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.

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.

Animal-based national surveillance for zoonotic disease: quality, limitations, and implications of a model system for monitoring rabies.

Surveillance for zoonotic diseases among wildlife is a research and public health challenge. The inherent limitations posed by the requisite human-animal interactions are often undefined and underappreciated. The national surveillance system for animal rabies in the United States was examined as a model system; reporting of animal rabies is legally mandated, each case of rabies is laboratory confirmed, and data have been consistently collected for more than 50 years. Factors influencing the monthly counts of animal rabies tests reported during 1992-2001 were assessed by univariate and multivariable regression methods. The suitability of passively collected surveillance data for determining the presence or absence of the raccoon-associated variant of rabies within states and within individual counties was assessed by determining critical threshold values from the regression analyses. The size of the human population and total expenditures within a county accounted for 72% and 67%, respectively, of the variance in testing. The annual median number of rabies tests performed was seven for counties without rabies, 22 for counties with non-raccoon rabies, and 34 for counties with raccoon rabies. Active surveillance may be required in locales with sparse human populations when a high degree of confidence in the status of rabies is required.

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.

Persistence of elevated rabies prevention costs following post-epizootic declines in rates of rabies among raccoons (Procyon lotor).

Determining the benefits to cost relationships among different approaches to rabies control and prevention has been hindered by the inherent temporal variability in the dynamics of disease among wildlife reservoir hosts and a tangible and objective measure of the cost of rabies prevention. A major and unavoidable component of rabies prevention programs involves diagnostic testing of animals and the subsequent initiation of appropriate public health responses. The unit cost per negative and positive diagnostic test outcome can be reasonably estimated. This metric when linked to methodologies subdividing the epizootic process into distinct temporal stages provided the requisite detail to estimate benefits derived from rabies control strategies. Oral rabies vaccine (ORV), for prevention of the raccoon-associated variant of rabies, has been distributed in Ohio and adjoining states in an effort to develop an immune barrier to the westward spread of epizootic raccoon rabies. The costs of ORV delivery have been quantified. Herein, the cost structures required to assess the benefits accrued by prevention were developed. A regression model was developed effectively predicting (r2=0.70) the total number of rabies diagnostic tests performed by 53 counties in five northeastern (NE) states from 1992 to 2001. Five temporal stages sufficed to capture the range of variability in the raccoon rabies epizootic process. Unit costs, dollars per diagnostic test outcome, were calculated for negative and positive results from published reports. Ohio counties were matched to NE counties based on similar socioeconomic characters. A "pseudo-epizootic" of raccoon rabies was introduced into Ohio and the costs savings from ORV were derived as the excess costs imposed by epizootic spread throughout the state. At 46 km/year (range modeled, 30-60 km/year), the pseudo epizootic spread, and reached the enzootic stage, in all Ohio counties by year 13 (range modeled, 11-17 years). Cumulative excess costs for Ohio ranged between $11 and $21 million; counties of low socioeconomic status experienced the greatest relative excess costs. The costs for rabies prevention activities reached apices during the epizootic stage of raccoon rabies (2.7-10.8 times baseline) an unforeseen finding indicated elevated costs persisted (1.7-7.2 times baseline) into the enzootic stage.

Serum bilirubin pigments covalently linked to albumin.

The serum levels of biliprotein (i.e., the bilirubin fraction covalently linked to albumin) were measured in patients with hepatobiliary disease by two analytical procedures: reverse-phase anion-exchange chromatography (Bond-Elut) and anion-exchange chromatography. Bond-Elut extracts contained tetrapyrroles, allowing the analysis of biliprotein, bilirubin, and bilirubin conjugates, whereas the anion-exchange method converted tetrapyrroles to azodipyrroles (protein-free), which were adsorbed on the resin, with the exception of azopyrrole of biliprotein (covalently linked to albumin), which passed through the resin and could be analyzed. In contrast, other cationic polymer columns did not completely bind the alkaline diazo derivatives of unconjugated bilirubin, conjugated bilirubin, or the azopyrrole of biliprotein not bound to albumin. Therefore, a comparison was made of these two methods using the original anion-exchange resin. Both procedures gave similar values for serum levels of biliprotein in patients with high levels of total bilirubin.

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.

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.

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.

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.

Role of liver plasma membrane fluidity in the pathogenesis of estrogen-induced cholestasis.

The role of liver plasma membrane (LPM) fluidity in the pathogenesis of intrahepatic cholestasis in rats was assessed by comparing the effects of ethinyl estradiol, a cholestatic agent, and spironolactone on membrane fluidity and bile flow. Spironolactone is a steroid that has some feminizing actions but that lacks the phenolic A ring necessary for estrogens to cause cholestasis. Bile flow was reduced 42% (p less than 0.01) by ethinyl estradiol and increased 22% (p less than 0.05) by spironolactone; however, both agents produced a significant reduction of membrane Na+, K+-ATPase activity (p less than 0.01) and fluidity (p less than 0.01). The decreased fluidity persisted in liposomes prepared from the total lipid extract as well as the phospholipid extract of these membranes. Both agents produced similar significant increases in the cholesterol ester content and cholesterol-to-phospholipid molar ratio of the membranes. In addition, ethinyl estradiol and spironolactone increased the membrane sphingomyelin content (15% and 11%, respectively); however, neither agent altered the fatty acid composition of the phospholipids. Because the decreased fluidity persisted in liposomes prepared from phospholipids extracted from the LPMs of treated rats, changes in membrane cholesterol are not the sole cause of the altered membrane fluidity. Rather, the increased sphingomyelin is at least partially responsible for these changes. Also, because ethinyl estradiol and spironolactone produce similar changes in LPM lipid composition and fluidity but disparate effects on bile flow, membrane fluidity as assessed by fluorescence polarization does not appear to be the rate-limiting determinant of bile flow in estrogen-induced cholestasis.

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.

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.

Biochemical aspects associated with an ethanol-induced fatty liver.

Isocaloric replacement of either the fat or carbohydrate content of the diet by ethanol (36% of the total caloric intake) produced fatty infiltration of the liver in rats. The increase in hepatic triglyceride content was associated with a decrease in both ATP and glycogen contents. Increased activity of mitochondrial Mg(2+)-stimulated adenosine triphosphatase paralleled the increase in the free P(i) content of the liver homogenate. During the regression of the fatty liver, glycogen contents returned to normal within 24h of the removal of ethanol from the diet. Not until the third day after the withdrawal of ethanol had the Mg(2+)-stimulated adenosine triphosphatase activity and free P(i) content of the homogenate returned to normal. A slow regression of the triglyceride content from the liver occurred and by the fifth day both ATP and triglyceride concentrations had returned to the values observed in the rats given the liquid control diet.