Ellen R. Gordon

Membrane fluidity and cholestasis

Editorial commentant le role de la membrane plasmatique hepatique dans la formation de la bile, et de la responsabilite de troubles de fonctionnement de cette membrane dans la pathogenese de la stase biliaire intrahepatique

Alcohol-Induced Mitochondrial Changes in the Liver

The chronic ingestion of ethanol results in liver-cell damage, and characteristic features of this injury are the marked alterations in both the functions and morphology of the mitochondria. Morphologically, the changes observed in human alcoholics and experimental animals appear similar. Bizarrely shaped mitochondria and megamitochondria are detected at the fatty liver stage and persist as the disease progresses. As yet, however, no correlation has been found between the severity of these morphological changes and the development of cirrhosis. Analysis of the mitochondrial membranes indicates that ethanol consumption produces changes in both the protein and lipid composition of the membrane. Profound decreases in the components of the respiratory chain have been detected, and these changes are associated with marked depressions in the activity of NAD+-linked dehydrogenases, cytochrome oxidase, and the ATP synthetase complex. On the other hand, no consistent pattern has emerged as to the effect of chronic ethanol consumption on the composition of the membrane phospholipids. Many of the changes appear to be dependent on the sex of the animal, the dietary status, and the duration of ethanol intake, and are suggestive of changes in fatty acid desaturase activity. Mitochondria isolated from ethanol-fed rats displayed impaired respiration and a lowered steady-state rate of ATP synthesis. Whether or not these functional changes are directly related to alterations in the physical properties of the membranes remains to be resolved. This marked depression of respiratory functions in isolated mitochondria was not reflected by a significant decrease in O2 consumption by the livers of ethanol-fed animals.

High-performance liquid chromatographic separation of bilirubin conjugates: The effects of change in molarity and pH

A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.

The effect of ethanol and calcium on fluid state of plasma membranes of rat hepatocytes

Basolateral (blLPM) and canalicular (cLPM) plasma membrane vesicles were isolated from rat liver to compare membrane fluidity, fluidity responses to membrane perturbants, and the relationship between fluidity and a membrane protein function such as carrier-mediated taurocholate transport. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as a probe. Uptake of [3H] taurocholate was measured by a rapid Millipore filtration technique. blLPM were more fluid than cLPM. Ethanol produced a concentration-dependent fluidizing effect on both membrane preparations, the change being greater in blLPM. Incubation with calcium for 2 hr at 37 degrees C rendered both membrane preparations more rigid, again the cLPM being more resistant to perturbation. There was a linear correlation between an increase in membrane fluidity and inhibition of taurocholate uptake into blLPM in the presence of increasing concentrations of ethanol. The data support the concept that membrane lipid fluidity is an important regulator of membrane protein functions and hence also of overall cellular activity.

Role of the physical state of the hepatic microsomal membrane in the formation of bilirubin diglucuronide

In vitro formation of bilirubin diglucuronide by rat hepatic microsomes proceeds efficiently only under specific conditions; i.e., a low level of bilirubin, temperature greater than 23-24 degrees C and treatment of the microsomes with a very specific level of perturbant (J. Biol. Chem., 258: 15028-15036). In the present study, the effect of temperature and detergents on the anisotropy of fluorescent probes in the rat hepatic microsomal membrane was used to determine the role of the physical state of the membrane in controlling the formation of bilirubin diglucuronide. A lipid phase separation that occurred at 23 +/- 2 degrees C was identified in these membranes indicating that bilirubin diglucuronide is formed efficiently only above the lipid phase separation when the phospholipids are in the liquid crystalline state. In addition, use of fluorescent probes for the surface and core of the membrane indicates that alterations in the physical state of the hydrophobic core rather than the phospholipid polar head group region of the membrane controls the in vitro formation of bilirubin diglucuronide.

Driving Forces for Taurocholate Transport across Basolateral and Canalicular Rat Liver Plasma Membrane Vesicles

Highly purified basolateral (bl LPM) and canalicular (cLPM) rat liver plasma membrane vesicles were simultaneously isolated from the same homogenate’ in order to directly evaluate and compare the driving forces for taurocholate transport across the two polar membrane domains of hepatocytes. The isolated LMP subfractions were minimally contaminated with intracellular organelles.’ Furthermore, cLPM were virtually free of basolateral contaminants as reflected by the absence of Na+K+-ATPase and glucagon-stimulatable adenylate cyclase activities in cLPM, which otherwise were 48-1 16-fold enriched over homogenate in the “canalicular markers” leucylnaphthylamidase, y-glutamyltranspeptidase, 5’-nucleotidase, alkaline phosphatase, Mg2’ATPase and alkaline phosphodiesterase I. in contrast, bl LPM vesicles were 34-fold enriched in Na’/K’-ATPase activity and exhibited considerable glucagon-stirnulatable adenylate cyclase activity. The various “canalicular marker” enzyme activities were 4-1 5-fold increased over homogenate. Examination by transmission electron microscopy revealed that cLPM and bl LPM were both composed of membrane vesicles although bl LPM still contained some unbroken lateral membrane sheets. 80% of the cLPM vesicles were of “right side out” orientation as determined by freeze fracture analysis. Equilibrium ( 1 20 minutes) uptakes of [‘4C]glucose, which did not bind to any extent to the vesicles, revealed intravesicular volumes of approximately 1 and 2 pl rng-’ protein for bLPM and cLPM, respectively. bl LMP vesicles accumulated taurocholate ( 1 p M ) threeto fourfold above equilibrium (overshoot) in the presence of an inwardly directed NaCl (100 mM) gradient. Taurocholate influx also was stimulated by Na’ when compared to K’ under ion-equilibrated conditions. Furthermore, preloading of the bl LPM vesicles with unlabeled taurocholate (20 p M ) stimulated uptake of extravesicular tracer taurocholate in the presence of Na’ but not in the presence of K’ (Na+-dependent countertransport, TABLE I A and B; and Na’-stimulated tracer exchange, TABLE 1 C). Valinomycin induced K’ diffusion potentials had no effects on Na+-stimulated taurocholate uptake into b I LPM vesicles. In contrast, in cLPM vesicles while taurocholate uptake also was stimulated by out-to-in NaCl as compared to KCI gradients (TABLE I ) , Na’ had no effect on taurocholate uptake under equilibrated or tracer exchange conditions (TABLE I F). Furthermore, in cLPM the phenomenon of transstimulation was seen in the presence of both Na’ and K’ gradients (TABLE I D and E) indicating the presence of an Na ‘-independent taurocholate “carrier” in canalicular LPM. This conclusion was further supported by the observation of an Na+-independent taurocholate efflux from

Assessment of Liver Function Before and After Liver Transplantation

Bilirubin is the end product of heme catabolism and has no physiological function, yet it has been the subject of intense investigation, with millions of dollars being spent yearly by clinicians and scientists [1]. From its structure (Fig.la), one would predict that it was very water-soluble; however, at pH 7.4 its solubility is in the nanogram range. Chemists explain this occurrence as a result of bilirubin forming a “ridge tile” through the diacid state with six intramolecular hydrogen bonds [2, 3]. This renders bilirubin insoluble (Fig.lb). Thus bilirubin is not secreted in urine, but is converted to conjugates within the liver. These appear in bile, mainly as bilirubin diglucuronide with minor components of bilirubin monoglucuronide and a diester containing monoglucuronide and monoglucoside [4, 5].