifestations of HE. Specifically, patients with HE show evidence of depressed neuronal activity manifested as lethargy, decreased cognitive function, and electroencephalographic slowing. 6.s Seizures are relatively rare, and neuronal degeneration is not a significant characteristic of this reversible, metabolic encephalopathy. 8'9 Moreover, because the mechanisms underlying the pathogenesis of HE caused by acute or chronic liver failure are not entirely the same, the involvement of quinolinic acid in patients with HE associated with chronic liver failure may not be relevant to patients with HE caused by acute or fulminant hepatic failure. In view of these incongruities, we have measured quinolinic acid levels in the plasma and selected brain regions of patients with either acute or chronic liver failure at several stages of HE. This may provide an insight into the role that quinolinic acid may play in the pathogenesis of acute episodes of HE associated with either acute or chronic liver failure.
Materials and Methods
T
Patient Characteristics The clinical characteristics of the 28 patients with acute liver failure, 30 patients with chronic liver failure, and 18 normal subjects are presented in Table 1. The manifestations of HE appeared acutely in the patients with chronic liver failure as well as the acute liver failure group. One of the patients with an acute presentation of Wilson's disease was included Abbreviations used in this paper: HE, hepatic encephalopathy. This is a U.S. governmentwork, There are no restrictionson its
use, 0016-5085/95/$0,00
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QUINOLINIC ACID AND HEPATIC ENCEPHALOPATHY 819
Table 1. Clinical Data on Patients With HE and Control Subjects Sex (M/F)
Age (yr)
Acute liver failure
16/12
29 _+ 2
Chronic liver failure
20/12
53 + 2
9/9
46 + 4
Group
Control subjects
Cause of death Acetaminophen overdose (19) Other drug overdose (2) Hepatitis (5) Wilson's disease (1) Alcoholic cirrhosis (11) Primary biliary cirrhosis (10) Cryptogenic cirrhosis (4) Hepatic carcinomatosis (2) Hepatitis (3) Wilson's disease (1) Ischemic heart failure (14) Motoneuron disease (1) Rheumatic heart disease (1) Lung cancer (2)
Prothrombin time
(seconds)
85 _+ 9
41 + 7
NOTE. The data represent the mean _+ SE.
in the acute liver failure group because the clinical course of the condition was identical to that of patients with acute liver failure rather than that of a patient with chronic liver failure. The primary cause of death for patients in the acute liver failure category was acetaminophen overdose (68%), whereas most patients with chronic liver failure had alcoholic cirrhosis (37%) or primary biliary cirrhosis (33%). The principal cause of death among the controls was ischemic heart failure (78%). Of the patients with hepatitis, 3 of 5 patients with acute liver failure had non-A, non-B hepatitis; 1 of 5 had ischemic hepatitis; and 1 of 5 had drug-induced hepatitis, whereas all 3 of the patients with chronic liver failure had hepatitis B. Some of the patients received ranitidine, cefuroxime, vitamin K, and parentrovite (containing 500 mg ascorbic acid, 900 mg glucose, 160 mg nicotinamide, 50 mg pyridoxine, 4 mg riboflavine, and 250 mg thiamine HC1 per 10 mL) as routine therapy. Furthermore, 9 patients received noradrenaline or adrenaline for hypotension. The severity of HE in each of these patients was graded according to the general criteria previously described, lo Patient plasma samples from only one stage of HE were included in subsequent analyses. There was no significant difference between the groups in the numbers of men and women studied. The age of the patients in the acute liver failure group was significantly less than either the control or chronic liver failure groups (P < 0.01; analysis of variance). However, the ages of the patients in the control and chronic liver failure groups were not significantly different from each other. Not all patients were sampled for both brain and plasma.
Quantification of Quinolinic Acid Plasma samples ( 3 - 1 5 mL) were taken from patients at various times during their hospital care in the United Kingdom with the first sample taken within 24 hours after admission. Brain samples were taken an average of 20 _+ 2 hours after death according to previously described techniques. 11 The samples were stored at - 8 0 ° C for 3 - 4 months and then shipped on dry ice for analysis in the United States, where they were analyzed within 1 month.
Aliquots of the plasma samples (20 btL) were diluted with 1 mL of 1 mol/L HC1, whereas brain samples (50 mg) were homogenized by sonication with 1 mL of 1 mol/L HC1. Both preparations were then centrifuged at 12,000g for 20 minutes at 4°C, and aliquots of the supernatant (300 }.tL of plasma and 800 btL of brain) were retained. Quinolinic acid standards were dissolved in equivalent volumes of deionized water. [iSO]Quinolinic acid (2 ng) was added as an internal standard to each tube. All tissue extracts and corresponding quinolinic acid standards were washed with 3 mL of chloroform, and equal volumes of the aqueous layer were collected. The samples and standards were then freeze-dried overnight. Quinolinic acid and [lSO]quinolinic acid were derivatized to their dihexafluoroisopropanol esters with complete retention of the isotopes, washed with 250 [.tL of water, extracted into 3 0 0 - 1 0 0 0 btL of heptane, and then quantified using a Hewlett-Packard 5988 mass spectrometer (San Fernando, CA) operated in the electron capture negative chemical ionization mode using methane as the reagent gas. Aliquots of each sample were injected into a 1 m X 0.53 m m (inside diameter) fused silica precolumn (on-column injection at 80°C) that had been sealed to a 15 m X 0.25 m m (inner diameter) DB5 analytical column (J & W Scientific, Folsum, CA). Column temperature was 112°C, and helium was the carrier gas. The molecular anions of quinolinic acid (m/z 467) and the [180]quinolinic acid internal standard (mass/charge = 471) were monitored and each peak area quantified at the appropriate retention time. All quantification was performed at a signal/noise ratio of >--20:1. The minimum detectability was 300 fg at a signal/noise ratio of -----5:1.
Statistics Intergroup comparisons were made using analysis of variance followed by post hoc multiple comparison analysis with Fisher's least significant difference test (Systat, Evanston, IL). Correlations were determined using the Pearson correlation matrix with Bonferroni-adjusted probabilities.
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BASILE ET AL.
GASTROENTEROLOGY Vol. 108, No. 3
tients with chronic liver failure were not significantly increased above control levels until stages II and III of HE, where the maximum concentrations reached approximately 5 btmol/L (Figure 1 and Table 1). Overall, plasma samples from 13 of 23 patients with chronic liver failure had quinolinic acid levels within the range of normal values. Analyzed by themselves, a strong correlation was found between the plasma concentration of quinolinic acid and the stage of HE (Pearson correlation coefficient = 0.75; P < 0.01) in patients with chronic liver failure. When the plasma quinolinic acid concentrations from both patients with acute and chronic liver failure were pooled, there was a moderate but significant correlation between the stage of HE and the quinolinic acid concentration (Pearson correlation coefficient = 0.33; P
18-
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16 14
12lo-
8 6-
o
o A
0
O
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:
2
B
Ct:rl
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oo , , , t a ~
o
i Stage
O
~o
o
A A
0
AA
O
4
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CY
8
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HE
Figure 1. Distribution of plasma quinolinic acid levels in controls (F1)
and patients with acute (©) or chronic (A) liver failure. Patients were sampled only once at any stage of encephalopathy. Approximately 35% of the patients with acute liver failure and 57% of the patients with chronic liver failure had quinolinic acid levels that fell within the control range, regardless of the stage of HE.
=
0.01).
The quinolinic acid concentrations in samples of cerebral cortex, caudate nucleus, or cerebellum from patients who died from chronic liver failure in stage IV HE were not significantly different from controls (Figure 2 and Table 3). However, mean quinolinic acid concentrations in patients who died from acute liver failure were elevated by approximately 1 0 0 % - 4 0 0 % above control levels in the cerebral cortex, caudate nucleus, and cerebellum (F(8,795 = 3.736; P < 0.01, P < 0.05, and P < 0.05, respectively). There was no significant difference in quinolinic acid concentrations between the brain regions from patients with acute liver failure (P = 0.06, caudate nucleus vs. cortex; P = 0.19 caudate nucleus vs. cerebellum). Approximately 35% of the patients with acute liver failure had brain quinolinic acid concentrations that fell within normal levels.
Results
Approximately 35% of the patients with acute liver failure had plasma quinolinic acid levels within the control range (<1.2 btmol/L). Overall, patients with acute liver failure had mean plasma quinolinic acid concentrations 11.3 times greater than control by stage I HE (F(8,51) = 2.29; P < 0.05), which remained significantly elevated throughout stages I I - I V of HE (Figure 1 and Table 2). The mean plasma concentration of quinolinic acid in patients with acute liver failure was stable at approximately 5 btmol/L through stages I - I V . There was no significant difference between the plasma quinolinic acid concentrations in patients with acute hepatic failure at various stages of HE, and there was no correlation with the severity of HE (Pearson correlation coefficient = 0.25; P = 0.15). Contrasting with the observations in patients with acute liver failure, plasma quinolinic acid levels in pa-
Discussion
Increased brain and plasma tryptophan levels are a hallmark of liver failure2'3 and may enhance the synthesis of metabolites such as serotonin and quinolinic acid. Elevated concentrations of these metabolites may then alter neuronal activity and contribute to the manifesta-
Table 2. Ouinolinic Acid Levels in Plasma From Patients With Acute and Chronic Liver Failure Stage of HE Group Control Acute liver failure Chronic liver failure
0
I
II
III
IV
0.57 (1)
5.4 _+ 3.0 a (4) 1.4 + 0.3 c (18)
5.0 + 1.4 a (5) 4.7 (2)
4.6 + 2.2 b (4) 5.2 + 0.7 a (3)
5.1 + 1.6 a (12)
0.44 + 0.11 (10)
NOTE. All values are mean _+ SE in micromolar. Values in parentheses represent the number of observations. "P < 0.05; bp < 0.01; significantly different from control values, analysis of variance followed by Fisher's least significant difference multiple comparison test. cSignificantly different from stage I acute liver failure, P < 0.05, analysis of variance followed by Fisher's least significant difference multiple comparison test.
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QUINOLINIC ACID AND HEPATIC ENCEPHALOPATHY
Table 3. Quinolinic Acid Levels in Brain Regions From
16-
E o
Humans With Acute and Chronic Liver Failure
A
Brain region
6o
@
o
og
L~ c
-6 v
5-
4: 3: 2:
0
OoO
n°
o~o Q"D
r-
o
~oo~on ~
nBo°~On
Control
Chronic
°1
0 0
5o
4:
O v
0
2:
1:
0
O0 Control
0~0 Chronic
BB OoO Acute
c
o
0 0 o
0 l__
3o
o
C= 0
cv
2"
0
%%
(EY
moO0O Control
000
Chronic
Group
Cerebral cortex
Caudate nucleus
Cerebellum
Control Acute liver failure Chronic liver failure
0.52 _+ 0.25 (12)
0.65 _+ 0.50 (11)
0.63 _ 0.42 (6)
1.9 + 0.592 (13)
3.4 _+ 1.3 b (11)
2.3 _+ 0.65 b (9)
0.48 _+ 0.12 (13)
0.54 _+ 0.13 (13)
0.53 ± 0.14 (3)
NOTE. The data represent the mean _+ SE of quinolinic acid concentrations in nanomole per gram wet weight. Values in parentheses represent the number of observations. ap < 0.01, bp < 0.05; significantly different from control values, analysis of variance followed by Fisher's least significant difference multiple comparison test.
Acute
' TB
.2
1
821
0
Acute
Figure 2. Quinolinic acid levels in (A) samples of cerebral cortex, (B) caudate nucleus, and (C) cerebellum from controls and patients who died from either acute or chronic liver failure in stage IV HE. The mean quinolinic acid levels in patients with chronic liver failure were not significantly different from control levels in any brain region. Although quinolinic acid levels in approximately 35% of the patients who died of acute liver failure in stage IV HE were within the control range, the mean quinolinic acid levels were significantly higher than controls in all three brain regions•
tions of HE. Indeed, increased turnover of serotonin 11'12 as well as elevated concentrations of quinolinic acid 5 are observed in patients with liver failure. However, the relationship of either brain or plasma quinolinic acid concentrations to the severity of HE are not known. In patients with either acute or chronic liver failure, plasma quinolinic acid levels were significantly increased above control levels to an average maximum of ~ 5 btmol/ L. However, elevated plasma quinolinic acid levels were not a reliable feature of liver failure because approximately 3 0 % - 5 0 % of the patients at all stages of HE had quinolinic acid levels in the normal range. This factor and the relatively stable elevation of quinolinic acid in the plasma of patients with acute liver failure regardless of the stage of HE may explain why the correlation between the plasma concentration of quinolinic acid and the severity of encephalopathy was only moderate. More important are the differences between the brain levels ofquinolinic acid in patients with acute vs. chronic liver failure. No significant changes in quinolinic acid levels were observed in the cortex, caudate nucleus, or cerebellum of patients who died in stage IV HE caused by chronic liver failure despite evidence of elevated plasma quinolinic acid concentrations. This observation contrasts with that of a previous report 5 indicating that quinolinic acid levels in the frontal cortex of patients with chronic liver failure (caused by hepatic cirrhosis or metastatic liver cancer) were increased approximately threefold, implying that CNS synthesis of quinolinic acid is not significantly enhanced in patients with chronic liver failure despite the presence of elevated concentrations of its precursor, tryptophan) This strongly suggests that quinolinic acid has a limited role in the pathogenesis of HE. In contrast, quinolinic acid levels were significantly increased in the cortex, caudate nucleus, and cerebellum of patients with acute liver failure arising primarily from drug overdose. The level of quinolinic acid in these re-
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BASILE ET AL.
gions was lower than the plasma level and was evenly distributed throughout the brain regions sampled, providing further evidence that the quinolinic acid measured in the CNS may be of peripheral origin. The origin of the quinolinic acid in the CNS of patients with acute liver failure is unclear. Normal brain does not convert tryptophan to quinolinic acid, 13 and quinolinic acid does not penetrate a normal blood-brain barrier. 14'15 Whereas quinolinic acid can be synthesized from tryptophan by activated macrophages, 16'1v it is not known whether macrophages invade the CNS of patients with acute liver failure. Thus, the plasma is the most probable source of the quinolinic acid we measured in the brain, entering through a permeabilized blood-brain barrier. ~8'~9 The dependence of the entry of quinolinic acid into the CNS on the permeability of the blood-brain barrier may explain the differences in CNS quinolinic acid concentrations between patients with acute vs. chronic liver failure because increased blood-brain barrier permeability is believed to play a role in the pathogenesis of HE complicating acute liver failure,9'~8'~9 whereas its role in chronic liver failure is unclear. 9'2° Plasma quinolinic acid levels may increase as a result of enhanced quinolinic acid synthesis in the liver resulting from glucocorticoid stimulation of hepatocytes2~ or by cytokine activation of sinusoidal endothelial cells as a result of inflammatory liver disease processes associated with both acute and chronic liver failure. 22-25 Increases in plasma and brain quinolinic acid levels similar to those observed in patients with acute liver failure are also observed in the galactosamine-treated rabbit and thioacetamidetreated rat models of HE due to fulminant hepatic failure. 26 The elevation in quinolinic acid concentrations is accompanied by increased activity of hepatic tryptophan2,3-dioxygenase (the rate-limiting enzyme for the hepatic synthesis of quinolinic acid from L-tryptophan) but not brain indoleamine-2,3-dioxygenase (the rate-limiting enzyme for extrahepatic conversion of tryptophan to quinolinic acid), 21 supporting the proposal that the primary source of quinolinic acid in hepatic failure is the liver. Thus, the evidence implicating quinolinic acid in the pathogenesis of HE should be reassessed. Not only are the outward manifestations of HE one of depressed CNS function and neuronal activity, but the correlation between plasma quinolinic acid concentrations and the stage of HE is only moderate. Indeed, patients with chronic liver failure show no evidence of increased CNS quinolinic acid levels during an acute episode of HE. Furthermore, the CNS concentrations of quinolinic acid observed in patients with acute liver failure are ~20% of those observed in patients with infectious or inflam-
GASTROENTEROLOGY Vol. 108, No. 3
matory diseases 2v'28 and are probably insufficient to cause a permanent neuronal lesion. Nonetheless, quinolinic acid may play a differential role in CNS function in patients with acute rather than chronic liver failure, and that role may be to facilitate seizure development. While seizures are not considered a principal manifestation of HE per se, they occur more commonly in patients with acute or fulminant hepatic failure29 than in those with chronic hepatic failure. The lack of increase in CNS quinolinic acid concentrations, the possible development of "tolerance" to ammonia, 3° and the generalized increase in ]'-aminobutyricergic neurotransmission in patients with chronic liver failure7 would act to reduce the frequency of seizures in this population. Given the ability of quinolinic acid to cause neuronal hyperexcitability and seizures, 1 it is possible that the generalized increase in CNS quinolinic acid levels observed in patients with acute liver failure may predispose them to convulsions, a condition that would be exacerbated by the rapid increase in CNS ammonia levels 31'32 and cerebral edema33 observed in fulminant hepatic failure.
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Received June 21, 1994. Accepted October 17, 1994. Address requests for reprints to: Anthony S. Basile, Ph.D., Laboratory of Neuroscience, Building 8, Room 111, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. Fax: (301) 402-2872.