Maria Luisa Zeneroli

Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy

Abstract: The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti‐benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti‐benzodiazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained ∼ 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased ∼ 10‐fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.

Globus pallidus alterations and brain atrophy in liver cirrhosis patients with encephalopathy: An MR imaging study

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.

Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma

Increased number of peripheral benzodiazepine receptors (PBRs) have been found in some tumors outside the liver. The present study was to verify whether the PBR system is altered in hepatocellular carcinoma (HCC). The levels of endogenous benzodiazepine-like compounds (BZDs), measured by radioreceptor binding technique after HPLC purification and the endogenous ligand for PBRs, termed diazepam binding inhibitor (DBI), measured by radioimmunoassay utilizing a specific antibody for human DBI, were studied in the blood of 15 normal subjects, 12 liver cirrhosis and 10 patients with HCC. The levels of BZDs in serum were increased hundred fold in liver cirrhosis patients and slightly elevated in HCC patients. DBI was found to be increased in HCC patients. The binding recognition sites for PBRs (Bmax) were increased 4 to 7 fold in HCC tissue in comparison with that found in non-tumoral liver tissue (NTLT). On the contrary the concentrations of DBI were found to be significantly decreased in HCC tissue in comparison with the respective NTLT. These results seem to suggest an implication of PBRs and of their putative endogenous ligands in the metabolism of these neoplastic cells and possibly in their proliferation. The up-regulation of PBRs found in HCC tissue seems to indicate an increased functional activity of these receptors and opens up the possibility of new pharmacological and diagnostic approaches while the changes in the circulating endogenous ligands for the above receptors might be envisaged as early markers of tumorigenesis in liver cirrhosis.

INCREASED EXPRESSION OF PERIPHERAL BENZODIAZEPINE RECEPTORS AND DIAZEPAM BINDING INHIBITOR IN HUMAN TUMORS SITED IN THE LIVER

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.

Endogenous benzodiazepine-like compounds and diazepam binding inhibitor in serum of patients with liver cirrhosis with and without overt encephalopathy

Background/Aim—Despite some controversy, it has been suggested that endogenous benzodiazepine plays a role in the pathogenesis of hepatic encephalopathy. The aim of the present study was to evaluate the concentrations of endogenous benzodiazepines and the peptide, diazepam binding inhibitor, in the blood of patients with liver cirrhosis with and without overt encephalopathy, and to compare these levels with those of consumers of commercial benzodiazepines. Subjects—Normal subjects (90), benzodiazepine consumers (14), and cirrhotic patients (113) were studied. Methods—Endogenous benzodiazepines were measured by the radioligand binding technique after high performance liquid chromatography (HPLC) purification. The presence of diazepam andN-desmethyldiazepam was assayed by HPLC-electrospray tandem mass spectrometry. Diazepam binding inhibitor was studied in serum by radioimmunoassay. Results—Endogenous benzodiazepines were below the limit of detection in 7% of patients with encephalopathy. When detectable, their levels were at least comparable with those of benzodiazepine consumers and correlated with the liver dysfunction but not the stage of encephalopathy. Serum levels of diazepam binding inhibitor tended to decrease when endogenous benzodiazepines levels increased. Conclusions—Endogenous benzodiazepines may accumulate in patients with liver cirrhosis during the course of the disease, and the phenomenon appears to be independent of the presence or absence of encephalopathy.

Prevalence of brain atrophy in liver cirrhosis patients with chronic persistent encephalopathy: Evaluation by computed tomography*

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 Childs 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.

Hyperhomocysteinaemia in chronic liver diseases: role of disease stage, vitamin status and methylenetetrahydrofolate reductase genetics

Abstract: Background/Aims: The liver plays a key role in sulphur aminoacid metabolism hence, homocysteine metabolism may be impaired in chronic liver diseases. The aim of this study was to investigate, in patients affected by chronic liver diseases, (1) the prevalence of hyperhomocysteinaemia and (2) the role of its determinants such as the stage and the aetiology of disease, vitamin status, genetic documented alterations (methylenetetrahydrofolate reductase deficiency) and presence/absence of documented malignant evolution (hepatocellular carcinoma).

Serum Ferritin as a Predictor of Treatment Outcome in Patients With Chronic Hepatitis C

OBJECTIVES:Antiviral treatment in chronic hepatitis C (CHC) involves ribavirin, a hemolytic agent. We planned a prospective study to evaluate whether drug-induced iron perturbation is clinically relevant as it relates to therapeutic outcome.METHODS:Iron variables were sequentially assessed in 206 CHC patients undergoing antiviral therapy and were correlated with pretreatment iron status and histology, hemolysis, and therapeutic outcome.RESULTS:At week 1 of therapy, serum iron (SI), transferrin saturation (TS), and serum ferritin (SF) increased markedly in all patients. All iron parameters correlated with hemolysis up to week 4; this correlation was lost for SF at later time points. SF rise during treatment was inversely related to baseline SF and iron deposits in hepatic mesenchymal/Kupffer cells. Both baseline SF and mesenchymal iron significantly correlated with fibrosis at multivariate analysis (P=0.015 and 0.008, respectively). Interestingly, baseline SF, despite good specificity (89%), had low sensitivity in predicting siderosis (25%). During therapy, SI, TS, and hemolysis parameters did not correlate with sustained virological response (SVR), whereas SF rise became an independent predictor of therapeutic response: a 2.5-fold increase of SF at week 12 associated with higher likelihood of SVR (odds ratio 1.91, P=0.032). Accordingly, lack of mesenchymal iron deposits at the baseline biopsy correlated with SVR (odds ratio 3.02, P=0.043).CONCLUSIONS:In CHC, SF is a useful marker for assessing disease duration and progression before starting treatment and for predicting therapeutic response while on therapy. SF rise during antiviral therapy is largely independent of hemolysis and likely indicates activation of macrophages in response to antivirals.

Management of hepatic encephalopathy: Role of rifaximin

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, which develops in patients with acute or chronic liver failure. It is widely accepted to be due to impairment of hepatic clearance of toxic products from the gut such as ammonia. Accumulation of ammonia induces a glutamate neurotoxicity leading to an increased tone of the γ-aminobutyric acid A (GABA-A) receptor system in the brain which results in HE. Factors either increasing the ammonia levels (protein load, constipation, sepsis, or gastrointestinal bleeding) or potentiating the functional activity of the GABAergic system [natural benzodiazepine-like compounds (NBZDs) or exogenous benzodiazepines] may act as precipitating factors of HE. NBZDs are present in trace amounts in the blood of normal subjects and have been found to be increased in the blood of patients with liver cirrhosis, with or without HE. These compounds may derive either from the diet since they have been found in plants, vegetables and animals or from gut bacteria. The observation that intestinal bacterial flora is involved in the production of both primary agent of HE (ammonia) and precipitating factors (NBZDs) suggests that the use of nonabsorbable antibiotics such as rifaximin may be useful in preventing episodes of HE in patients with liver cirrhosis.

Alterations of GABA-A and dopamine D-2 brain receptors in dogs with portal-systemic encephalopathy

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.