Natural endogenous ligands for benzodiazepine receptors in hepatic encephalopathy.

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.

Tumour markers in internal medicine: a low-cost test or an unnecessary expense? A retrospective study based on appropriateness.

OBJECTIVE AND BACKGROUND: In the last 35 years tumour markers (TM) have gained currency in clinical practice. However, in the light of indications by international guidelines, their use is often unjustified. Our aim was to quantify the use of some of the most common TM, assessing their appropriateness and their efficacy in an Internal Medicine Unit. METHODS: In the three Internal Medicine Units of the Department of Internal Medicine of Policlinico of Modena we have carried out a retrospective analysis of the assessment of the main TM (CEA, CA19.9, CA 125, CA 15.3, NSE). The analysis was divided into two distinct phases: (I) quantitative phase, in order to assess the scale of the problem in economical terms; (II) qualitative phase, in order to assess the efficacy of the tests and the appropriateness of their use. RESULTS: (I) At last one of the considered TM was requested in 5102 out of the 8253 admitted patients (62%) (period 2001-2003). The trend was similar in all three units examined. (II) The qualitative analyses revealed: (1) the most common motivation for their use (79%) was diagnostic, mostly prior to any other test; (2) a mere 5% of the requests were appropriate according to the international literature; and (3) TM showed a low positive predictive value when used for diagnosis in an unselected population such as that of an Internal Medicine unit. CONCLUSIONS: The results of our study showed that TM determination represents an overall cost for Internal Medicine units and that there is a high inappropriateness in their use compared to what it is suggested by international guidelines. Though the TM is a low-cost test when used correctly, it seems an unnecessary expense if not adequately incorporated into the decision making process.

Evaluation of rifaximin, placebo and lactulose in reducing the levels of benzodiazepine-like compounds in patients with liver cirrhosis: a pilot study.

Benzodiazepine-like compounds (BZDs), either taken with the diet or synthesized by intestinal bacterial flora, may represent a precipitating factor for hepatic encephalopathy (HE) in cirrhotic patients. We evaluated whether a diet and/or treatment with rifaximin or lactulose can reduce serum concentrations of BZDs in 18 cirrhotic patients without HE. Patients were given a standard diet for 7 days to keep the dietary intake of BZDs constant and were then randomized to a 7-day treatment with rifaximin 1,200 mg/day, lactulose 10-20 g three times daily, or placebo. Blood samples were collected at enrollment, at the end of the diet and drug treatment periods, and 7 days after the drug was stopped (follow-up). Serum concentrations of BZDs were measured by a radioligand binding technique after high-performance liquid chromatography extraction and purification and were expressed as diazepam equivalents (DE). No change in serum BZD concentrations was observed during the diet, while a statistically significant decrease from 105.6 +/- 66.5 to 63.5 +/- 49.5 pmol DE/ml was achieved in rifaximin-treated patients (p < 0.05) but not in patients treated with lactulose or placebo. During the followup, serum BZD concentrations returned to 104.5 +/- 74.0 pmol DE/ml in rifaximin-treated patients (p < 0.05 vs. end-treatment values), while no significant change was observed in the lactulose- and placebo-treated patients. These data indicate that control of bacterial flora with cyclic administration of rifaximin plays a pivotal role in avoiding increased plasma concentrations of BZDs, which represent a precipitating factor for HE inpatients with severe liver disease.

Ammonia and endogenous benzodiazepine-like compounds in the pathogenesis of hepatic encephalopathy.

BACKGROUND: Ammonia and endogenous benzodiazepines (BDZs) are two of the most important agents among those taken into consideration in the pathogenesis of hepatic encephalopathy (HE). METHODS: Venous ammonia and endogenous BDZs sera levels were assayed in 58 liver cirrhosis patients (34 male, 24 female) free of commercial BDZs. Endogenous BDZs were measured by binding assay after high-performance liquid chromatography purification. Ammonia was assessed by colorimetric test. RESULTS: Endogenous BDZs and ammonia were significantly higher in Child-Pugh class C than in class B and class A (P < 0.05), correlating to the severity of the liver dysfunction but not with the degree of HE. A significant difference, in fact, was noted between degree 0 (no HE) versus III-IV of HE (P < 0.05), but not between degrees I-II versus III-IV. Regression analysis performed to find a correlation between the ammonia and BDZ levels in HE resulted negative. CONCLUSION: Clinical evidence is provided in cirrhotic patients that ammonia and endogenous BDZ levels do not correlate with each other in the outcome of HE.

Endogenous benzodiazepines.

The existence of endogenous benzodiazepines such as diazepam and nordiazepam has been provided in human blood and brains as well as in medicinal plants and foods. It must be stressed, however, that in plasma and brain tissue there are also other benzodiazepine-like compounds termed 'endozepines' which are not halogenated. A synthetic pathway for the production of benzodiazepine-like compounds and endozepines has not yet been found, hence it may be surmised that these compounds could be of exogenous source. Changes in the level of endogenous circulating benzodiazepines due to food or drug ingestion could be responsible for pathological conditions. Clinical experiments were designed in order to study the levels of the endogenous benzodiazepines in vegetables and in the blood of control subjects and of cirrhotic patients. These patients accumulate benzodiazepines because of decreased liver metabolization capacity and impaired renal secretion, reaching plasma concentrations similar to those recorded in commercial benzodiazepine consumers.

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.

May plasma cholesterol level be considered a neoplastic marker in liver disease from cirrhosis to hepatocellular carcinoma?

BACKGROUND: Although the role of cholesterol in tumourigenesis is unclear, it is used by the tumoural cells for biosynthetic processes and for steroid synthesis. AIM: To accertain whether plasma cholesterol levels might be a reliable neoplastic marker of a developing hepatocellular carcinoma in patients with liver cirrhosis. PATIENTS: Plasma cholesterol has been studied in 287 liver cirrhosis patients without hepatocellular carcinoma and in 132 patients with hepatocellular carcinoma. RESULTS: Cholesterol (mean +/- SEM) was higher in hepatocellular carcinoma patients when compared with age-, sex- and Child-Pugh class matched cirrhotic controls. In Child-Pugh class A, B and C with uncomplicated liver cirrhosis these values were, respectively, 142.0 +/- 2.5, 117.3 +/- 2.5, 97.4 +/- 2.9 vs 172.5 +/- 4.7, 163.8 +/- 7.9, 153.5 +/- 8.0 +/- mg/dl in patients with hepatocellular carcinoma (p < 0.001). A significant increase of cholesterol (p < 0.001) has been reported in the patients with liver cirrhosis when complicated by hepatocellular carcinoma and it was not related to cholestasis. CONCLUSIONS: This observation seems to suggest that the enhanced cholesterol biosynthesis by tumoural cells leads to a rise in plasma cholesterol of patients with cancer, and, moreover, that, this increase may be used as a neoplastic marker indicating the development of a tumour in patients with liver cirrhosis.

Tryptophan metabolism and hepatic encephalopathy. Studies on the sedative properties of oxindole.

Oxindole administration (1-100 mg/kg i.p.) to mammals decreases locomotor activity, reduces muscular tone and blood pressure and at larger doses causes coma and death. Utilizing both HPLC and GC/MS, we showed that oxindole is present in the blood, brain and other organs of several animal species, including humans. We demonstrated that oxindole is a tryptophan metabolite able to significantly decrease neuronal excitability by modifying the function of voltage-operated sodium channels. Its synthesis requires the availability of indole, which is formed in the gut. When liver function is impaired, a sufficient amount of indole reaches systemic circulation and is oxidized into oxindole, which seems to be one of the responsible agents for the neurological symptoms found in the course of liver impairment.

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.

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 and N-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.

Diazepam binding inhibitor and total cholesterol plasma levels in cirrhosis and hepatocellular carcinoma.

Cholesterol is used by cells for biosynthetic processes and for steroid synthesis. Although the role of cholesterol in tumorigenesis is not clear it is known that steroids are important factors in human carcinogenesis. A polypeptide, diazepam binding inhibitor (DBI), which is an endogenous ligand for peripheral benzodiazepine receptors enhances steroidigenesis by promoting cholesterol delivery to the inner mitochondrial membrane which represents the rate-limiting step of steroid biosynthesis. We have assayed the total cholesterol (TC) and the DBI plasma concentrations in patients with liver cirrhosis complicated by hepatocellular carcinoma (HCC) in comparison with those of uncomplicated liver cirrhosis. TC and DBI levels have been studied in 73 cirrhotic patients and in 23 patients with HCC. Both TC and DBI levels were higher in HCC patients when compared with age, sex and Child-Pugh class matched cirrhotic controls. The values (mean+/-S.D.) in patients in Child-Pugh class B and C with and without HCC were respectively 128+/-30 mg/dl vs. 106+/-27 mg/dl (P < 0.01) and 2.05+/-0.78 pmol/ml vs. 0.78+/-0.84 pmol/ml (P < 0.0001). The data may be the result of the metabolic influence of tumors that enhances steroid biosynthesis during tumor proliferation.

Benzodiazepine-like compounds in the plasma of patients with fulminant hepatic failure.

BACKGROUND: Benzodiazepine-like compounds have been implicated in the pathogenesis of encephalopathy after fulminant hepatic failure. METHODS: The levels and the nature of benzodiazepine-like compounds were determined in six cases of fulminant hepatic failure during the course of the disease. Blood samples were collected on admission and a few days later, when the neurologic status had improved in five cases and immediately before death in one case. The compounds were measured in sera with a binding technique after high-performance liquid chromatography purification and analyzed with mass spectrometry. RESULTS: Their levels were highly variable in those with severe encephalopathy and were still increased on awakening in some cases. Diazepam and N-desmethyldiazepam were inconsistently present. CONCLUSIONS: The inconsistent presence of benzodiazepine-like compounds in encephalopathy after fulminant hepatic failure and their persistence, in some cases, at high levels on awakening from coma seem to indicate that the encephalopathy is not strictly dependent on the levels of these compounds.

Mesenteric vein thrombosis: a rare cause of abdominal pain in cirrhotic patients--two case reports.

Mesenteric vein thrombosis is a rare disorder which can develop rapidly with intestinal infarction or subacutely with abdominal pain due to intestinal ischemia. Despite the availability of modern diagnostic tools, which allow an early diagnosis in most cases, the mortality from this disease has not significantly diminished over the years. The problem is that the syndrome is rare and unusual and the clinical presentation is usually vague or confusing. Particularly in cirrhotic patients, this diagnosis requires the exclusion of several other complications of liver disease, like spontaneous bacterial peritonitis, tense ascites or portal thrombosis. Here, we report the occurrence of acute mesenteric vein thrombosis in two patients with liver cirrhosis. Severe subcontinuous abdominal pain out of proportion to the physical findings and abdominal distension were the major symptoms in both patients. Magnetic resonance imaging in one case and ultrasound scan with color Doppler followed by computed tomography in the other patient confirmed the diagnosis and enabled an appropriate early therapy to be undertaken.

Adult celiac disease and primary sclerosing cholangitis: two case reports.

The association of primary sclerosing cholangitis and celiac disease is uncommon. Herein, we report on 2 different cases which developed this association. Case 1 was a 59 year-old female who firstly complained of symptoms of cholestasis. The diagnosis of primary cholangitis was made on liver biopsy, and the endoscopic retrograde cholangiopancreatography (ERCP) showed narrowing and irregularity of the extra- and intrahepatic bile ducts. The results were positive for antiendomysial antibodies and the jejunal biopsy confirmed the coexistence of celiac disease, which was asymptomatic until that moment. The gluten-free diet ameliorated the index of cholestasis. Case 2 was an old man suffering from undiagnosed celiac disease for at least 5 years prior to admission at our Department. The diagnosis was based on the histological examination of a jejunal biopsy. The patient did not follow the gluten-free diet and was again admitted to our Department 6 years later with symptoms of cholestasis. The liver biopsy and ERCP confirmed the diagnosis of primary sclerosing cholangitis.

Antibacterial activity of rifaximin reduces the levels of benzodiazepine-like compounds in patients with liver cirrhosis.

Benzodiazepine-like compounds are present in trace amounts in the blood of normal subjects and increase in liver cirrhotic patients with or without encephalopathy. Their increased presence may, however, represent an occasional precipitating factor of hepatic encephalopathy. The source of these compounds is still unknown, but they are constituents of our diet since benzodiazepine receptor ligands have been described in plants, vegetables and in animals. They may also be synthesized, at least in part, by intestinal bacterial flora. In this article we report that the level of these compounds in the blood decreased by 40% after therapy with rifaximin, which reduces the aerobic and anaerobic intestinal bacterial flora. This observation indicates that intestinal bacterial flora is involved in the production of these compounds and that repeated short-term medications with this non-absorbable antibiotic may be useful in reducing the levels of benzodiazepine-like compounds in patients with liver cirrhosis.

Changes in endogenous benzodiazepine-like compound levels during the course of fulminant hepatic failure: potential effects of decreased renal function.

The pathogenetic agents which cause encephalopathy due to fulminant hepatic failure are still under debate. Ammonia and benzodiazepine-like compounds are two of the most important agents considered, so far. Herein, we report the levels of benzodiazepine-like compounds in serum and in urine and of venous ammonia measured during the course of the disease (30 days). The patient rapidly developed stage IV encephalopathy with high levels of ammonia and with only a slight increase of benzodiazepine-like compounds. At that moment, the levels of these compounds were similar to those recorded in the blood when the patient regained full consciousness 28 days later. During the course of the disease, there was a 10-fold increase of benzodiazepine-like compounds in serum which was recorded in parallel with an impaired excretion due to oliguria. This observation seems to indicate that encephalopathy may develop in the absence of significantly increased levels of these compounds and that their episodic increase during fulminant hepatic failure may be an epiphenomenon linked with several factors such as impaired renal function.

Hepatic encephalopathy in liver transplant recipients precipitated by benzodiazepines present in transfused blood.

The observation that there are episodes of encephalopathy in liver cirrhosis patients after orthotopic liver transplantation, despite a well functioning graft and despite the lack of cerebral complications, prompted us to investigate the potential role of circulating benzodiazepine-like compounds in these episodes. The plasma levels of benzodiazepines were examined in 14 liver cirrhotic patients before and after transplantation. The benzodiazepines in the fluids infused during surgery and in individual bags of blood administered after surgery to 4 of these patients were also assayed. Herein we report that benzodiazepines accumulating in the blood of some transplanted patients appear to derive from blood transfusions utilized during surgery. The analysis of the types of benzodiazepines present in the blood utilized for transfusions suggests the use of commercial benzodiazepines by the donors. These compounds seem to be able to precipitate hepatic encephalopathy in patients with preexisting encephalopathy. Hence we suggest not using benzodiazepine consumers as blood donors, at least for patients with encephalopathy undergoing to liver transplantation.

Evaluation of the efficacy and safety of flumazenil in the treatment of portal systemic encephalopathy: a double blind, randomised, placebo controlled multicentre study.

BACKGROUND: Portal systemic encephalopathy (PSE) is a complex neuropsychiatric syndrome associated with hepatic failure. Small scale studies have shown the benzodiazepine receptor antagonist flumazenil to be effective in ameliorating PSE. AIMS: To determine the efficacy of flumazenil in patients with non-comatous mild to moderate PSE (stages I to III) due to severe chronic liver disease. PATIENTS: 49 male and female adults without symptoms of severe bleeding and sepsis and who screened negative for benzodiazepine in both blood and urine, were included in the study. METHODS: Patients were randomised to receive either three sequential bolus injections of flumazenil (0.4, 0.8, and 1 mg) or placebo at one minute intervals, followed by intravenous infusions of either flumazenil (1 mg/h) or placebo for three hours. Clinical PSE grading and vital signs were assessed hourly during baseline and post-treatment periods and half hourly during treatment. The main outcome measures were improvement in group average PSE score and reduction of two points in individual PSE score (clinically relevant improvement). RESULTS: The mean average improvement in the PSE score in the subjects treated with flumazenil was not statistically significantly different from placebo. However, for patients showing clinically relevant improvement, the difference between flumazenil and placebo was statistically significant (seven of 28 v none of 21; p = 0.015). Flumazenil was well tolerated. CONCLUSIONS: A subgroup of patients with PSE resulting from chronic liver disease may benefit from the administration of flumazenil.

Increased brain concentrations of polyamines in rats with encephalopathy due to a galactosamine-induced fulminant hepatic failure.

Polyamine concentrations including putrescine, spermidine and spermine were documented in two brain areas of rats with mild and severe stages of hepatic encephalopathy (HE) due to fulminant hepatic failure induced by galactosamine HC1 injection (3 g kg-1 i.p.). In the mild stage of HE putrescine increased by 3-4 times whereas spermidine and spermine showed a slight increase. The scenario, however, was found to be changed going from the mild to the severe stage of HE, since in this last stage spermidine and spermine showed a further rise while putrescine was found to be significantly lower than in the mild stage of HE in both the brain areas studied. The changes in the ratio among the three polyamines with an enhanced prevalence in the severe stage of HE of spermidine and spermine are likely to be related to the exhaustion of the synthetic pathway of putrescine or to a reduction of the interconversion to this polyamine from spermidine and spermine. Considering that these last two polyamines potentiate the N-methyl-D-aspartate glutamate receptor mediated toxicity and that they might exert neurotoxic effects per se, there are clear reasons for suspecting an implication of the described changes of polyamines in the neurochemical mechanism which sustain HE and to surmise a potential therapeutic effect in this pathology of non-competitive antagonists of polyamine-site on N-methyl-D-aspartate glutamate receptors.