Original Article
Elevated Brain Concentrations of 1,4-Benzodiazepines in Fulminant Hepatic Failure
N Engl J Med 1991; 325:473-478August 15, 1991
- Abstract
Background.
Increased γ-aminobutyric acid (GABA) neurotransmission has been implicated in the pathogenesis of hepatic encephalopathy. The mechanism by which GABA-ergic activity is increased in hepatic failure is unclear, but recent studies in animals with encephalopathy due to fulminant hepatic failure suggest that GABA-ergic neurotransmission may be increased by the presence of elevated concentrations of benzodiazepine agonists such as diazepam and N-desmethyldiazepam.
Methods and Results.
Samples of frontal cortex were obtained at autopsy from 11 patients with hepatic encephalopathy who died of acetaminophen-induced fulminant hepatic failure and 8 patients who died of cardiovascular disease or trauma. None of the 19 patients had received benzodiazepines while hospitalized. Chromatographic analyses of extracts of these samples revealed 4 to 19 peaks representing substances that inhibited the binding of a Radio-labeled imidazobenzodiazepine ([3H]flumazenil) to its receptors. Several of these peaks had retention times corresponding to those of known 1,4-benzodiazepines. Ultraviolet- and mass-spectroscopic analysis confirmed that two of these peaks represented diazepam and N-desmethyldiazepam. The patients who died of fulminant hepatic failure could be divided into two groups: six who had had significantly elevated brain concentrations (2-fold to 10-fold higher than normal) of substances inhibiting the binding of [3H]flumazenil and five who had normal concentrations.
Conclusions.
Brain concentrations of substances inhibiting the binding of [3H]flumazenil to its receptors are increased in some patients with hepatic encephalopathy due to fulminant hepatic failure. The origin of these substances is unknown, but these findings provide a rational basis for trials of benzodiazepine-receptor antagonists in the management of this disorder. (N Engl J Med 1991; 325:473–8.)
Media in This Article
Figure 1
Representative Chromatograms of Extracts of Samples of Frontal Cortex from a Control Patient and a Patient with Hepatic Encephalopathy and the Elution Profile of the Benzodiazepine Standards.Figure 2
Ultraviolet and Mass Spectra of the Fractions with Retention Times of 50.5 to 51.5 Minutes (Top Panels) and 53.0 to 53.5Article Activity
- Article
Increased activity of the γ-aminobutyric acid [GABA] neurotransmitter (GABA-ergic) system may have a fundamental role in the pathogenesis of hepatic encephalopathy,1 , 2 a complex neuropsychiatrie disorder that complicates acute or chronic liver failure.3 Electrophysiologic,4 5 6 neurochemical,7 8 9 10 11 12 and behaviora15 , 6 , 13 evidence from several animal models of hepatic encephalopathy due to fulminant hepatic failure indicates that a major factor enhancing GABA-ergic tone is an elevation of the concentrations of substances that inhibit the binding of a Radio-labeled imidazobenzodiazepine ([3H]flumazenil) to its receptors (benzodiazepine-receptor ligands) that have agonist (i.e., diazepam-like) properties. The ability of benzodiazepines to augment GABA-ergic tone through a receptor-mediated mechanism is well established.14
Despite the presence of such compounds in animals with hepatic encephalopathy due to fulminant hepatic failure, the relevance of this mechanism to the pathogenesis of hepatic encephalopathy in humans is not known. In this study, we examined extracts of brain specimens obtained at autopsy from patients with hepatic encephalopathy due to acetaminophen-induced fulminant hepatic failure, and from control patients who died of cardiovascular disease or trauma, for the presence of benzodiazepine-receptor ligands. Diazepam and N-desmethyldiazepam were identified in these extracts by ultraviolet and mass spectroscopy. Significant elevations in the concentrations of these compounds and other unidentified substances with the pharmacologic and physicochemical properties of benzodiazepines were present in a subpopulation of the patients with hepatic encephalopathy. These findings provide a rational basis for the use of benzodiazepine-receptor antagonists in the management of hepatic encephalopathy due to fulminant hepatic failure.
Methods
The salient clinical characteristics of the 19 patients studied are shown in Table 1.Table 1
Clinical Data on Patients with Hepatic Encephalopathy Due to Acetaminophen-Induced Fulminant Hepatic Failure and Control Patients.* All 11 patients with acetaminophen-induced fulminant hepatic failure had grade IV hepatic encephalopathy (coma and loss of response to pain)15 at the time of their death. Histologic examination of the liver showed subtotal massive hepatocellular necrosis with areas of confluent zonal necrosis. All eight control patients died of nonhepatic diseases: five died of myocardial infarction (one with diabetes mellitus), one of dissecting aortic aneurysm, one of arrhythmia with cardiomyopathy, and one of chest injury. All died suddenly, with no preceding coma, and the gross appearance of their livers was normal. Careful review of the histories, clinical records, and drug charts of all 19 patients showed that none had received any benzodiazepines immediately before their last hospital admission or at any time during their stay (2 to 24 days).Measuring Levels of Benzodiazepine-Receptor Ligands
Since the neuropsychiatric manifestations of hepatic encephalopathy suggest impaired function of the frontal cortex,2 , 3 we studied samples of tissue from this area. Samples of frontal cortex anterior to the precentral gyrus were obtained at autopsy (approximately 5 g, 6 to 28 hours post mortem) and frozen at –25°C. Portions of the samples (0.1 to 2.5 g) were homogenized, digested in protease, and subjected to liquid-phase extraction according to a previously described modification7 , 9 of the techniques of Osselton16 and Foerster et al.17
The extracts were purified with a Novapak C18 reverse-phase cartridge in an RCM-100 radial-compression module (Waters/Millipore, Milford, Mass.). The column was eluted with a gradient of 90 percent 10 mM ammonium formate in 10 percent methanol at pH 4 (solution A), and 10 percent 10 mM ammonium formate in 90 percent methanol (solution B); the gradient was developed at a rate of 20 to 90 percent of solution B over a 70-minute period, at a flow rate of 1 ml per minute. Fractions were collected every 30 seconds. Absorbance was monitored at 229 nm and at the wavelength of maximal absorption with a diode array detector. The ultraviolet spectra of the unknown peaks were determined over the wavelengths of 210 to 400 nm. The column was washed with 100 percent methanol after each sample run and re-equilibrated with 60 ml of the starting mobile phase. All fractions were dried under vacuum at 45°C and subsequently stored at –20°C until assayed.
The levels of benzodiazepine-receptor ligands in the fractions collected from each Chromatographic run were quantitated in a radioreceptor assay using [3H]flumazenil (New England Nuclear, Boston).10 In fractions whose retention times corresponded to that of diazepam or N-desmethyldiazepam, and in which the presence of either diazepam or N-desmethyldiazepam was identified positively with mass-spectroscopic and ultraviolet-spectroscopic techniques, the quantity of diazepam or N-desmethyldiazepam was determined by extrapolation from standard curves for known concentrations of diazepam and N-desmethyldiazepam assayed under identical conditions. The recovery of these compounds was determined from the ratio of the known extraction efficiency of the particular benzodiazepine to the measured extraction efficiency of the internal standard, prazepam. The concentration of material determined by the radioreceptor assay was then corrected for this recovery. The mean (±SE) extraction efficiencies for 25 to 100 ng of diazepam, N-desmethyldiazepam, and prazepam from 1 to 2 g of normal rat brain were 78±12, 54±10, and 34±17 percent (four determinations), respectively.
The total benzodiazepine-receptor inhibitory activity was calculated by determining the inhibitory activity in each fraction, converting it to the equivalent amount of diazepam present, and then summing the amount in all the fractions. Because the chemical identity of all the components of total benzodiazepine-receptor inhibitory activity is unknown, the absolute quantity of substances inhibiting the binding of [3H]flumazenil to benzodiazepine receptors was not determined, since this would have required a correction based on the extraction efficiencies of the unknown compounds in relation to that of the internal standard.
Fractions with retention times similar to those of specific 1,4-benzodiazepine standards were combined for qualitative mass-spectroscopic analysis. N-Desmethyldiazepam-d 5 and diazepam-d 5 standards (70 ng) were added to the pooled fractions with retention times similar to those of N-desmethyldiazepam and diazepam standards, respectively. These samples were dried and sealed under nitrogen, then exposed at 80°C for one hour to 20 μl of 50 percent (t-butyldimethylsilyl)-trifluoroacetamide in acetonitrile. A 5-μl aliquot of the solution was then injected onto a DB-1 gas-chromatography column, 0.32 mm by 30 m, whose temperature was programmed to rise from 70°C to 290°C, first at a rate of 15°C per minute to 200°C and then at a rate of 10°C per minute to 290°C. The mass spectrometer (Finnigan 4500 GC-MS) was operated in chemical-ionization mode, with ammonia as the reagent gas. The compounds were identified and measured by integrating each of their protonated parent ions (both 35Cl and 37Cl) for 0.1 second, and the results were confirmed by comparing full-scan spectra (mass/negative charge [m/z—], 150 to 470) with standards.
Statistical Analysis
Initially, two-way analysis of variance was performed to assess the variance associated with concentrations of benzodiazepine-receptor ligands in the brain extracts from the two groups of patients. Then, the data were analyzed for homogeneity of variances with Cochran's C and the Bartlett—Box F tests. The results of these analyses indicated that the significance of the difference in mean benzodiazepine-receptor–ligand concentrations would be most appropriately analyzed with the nonparametric Mann-Whitney U test. A two-tailed Mann—Whitney U test was performed on the data as total diazepam equivalents. The results of this analysis indicated that subsequent tests of the differences between benzodiazepine levels could be performed with a one-tailed test. All data analyses were conducted with the SPSSX package (SPSS, Chicago).
Results
Analysis of the fractions collected during reverse-phase high-performance liquid chromatography of brain extracts from the patients with hepatic encephalopathy due to fulminant hepatic failure revealed the presence of 4 to 19 peaks containing substances that inhibited the binding of [3H]flumazenil (Fig. 1Figure 1
Representative Chromatograms of Extracts of Samples of Frontal Cortex from a Control Patient and a Patient with Hepatic Encephalopathy and the Elution Profile of the Benzodiazepine Standards.). Several of these fractions contained activity that had retention times (mean ±SE, 44.9±0.2, 46.7±0.2, 52.0±0.3, and 54.8±0.2 minutes; 2 to 11 determinations) corresponding to those of the standards deschlorodiazepam (44.8±0.2 minutes; 10 determinations), oxazepam (46.2±0.1 minutes; 10 determinations), N-desmethyldiazepam (52.2±0.1 minutes; 10 determinations), and diazepam (54.7±0.1 minutes; 10 determinations), respectively (Fig. 1). The prazepam internal standard was eluted with a retention time of 63.9±0.1 minutes. When 100 ng each of N-desmethyldiazepam, diazepam, and oxazepam were added to the extracts, these compounds comigrated with the unknown fractions as single peaks on high-performance liquid chromatography (determined by monitoring the absorption of ultraviolet radiation at 229 nm; data not shown).The ultraviolet-absorption spectra (scanned from 210 to 400 nm) of peaks with retention times of 52.0 and 54.8 minutes closely corresponded to the spectra of N-desmethyldiazepam and diazepam, respectively (Fig. 2Figure 2
Ultraviolet and Mass Spectra of the Fractions with Retention Times of 50.5 to 51.5 Minutes (Top Panels) and 53.0 to 53.5). The maximal absorption values in the unknown fractions were found at 229.2 to 230.4 nm and 316.8 to 318.0 nm, with an inflection point at 252.0 to 255.6 nm - findings characteristic of the absorption spectra of C-ring—substituted 1,4-benzodiazepines. The fractions associated with the peaks that had retention times of 52.0 and 54.8 minutes were collected, pooled, dried and derivatized, and analyzed by gas chromatography—mass spectroscopy. Mass spectroscopy of the peak with a retention time of 52 minutes revealed a cluster of four peaks in which the largest mass was at 343 m/z—. Of the four mass peaks, the height of the third peak was 47 percent of that of the primary peak, a pattern characteristic of chlorine. This cluster is consistent with that of the trimethylsilyl-N-desmethyldiazepam derivative. Furthermore, the parent ion cluster (N-desmethyldiazepam) was found at 271 m/z—. Mass-spectroscopic evidence of the presence of N-desmethyldiazepam was found in the brain extracts from seven patients with hepatic encephalopathy and in extracts from two control patients. Mass-spectroscopic analysis of the peak with a retention time of 54.8 minutes revealed a cluster at 285 m/z—. No trimethylsilyl derivative was found. This spectrum is consistent with that of diazepam, which was found in the brain extracts from all 11 patients with hepatic encephalopathy (>1 ng of diazepam per gram of tissue [wet weight], 8 patients) and 7 of the 8 control patients (>1 ng per gram, 4 patients).The concentration of total benzodiazepine-receptor ligands in the brain samples from the patients with hepatic encephalopathy was more than five times higher (P = 0.016 by Mann-Whitney U test) than that in the samples from the control patients (Table 2Table 2
Benzodiazepine Concentrations (Mean ±SE) in Extracts of Frontal Cortex from Patients with Hepatic Encephalopathy and Control Patients.* and Fig. 3Figure 3
Distribution of Levels of Total Benzodiazepine-Receptor–Ligand Activity, N-Desmethyldiazepam Concentrations, and Diazepam Concentrations among 8 Control Patients (Open Triangles) and 11 Patients with Hepatic Encephalopathy (Solid Triangles).), as determined by quantitative radiometric analysis. The mean concentrations of N-desmethyldiazepam and diazepam in the brain extracts from the patients with hepatic encephalopathy were 10 times and twice as great (P = 0.013 and 0.042, respectively) as those from the control patients (Table 2 and Fig. 3).The variance in the concentrations of benzodiazepine-receptor ligands in the patients with hepatic encephalopathy was large. Cochran's C and the Bartlett—Box F tests of variance homogeneity demonstrated that the group with hepatic encephalopathy was not homogeneous with respect to either the total concentration of benzodiazepine-receptor–ligand activity or the N-desmethyldiazepam concentration (P<0.001). The total levels of benzodiazepine-receptor ligands were below the lower limit of the 95 percent confidence interval for the group mean (confidence interval, 206 to 604 ng per gram of tissue [wet weight]) in 5 of these 11 patients, as were the levels of N-desmethyldiazepam, in 4 patients (confidence interval, 22 to 252 ng per gram). The levels of benzodiazepine-receptor ligands in this subgroup did not differ significantly from those in the control patients. No association was found between the levels of N-desmethyldiazepam, diazepam, or total benzodiazepine and the duration of coma, the presence of cerebral edema, dialysis treatment, the longest prothrombin time, or the prothrombin time at death.
Discussion
Converging lines of evidence indicate that increased GABA-ergic neurotransmitter activity contributes to the global depression of central nervous system activity found both in animal models of hepatic encephalopathy and in humans.2 , 15 This study indicates that the increase in GABA-ergic tone in hepatic encephalopathy may be mediated by elevated concentrations of benzodiazepines and related substances. The presence of diazepam and N-desmethyldiazepam in the frontal cortex of patients with hepatic encephalopathy was confirmed by mass and ultraviolet spectroscopy. The concentrations of these compounds, as well as of other unidentified benzodiazepine-receptor ligands, were significantly higher in the samples from patients with hepatic encephalopathy than in those from control patients. These results are consistent with previous reports of elevated concentrations of unidentified substances inhibiting the binding of benzodiazepine to its receptors in the plasma and cerebrospinal fluid of patients with hepatic encephalopathy who had been carefully evaluated to ensure that they had not taken any exogenous benzodiazepines.18 , 19
The average total concentration of benzodiazepine-receptor ligands in the brains of the patients with hepatic encephalopathy was in the range that would be expected after the administration of low (anxiolytic) doses of diazepam.20 , 21 Although the concentrations appeared to be lower than those required to cause overt encephalopathy, the action of these agents may be enhanced by other changes accompanying fulminant hepatic failure, such as increased concentrations of GABA in the central nervous system,22 altered glutamate metabolism,23 or both. Furthermore, the concentrations of benzodiazepine-receptor ligands vary from one discrete brain region to another in animals with hepatic encephalopathy.8 Finally, the ability of benzodiazepine-receptor antagonists to decrease the clinical and electrophysiologic manifestations of hepatic encephalopathy in a majority of patients24 25 26 27 suggests that the concentrations of benzodiazepine-receptor ligands are sufficiently elevated to play a part in the neuropsychiatric manifestations of this syndrome.
The results in the patients with encephalopathy showed that they fell into two groups. Fifty-five percent had elevated concentrations of benzodiazepine-receptor ligands, whereas the remaining 45 percent did not. These results indicate that other mechanisms may be involved in the evolution of hepatic encephalopathy in the latter group, while providing an explanation for the finding that encephalopathy in approximately 75 percent of patients with fulminant hepatic failure is ameliorated by the administration of benzodiazepine-receptor antagonists such as flumazenil.24 25 26 27
The origin of the benzodiazepines in the frontal cortex of these patients is unknown. These substances could have pharmaceutical or environmental sources, since the levels of these compounds in half the control patients were higher than the levels in human brains preserved before the availability of 1,4-benzodiazepine drugs.28 , 29 Although we found no evidence that any of the patients we studied had received benzodiazepines, the possibility of surreptitious self-administration of these drugs cannot be ruled out. However, the pattern of metabolites detected in these samples (the variability of the presence of N-desmethyldiazepam, coupled with the absence of oxazepam) is inconsistent with the exogenous administration of benzodiazepines. The lack of compelling evidence that halogenated 1,4-benzodiazepines can be synthesized by mammalian tissues, as well as the association of the administration of antibiotics and lactulose30 with amelioration of hepatic encephalopathy in patients with chronic liver disease, suggests that the most likely sources of the benzodiazepines detected in the brain in this study were the patients' diet,31 32 33 34 enteric flora,35 or both.
Although it is uncertain whether elevated concentrations of benzodiazepine-receptor ligands are involved in the pathogenesis of hepatic encephalopathy due to causes other than acute drug overdose (e.g., chronic liver failure or viral hepatitis), our results suggest that enhanced GABA-ergic neurotransmission, mediated by such benzodiazepine-receptor ligands, has an important role in the pathogenesis of hepatic encephalopathy in a subpopulation of patients with fulminant hepatic failure. The results also provide a rational basis for trials of benzodiazepine-receptor antagonists (such as flumazenil) in the management of hepatic encephalopathy.
We are indebted to Dr. M. Kato for his assistance in collecting the brain samples.
Source Information
From the Laboratory of Neuroscience (A.S.B., P.S.), the Laboratory of Bioorganic Chemistry (Y.M., L.P.), and the Liver Diseases Section (E.A.J.), Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Md., and the Liver Unit (R.D.H., P.M.H., R.W.), King's College School of Medicine and Dentistry, London. Address reprint requests to Dr. Basile at the Laboratory of Neuroscience, Bldg. 8, Rm. 111, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
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