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Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.

Brain magnetic resonance (MR) was performed in 29 liver cirrhosis patients without (N = 10) and with hepatic encephalopathy (HE) of chronic recurrent (N = 10) and of chronic persistent (N = 9) type. Sixty percent of the patients with chronic recurrent HE and 100% of the patients with chronic persistent HE showed a bilateral and symmetrical hyperintensity of the globus pallidus in the T1-weighted images while the T2-weighted images were normal, suggesting the possibility of the accumulation of a paramagnetic compound in this brain area during HE. Other findings of the study were evidence of brain atrophy of mild or moderate degree in 70% of patients with chronic recurrent HE and in 77% with chronic persistent HE and patients with liver cirrhosis without HE appeared normal on MR examination.

The binding characteristics of gamma-aminobutyric acid-A (GABA-A) receptors and the kinetic characteristics of the target enzyme of GABA synthesis in nerve terminals, glutamic acid decarboxylase (GAD), were studied in a dog model of portal-systemic encephalopathy obtained by porta-caval shunt performed in dimethylnitrosamine pretreated animals. Furthermore the properties of dopamine receptors and the levels of catecholamines of encephalopathic dogs were investigated. The mild stage of encephalopathy was characterized by an up-regulation of the inhibitory GABA-A receptors probably related to a decrese of GABA in nerve terminals since GAD was decreased and by a slight decrease of catecholamines and by an increased synthesis of octopamine associated with a decreased affinity of dopamine receptors. In the severe stage there was a selection of high affinity GABA-A receptors with an increased number of benzodiazepine recognition sites which were supersensitive to GABA stimulation, a decreased number of Dopamine D-2 receptors and a marked reduction of catecholamines. These data seem to suggest that the neurological disturbances of experimental portal-systemic encephalopathy might be the result of an imbalance between inhibitory and excitatory systems leading to a prevalence of the first one.

Since zinc is an important factor in membrane stability, we assayed the levels of zinc in several brain areas during the development of hepatic encephalopathy due to D-galactosamine-HCl in rat. We now report that zinc is significantly reduced in all tested brain areas. This finding seems to indicate an involvement of zinc in the changes of membrane properties that lead to alteration of GABA receptors in hepatic encephalopathy.

Brain computerized axial tomography scans were performed in 18 consecutive liver cirrhosis patients with chronic persistent encephalopathy (8 alcoholic and 10 nonalcoholic) in order to evaluate the incidence of brain atrophy in this pathological condition. Fifteen patients of similar age with liver cirrhosis of Child's class B but with acute episodic hepatic encephalopathy were studied in parallel. Brain atrophy was detected in 87.5% of alcoholic and in 50% of nonalcoholic liver cirrhosis patients with chronic persistent encephalopathy, whereas the patients with acute episodic encephalopathy were normal. The high frequency of brain atrophy in alcoholic patients with chronic encephalopathy can be attributed at least in part to the toxic effect of alcohol. The presence of brain atrophy in nonalcoholic liver cirrhosis patients with chronic persistent encephalopathy seems to indicate that the chronic exposure to toxins which are involved in the pathogenesis of hepatic encephalopathy leads to neuronal alterations which develop mainly in the cortex and which can be demonstrated by in vivo imaging only in a long-lasting state of coma. Moreover, alcohol and toxins causing hepatic encephalopathy seem to potentiate each other in inducing brain atrophy.

Clinical observation has indicated a supersensitivity to morphine in patients with hepatic encephalopathy. With the aim of clarifying the issue, radioreceptor binding studies of opiate receptors were performed in frontal cortex and hypothalamus of 6 dogs with mild portal-systemic encephalopathy induced by chronic treatment with dimethylnitrosamine followed by porto-caval shunt end-to-side. beta-Endorphin assays were performed in the same areas with radioimmunoassay. Opiate receptors labeled with [3H]naloxone in both areas showed a significant increase in the receptor densities (Bmax) without changes in the dissociation constant (KD). In parallel beta-endorphin levels showed a decline during the development of encephalopathy in both areas. The increased densities of opiate receptors in the mild stage of encephalopathy may explain the supersensitivity to morphine in patients with liver diseases.

The effects of the acute administration of 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one on diazepam-induced behaviour and electrophysiological activity were studied in rat. The compound, in doses of 5-10 mg/kg (i.p.), which per se did not induce alterations in spontaneous locomotor activity, antagonised the sedative effect induced by 5-10 mg/kg (i.p.) of diazepam. The injection of diazepam in rats, induced a profound reduction in the first negative wave of the recording of the visual evoked potential used as a sensitive electrophysiological test, in vivo. 2-Phenylpyrazolo[4,3-c]quinolin-3(5H)-one (10 mg/kg, i.p.) caused a recovery of the amplitude of the first negative wave within a few minutes. This result was confirmed by the finding that 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one, injected acutely in rats, pretreated with diazepam exhibited the capacity to antagonise the binding of [3H]diazepam determined in vitro on synaptic membrane preparations from cortex. The comparison of the pattern of the visual-evoked potential, recorded after the injection of 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one (50 mg/kg) with the patterns recorded after the injection of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazolo(1,5a) (1,4)benzodiazepine-3-carboxylate (50 mg/kg) and ethyl-beta-carboline-3-carboxylate and 1-methyl-beta-carboline demonstrated that 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one is devoid of intrinsic activity.

1. In the search to identify peripheral toxins which could be responsible for the supersensitivity of brain benzodiazepine receptors in experimental models of hepatic encephalopathy, [3H]diazepam-binding studies have been performed on brain tissues of normal rats treated with ammonium chloride, dimethyldisulphide and octanoic acid administered alone or in combination. 2. The subacute administration of the three toxins in combination induced a 30% increase in the number of benzodiazepine recognition sites. The administration of these toxins alone or in combination showed that this increase was mainly linked to the synergistic action of dimethyldisulphide or octanoic acid with ammonia, since dimethyldisulphide plus octanoic acid was ineffective. 3. These observations seem to reinforce the suggestion that these three toxins are able to induce neurochemical derangements similar to those described in experimental hepatic encephalopathy.

Kinetic studies of [3H]gamma-aminobutyric acid ([3H]GABA) after an intravenous injection were performed in normal rats and in rats with severe degree of hepatic encephalopathy due to fulminant hepatic failure induced by galactosamine. Moreover, plasma and brain GABA levels, and GABA and glutamic acid decarboxylase activity were studied in some brain areas. After intravenous injection, [3H]GABA disappeared very rapidly in the blood of normal rats, with a prompt increase of 3H metabolites. In comatose rats, a delayed disappearance of [3H]GABA was parallelled by a lower amount of metabolites, indirectly indicating a peripheral decrease of GABA-transaminase activity. The amount of [3H]GABA in brain was lightly but constantly lower in comatose rats than in controls, indicating that the change in permeability of the blood-brain barrier in hepatic encephalopathy does not affect the [3H]GABA uptake of the brain. Furthermore, the assay of endogenous GABA in blood, whole brain, and brain areas did not show any significant difference in any of the two groups. The finding that glutamic acid decarboxylase activity in brain was reduced, together with the indirect evidence of a reduction in GABA-transaminase, may account for the steady state of GABA in hepatic encephalopathy. However, the reduction in glutamic acid decarboxylase activity is in favor of a functional derangement at the GABA-ergic nerve terminals in this pathological condition.