Membrane fluidity and cholestasis.
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Cholestasis↗
Biomedical subjects
E R Gordon
Publications and source records attributed to E R Gordon.
Taurocholate uptake by isolated skate hepatocytes: effect of albumin.
Taurocholate transport was analyzed in a well characterized, polarized hepatocyte preparation from the small skate (Raja erinacea), an analbuminemic species that does not synthesize bile acids. In addition, the effect of sodium omission, bovine albumin, ovalbumin, and bovine gamma-globulin on the uptake of taurocholate was studied. Uptake could be divided into nonsaturable (0.48 pmol X min-1 X mg protein-1. microM-1) and saturable components (Km, 32.5 microM and Vmax 110 pmol X min-1 X mg protein-1). No evidence for sodium dependence could be obtained. Glycocholate competitively inhibited taurocholate uptake. The initial rate of taurocholate uptake was also inhibited by sulfobromophthalein, N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate, and 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid but not lactate or alanine. Hepatic uptake of taurocholate in albumin solutions (2.5%) was twice as great as expected from estimates of the free taurocholate concentration. In contrast, equimolar concentrations of ovalbumin or bovine gamma-globulin had no effect on measured rates of taurocholate uptake and no evidence for a specific albumin receptor could be found on these hepatocytes by 125I-albumin binding. These studies indicate that a carrier-mediated, sodium-independent transport system for taurocholate uptake is present in skate hepatocytes that is not driven solely by the free concentration of taurocholate.
Albumins↗
Mechanism and subcellular site of bilirubin diglucuronide formation in rat liver.
Two different subcellular sites and mechanisms have been proposed for the hepatic conversion of bilirubin monoglucuronide to bilirubin diglucuronide: a microsomal system requiring UDP-glucuronate and a UDP-glucuronate-independent transglucuronidation or dismutase reaction localized at the bile canalicular plasma membrane. To further define these, canalicular plasma membranes were highly purified from rat liver, and the capacity of these to form bilirubin diglucuronide was compared with that of simultaneously isolated hepatic microsomes. The canalicular liver plasma membranes were 48-116-fold enriched over homogenate in various canalicular marker enzyme activities; microsomal contamination was less than 10% based on the NADPH-cytochrome c reductase activity. No evidence of any conversion of highly purified bilirubin IX alpha monoglucuronide to bilirubin diglucuronide was found with canalicular liver plasma membranes either in the absence or presence of UDP-glucuronate. In contrast, digitonin-treated microsomes isolated under similar conditions converted 31% of added bilirubin monoglucuronide (9.4-17.1 microM) into bilirubin diglucuronide in 30 min, the reaction being dependent on UDP-glucuronate. When bilirubin (12.5 microM) was added to the microsomes, 42.3% was converted to bilirubin monoglucuronide and 40.9% to bilirubin diglucuronide in 30 min. These data establish that the endoplasmic reticulum and not the canalicular liver plasma membranes forms bilirubin diglucuronide from bilirubin monoglucuronide and that the reaction requires UDP-glucuronate.
Animals↗
Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids.
In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa3 content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa3 may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 +/- 0.03 mumol of phospholipid phosphorus/mg of protein vs. 0.32 +/- 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acids and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A2. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.
Animals↗
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.
Adenosine Diphosphate↗
Irreversible binding of conjugated bilirubin to albumin in cholestatic rats.
A diazo-positive fraction of serum bilirubin that is irreversibly bound to albumin has been shown to accumulate in serum of patients with cholestasis. In the present study, a cholestatic animal model was used to determine the chemical nature of the bilirubin species involved in its formation. The data indicate that conjugated bilirubin is the precursor of "albumin-bound bilirubin" and that the presence or absence of light does not affect its formation. An albumin-bound bilirubin-complex indistinguishable from the complex detected in cholestatic sera from patients or in bile duct-ligated Sprague-Dawley rats can be formed in vitro in sera enriched in conjugated bilirubin at 37 degrees C, pH 7.4.
Animals↗
The hepatic microsomal formation of bilirubin diglucuronide.
Although the formation of bilirubin monoglucuronide by hepatic microsomes has been easily demonstrable, that of bilirubin diglucuronide, the principal conjugate of bile, has been more difficult. Therefore, an examination of the uridine diphosphate glucuronate-dependent microsomal formation of these two conjugates has been made utilizing a high performance liquid chromatographic method which quantitates the isomeric forms of the products. Initial studies indicated that at high starting bilirubin concentrations, only bilirubin monoglucuronide was formed; whereas at lower concentrations (approximately 15 microM), bilirubin diglucuronide could be formed efficiently, but only under very specific conditions. Untreated microsomes and microsomes treated with Triton X-100 did not differ; each formed monoglucuronide efficiently, yet formed diglucuronide poorly. Digitonin or UDP-N-acetylglucosamine pretreatment, in contrast, was found to facilitate bilirubin diglucuronide formation, the former much more than the latter. The activity of mannose 6-phosphatase, an enzyme located on the inner surface of the microsomal vesicles, did not correlate well with the bilirubin diglucuronide formation. Time course studies with digitonin and UDP-N-acetylglucosamine indicated a precursor-product relation between bilirubin monoglucuronide and bilirubin diglucuronide, and product isomer composition studies indicated that the bilirubin tetrapyrroles were stable (no random dipyrrolic exchange had occurred). Temperature studies with the digitonin-treated preparation demonstrated an increase in monoglucuronide-forming activity over the 0-25 degrees C range, whereas diglucuronide formation increased dramatically over the range from 25 to 35 degrees C. The results indicate that microsomal diglucuronide-forming activity differs characteristically from monoglucuronide-forming activity, and that it is intensely sensitive to the manipulation of its microsomal membrane environment.
Animals↗
Effects of chronic ethanol feeding and acetaldehyde metabolism on calcium transport by rat liver mitochondria.
The prolonged feeding of ethanol to rats alters in vitro mitochondrial transport of calcium. Hepatic mitochondria isolated from rats fed ethanol for 7 weeks exhibited decreased retention of calcium in the presence of 4mM-Pi. This defect was associated with enhanced efflux of calcium when mitochondria were incubated with EGTA. Acetaldehyde at low, "physiological" concentrations (100 microM) enhanced calcium retention by mitochondria but this response was blunted after chronic ethanol administration. The in vitro actions of acetaldehyde appear to be mediated, in part, by its metabolism in mitochondria since pretreatment of rats with cyanamide (an aldehyde dehydrogenase inhibitor) prevents this effect.
Acetaldehyde↗
Uptake of monohydric alcohols by liver: demonstration of a shared enzymic space.
Multiple-indicator dilution studies of the hepatic uptake of straight-chain C1-C5 monohydric alcohols were carried out in anesthetized dogs, with either no preceding or saturating infusions of ethanol, and at different steady-state levels for the C2 experiments. Labeled red cells were utilized as a vascular reference, and labeled water was used as a second reference entering liver cells. Kinetic analysis of the data provided estimates of both an uptake rate constant and the space of distribution available to label. From the decrease in the uptake rate constant for labeled ethanol with bulk concentration, we calculated a maximal removal rate of 0.025 mumol X s-1 X (ml liver water)-1 and a Michaelis constant (Km) of 0.32 mM. Especially for the labeled C3-C5 alcohols, a space in excess of that available to water was found, and the bulk of this was dissipated by ethanol infusion. The increment, the "shared enzymic space", which varies with enzymic concentration and inversely with Km, was used to calculate Km values for the other alcohols.
Alcohol Dehydrogenase↗
Effect of ethanol administration on the metabolism of ethanol in baboons.
To determine the mechanism whereby alcohol consumption accelerates ethanol metabolism, baboons were fed a diet containing ethanol (50% of calories) or an isocaloric control diet for 2 to 7 years. In alcohol-fed animals, the rate of ethanol metabolism per kilogram of body weight was accelerated by 34% at 50 mM concentrations of blood ethanol and liver size (estimated radiologically) increased by 23%. However, the rate of ethanol metabolism per gram of liver was not significantly increased. There was a 35% decrease in alcohol dehydrogenase (ADH) activity expressed per gram of liver and a 19% decrease per kilogram of body weight. Furthermore, the mitochondrial capacity to handle reducing equivalents was strikingly decreased as assessed by the rate of oxygen consumption at state 3 with glutamate and by the decrease in the activities of NADH and glutamate dehydrogenases. Thus, both factors presumed to be rate limiting for the ADH pathway (ADH activity and NADH reoxidation) were found to be decreased after chronic alcohol feeding. Therefore, even taking the increased liver size into consideration, the ADH pathway could not account for the rate of ethanol metabolism. By contrast, it was found that the activity of the microsomal ethanol-oxidizing system increased significantly after chronic alcohol consumption (by 22% expressed per gram of liver and by 54% expressed per kilogram of body weight). There were no significant changes in the content of cytochrome P-450.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Oxidoreductases↗
Lack of correlation between hepatic mitochondrial membrane structure and functions in ethanol-fed rats.
A current hypothesis suggests that alterations in the chemical composition and the subsequent changes in the structure of the membrane could account for the functional derangements observed in the hepatic mitochondria of animals fed ethanol for extended periods. An examination of this hypothesis reveals that the liver mitochondria of ethanol-fed rats show a dissociation between the respiratory functions and the lipid composition and microviscosity of the membranes.
Animals↗
A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile.
A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile has been developed. It is based on the use of ion-pair reverse-phase chromatography, utilizing heptanesulfonic acid in an acetate buffer, with a progressively increasing gradient of acetonitrile. The method resolves the following bilirubin IX alpha conjugates from bile: bilirubin diglucuronide, the two isomeric forms of bilirubin monoglucoside monoglucuronide diester, the two isomeric forms of bilirubin monoglucuronide, and bilirubin diglucoside. Unconjugated bilirubin is then eluted from the column by increasing the flow rate. The method also resolves the bilirubin XIII alpha and III alpha isomers of both bilirubin and the conjugates. In each case, the bilirubin XIII alpha precedes and the bilirubin III alpha follows the bilirubin IX alpha component. The high performance chromatographic run is completed in 35 min. The identity of the conjugates was ascertained by use of reference compounds isolated by thin-layer chromatography and by recollection of chromatographic peaks with identification of their diazo derivatives. The method was shown to have sufficient sensitivity that in vitro conjugating systems can be explored. Recollection and reinjection indicated that no isomeric scrambling occurs during the analytical procedure.
Animals↗
Partial characterization of hepatic aldehyde dehydrogenase from the baboon.
The activities, isozymic forms, response to disulfiram and some physical and kinetic properties of hepatic aldehyde dehydrogenase (A1DH, [EC.1.2.1.3]) from baboons were investigated. A1DH specific activity was greater in mitochondria than in cytosol but cytosolic A1DH was more sensitive to disulfiram than mitochondrial A1DH. Gel isoelectric focusing revealed three major isozyme groups focusing between pH 4 and 7, two in mitochondria and one in cytosol. Isozyme profile differences between control and ethanol-fed baboons appeared chiefly in the cytosol. Cytosolic A1DH from a control baboon was purified 42 fold with a 21% yield by ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography. The enriched enzyme displayed non-linear kinetics with 10% of the total activity having a Km of 0.3 microM and 90% having a Km of 160 microM. pH for optimum activity was 9.0, molecular weight 290,000 and, between pH 6.8 and 8.0, Mg++ ions inhibited the A1DH activity in the cytosol.
Aldehyde Dehydrogenase↗
Effect of chronic ethanol consumption on aldehyde dehydrogenase activity in the baboon.
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Alcoholism↗
Determinants of plasma free acetaldehyde levels during the oxidation of ethanol: effects of chronic ethanol feeding.
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Acetaldehyde↗
Ethanol-induced lipid peroxidation: potentiation by long-term alcohol feeding and attenuation by methionine.
Lipid peroxidation has been incriminated in some types of drug-induced liver injury, but it is unclear whether it contributes to or is present in alcoholic liver injury. In order to study this question, hepatic lipid peroxidation (measured as formation of diene conjugates) and hepatic GSH were assessed in baboons and rats after short- and long-term ethanol administration. Compared to controls, baboons fed alcohol for 1 to 4 years (chronic administration) had increased hepatic diene conjugates (16.9 +/- 4.8 OD/gm of liver) and depressed GSH (3.8 +/- 0.6 VS. 6.3 +/- 0.8 mumol/gm of liver; p less than 0.01) after an overnight withdrawal from ethanol. Administration of 1.6 gm/kg ethanol over 6 hr (acute administration) increased diene conjugates (17.6 +/- 4.3) and decreased GSH (3.2 +/- 0.5; p less than 0.01) in control animals and had an even greater effect in animals chronically fed alcohol (diene conjugates 48.5 +/- 9.2; GSH 1.8 +/- 0.3; p less than 0.001). In six rats fed alcohol for 5 to 6 weeks (chronic administration), an increase in diene conjugates was detected in microsomes (0.343 +/- 0.210 OD/mg of lipid) and mitochondria (0.143 +/- 0.061), accompanied by decrease in arachidonic acid and C22 polyenes, after acute ethanol administration (3 gm/kg p.o.) but no significant change in GSH. Simultaneous administration of methionine attenuated diene conjugate formation (0.107 +/- 0.058 and 0.035 +/- 0.020 OD/mg of lipid, respectively) and fatty acid changes. Thus chronic alcohol feeding potentiates lipid peroxidation produced by an acute dose of ethanol; these changes are not dependent on GSH depression but may be potentiated by it.
Animals↗
The formation of bilirubin diglucuronide by rat liver microsomal preparations.
Bilirubin transformation in vitro to bilirubin conjugates in the presence of activated rat liver microsomal preparations and UDPglucuronate was assessed with a method involving isolation of the products as tetrapyrroles. The proportions of bilirubin monoglucuronide and diglucuronide formed by the microsomal bilirubin UDPglucuronosyltransferase were found to be governed by the concentration of bilirubin present and the nature of the activation of the microsomal membrane. Activation of the microsomal preparations with the nonionic detergents Triton X-100 or Emulgen 911, or with digitonin for 24 h, produced bilirubin monoglucuronide as the only product at all concentrations of bilirubin investigated. In contrast, bilirubin diglucuronide was the only conjugate formed when hepatic microsomal preparations were activated with digitonin for periods of less tha 2 h and the concentration of bilirubin was 20 microM. Increasing the concentration of bilirubin utilized in this assay system changed the relative amounts of bilirubin monoglucuronide and diglucuronide formed. As the level of bilirubin was increased from 20 to 166 microM, the proportion of bilirubin diglucuronide decreased and that of bilirubin monoglucuronide increased, until at levels of 108 and 166 microM bilirubin only bilirubin monoglucuronide was formed. No evidence was found with liver plasma membranes that transglucuronidation plays a major role in the formation of bilirubin diglucuronide from bilirubin monoglucuronide.
Animals↗
Effect of diet and drugs on the qualitative and quantitative distribution of cytochromes P-450 in rat liver.
Male Sprague-Dawley rats were fed either Purina chow or a nutritionally adequate liquid control diet containing either 4 or 38% of the total caloric intake as fat. The hepatic cytochromes -450 were induced in these animals either by the administration of phenobarbital, beta-naphthoflavone, or isocaloric replacement of the carbohydrate of the liquid diets by ethanol at a level of 36% of total calories for 4 or 6 weeks. Three distinct groups of cytochromes P-450 could be efficiently separated by ion-exchange chromatography of microsomal preparations from these rats. The concentration of each group of cytochromes P-450 was markedly affected by variations in lipid and carbohydrate content of the diet as well as by administration of drugs.
Animals↗