Journal zyxwvutsrqpon of Neurochemistry zyxwvutsrqpon Raven zyxwvutsrqponmlk Press, Ltd., New York zyxwvutsrqpon 0 1990 International Society for Neurochemistry Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy Marjut Olasmaa, *Jeffrey D. Rothstein, Alessandro Guidotti, ?Richard J. Weber, $Steven M. Paul, $Sydney Spector, "Maria L. Zeneroli, "Mario Baraldi, and Erminio Costa FIDIA-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, Washington, D.C., *Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, and 7Neuroimmunology Unit, Laboratory of Medicinal Chemistry, NIDDK, and +NIMH, NIH, Bethesda, Maryland, and $Department of Pharmacology and Psychiatry, Vanderbilt University, Nashville, Tennessee, U.S.A.; and "Departmentof Pharmacology and Pharmacognosy, University of Modena, Modena, Italy Abstract: The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalop- athy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and pu- rification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were camed out using both radioreceptor binding assays and radioimmu- noassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and des- methyldiazepam, based on elution profiles and anti-benzo- diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained zyxwvuts - 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver dis- ease. 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 sig- nificant and may contribute to the pathogenesis of hepatic encephalopathy. Key Words: Benzodiazepine-y-Amino- butyric acid-Hepatic encephalopathy-Diazepam binding inhibitor-Liver-Cerebrospinal fluid. Olasmaa M. zy et al. Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy. J. Neurochem. 55, 20 15- 2023 (1990). Hepatic encephalopathy is a neuropsychiatric dis- order resulting primarily from metabolic abnormalities caused by acute or chronic liver disease (Jones and Shafer, 1986; Lockwood, 1987; Zieve, 1987). Even though the neurochemical pathogenesis of hepatic en- cephalopathy is still unknown, it is generally held that multiple factors underlie the clinical manifestations of this disorder, e.g., accumulation of ammonia, mercap- tans, and branched-chain amino acids, and that their adverse effects on central nervous system function may be synergistic (Zieve, 1987). Alterations in GABAergic neurotransmission have also been implicated as one of the factors affecting the level of consciousness of pa- tients suffering from hepatic encephalopathy (Baraldi and Zeneroli, 1982; Schafer and Jones, 1982; Baraldi et al., 1984). y-Aminobutyric acid (GABA), the principal inhib- itory neurotransmitter in brain, interacts with at least two receptors. One GABA receptor subtype, the GA- BAAreceptor, is modulated allosterically by sedative/ hypnotic drugs such as the benzodiazepines and bar- biturates and the endogenousfamily of peptides derived from a precursor polypeptide, diazepam binding in- hibitor (DBI) (Costa and Guidotti, 1987; Barbaccia et al., 1989). Several investigators have reported changes in the content of these allosteric modulators in tissues from animals and humans with hepatic encephalopa- thy. For example, Rothstein et al. (1989) have recently Received March 22, 1990; revised manuscript received May 22, 1990; accepted May 25, 1990. Address correspondence to Dr. J. D. Rothstein at Department of Neurology, The JohnsHopkins University School of Medicine, Meyer 5-1 19, zyxwvutsrqponm 600 N. Wolfe St., Baltimore, MD 21205, U.S.A. Address reprint requests to Dr. E. Costa at FIDIA-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, 3900 Reservoir Rd. N.W., Washington, zyx DC 20007, U.S.A. Abbreviations zyxwv used: DBI, diazepam binding inhibitor; DE, diaze- pam equivalents; GABA, y-aminobutyric acid TFA, trifluoroacetic acid. 2015