[en] The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associated with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction

[en] A breath test to assess hepatic mitochondrial function in vivo was evaluated in rats. Following the i.p. administration of [1-¹⁴C]-alpha-ketoisocaproic acid, ¹⁴CO₂ exhalation reached a peak within 10 to 20 min and then declined exponentially, with a half-life of 14.3 min. Control animals exhaled 38.6% of the administered radioactivity within 1 hr. In functionally anhepatic animals, ¹⁴CO₂ in breath amounted to 23% of that in control animals, indicating that alpha-ketoisocaproic acid decarboxylation reflects mainly hepatic mitochondrial function in vivo. Ethanol (3 gm per kg) significantly decreased alpha-ketoisocaproic acid decarboxylation (21.8% of the dose appearing in breath in 1 hr), probably due to the ethanol-induced shift in the NAD+:NADH ratio. In contrast, an uncoupler of mitochondrial respiration, sodium salicylate (375 mg per kg), increased the decarboxylation of alpha-ketoisocaproic acid (56.3% of the dose recovered as ¹⁴CO₂ in 1 hr). Mitochondrial damage induced by 4-pentenoic acid decreased the decarboxylation of alpha-ketoisocaproic acid but did not affect the microsomal metabolism of antipyrine. The present data indicate that the alpha-ketoisocaproic acid breath test provides a noninvasive estimate of hepatic mitochondrial function in vivo which, when applied to man, might yield clinically useful information

[en] The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [³H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [³H]flunitrazepam and [³H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [³H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

[en] In order to study the relationship between amiodarone-induced hepatic phospholipidosis and liver disease, liver biopsies obtained from 13 patients treated with amiodarone for 4 months to 15 years were investigated by light and electron microscopy. Light microscopy showed pseudoalcoholic liver lesions that were probably related to amiodarone in four cases, various alterations (i.e. cirrhosis, three cases; steatosis and fibrosis, two cases; chronic venous congestion, one case; acute hepatitis, one case) that could be explained by another cause than amiodarone in seven cases and normal liver in two cases. In all cases, electron microscopy showed intralysosomal myelin figures suggestive of phospholipidosis. These myelin figures were associated with intralysosomal electron-dense deposits. In the four cases in which analysis by electron microprobe was performed, it demonstrated large amounts of iodine in the electron-dense deposit-containing lysosomes, indicating the accumulation of amiodarone. These results show that hepatic phospholipidosis is constantly observed in amiodarone-treated patients, whether or not pseudoalcoholic liver lesions are present. This phospholipidosis, which could be only a morphological marker of intrahepatic accumulation of the drug, should not therefore be considered grounds for attributing liver disease to the drug

[en] Sucrose and ethanol inhibit albumin synthesis; sucrose via an osmotic mechanism and ethanol during its metabolism. The present study was undertaken to compare the effects of both of these agents on albumin synthesis and secretion, and to see if ethanol inhibition could be related to an osmotic effect. Male, fed rabbits served as liver donors in all studies. There were a total of 35 studies: 13 control; 10 ethanol (39 to 52 mM); 4 cycloheximide (0.5 mM), and 8 sucrose (1%). Plasma volume was measured with ¹²⁵I-albumin (human) and extracellular volume measured with either /sup 99m/Tc diethylenetriamine pentaacetic acid or [¹⁴C]sucrose. During perfusion, rabbit albumin content in the perfusate was measured immunologically every 15 to 30 min for 225 min. Interstitial albumin efflux was measured by the rate of appearance in the perfusate of ¹²⁵I-albumin given to 10 other rabbits 3 days prior to hepatic removal and perfusion. During the initial 75 min of perfusion, 74% of the in vivo equilibrated exchangeable ¹²⁵I-albumin appeared in the perfusate, and during this period the rabbit albumin that entered the perfusate was taken to represent efflux from the interstitial volume plus synthesis. Rabbit albumin appearing in the perfusate during the later period of 150 min was taken to represent mainly synthesis and was used to calculate the amount of albumin that would be synthesized in 75 min. The difference between these two values would be hepatic interstitial albumin appearing in the perfusate

[en] The role of rat liver cell organelles in retinoid uptake and processing was studied by electron microscopic autoradiography. [³H]Retinoids were administered either orally, to make an inventory of the cell organelles involved, or intravenously as chylomicron remnant constituents to study retinoid processing by the liver with time. No qualitative differences were observed between the two routes of administration. Time-related changes in the distribution of grains were studied using chylomicron remnant [³H]retinoids. The percentages of grains observed over cells and the space of Disse at 5 and 30 min after administration were, respectively: parenchymal cells, 72.6 and 70.4%; fat-storing cells, 5.0 and 18.1%, and the space of Disse, 14.4 and 8.9%. Low numbers of grains were observed over endothelial and Kupffer cells. The percentages of grains observed over parenchymal cell organelles were, respectively: sinusoidal area, 59.6 and 34.4%; smooth endoplasmic reticulum associated with glycogen, 13.8 and 13.4%; mitochondria, 5.4 and 13.6%; rough endoplasmic reticulum, 4.2 and 7.3%, and rough endoplasmic reticulum associated with mitochondria, 3.7 and 6.5%. It is concluded that chylomicron remnant [³H]retinoids in combination with electron microscopic autoradiography provide a good system to study the liver processing of retinoids in vivo. These results, obtained in the intact liver under physiological conditions, further substantiate that retinoids are processed through parenchymal cells before storage occurs in fat-storing cell lipid droplets, that retinoid uptake is not mediated through lysosomes and that the endoplasmic reticulum is a major organelle in retinoid processing

[en] One drawback to using perfluorochemical emulsions as blood substitutes is that perfluorochemical particles are cleared from the blood by the reticuloendothelial system, primarily liver and spleen. The authors measured the impact of two perfluorochemical emulsions on clearance of colloidal carbon (less than 1 microns) and 51Cr-sheep red blood cells (about 8 microns) by the reticuloendothelial system in vivo and in the isolated perfused liver. Male rats were injected with 2 ml/100 gm body wt of Fluosol-DA or Oxypherol-ET for 4 consecutive days. Carbon (1 ml/100 gm body wt) or sheep red blood cells (0.05 ml of 5% vol/vol/100 gm body wt) were then injected intravenously (in vivo) or added to perfusate. Samples were taken at several time points for 1 hr. In the isolated perfused liver, carbon clearance was depressed by 25% 1 day after treatment. Rates returned to control levels by 12 days in Fluosol-DA-treated rats but remained depressed by 67% in Oxypherol-ET-treated rats. Sheep red blood cell (8 microns) clearance was two to five times slower than carbon clearance and depressed by 40% in livers from Fluosol-DA rats 1 day and 12 days after treatment. Added serum did not improve phagocytosis. In vivo carbon clearance remained normal in Fluosol-DA-treated rats but decreased by 74% in Oxypherol-ET-treated rats 1 day after treatment, returning to normal by 12 days. Clearance rates were similar in control rats in vivo and in the perfused liver. They conclude that the isolated perfused liver is a good model to measure liver clearance function. Although low doses of perfluorochemical emulsions may depress Kupffer cell phagocytosis, general reticuloendothelial system function is not significantly compromised

[en] [³H]glutamate, [³H]alpha-ketoglutarate or [³H]aspartate was injected in physiological concentrations into antegrade (from portal to hepatic vein) or retrograde (from hepatic to portal vein) perfused rat liver, and the tissue distribution of radioactivity was studied by histoautoradiography. Independent of the direction of perfusion, radioactivity was accumulated in a small perivenous liver parenchymal cell population, which surrounded the terminal hepatic venules as a layer of about two to five cells thick. In contrast, accumulation of radioactivity in periportal hepatocytes was low and sometimes not detectable. This distribution pattern roughly resembled that described for the immunohistochemical distribution of glutamine synthetase in liver. The present histoautoradiographic findings demonstrate a predominant uptake of vascular glutamate, aspartate and ketoglutarate into a small perivenous cell population. They confirm previous label incorporation studies in the metabolically intact liver, demonstrating an almost exclusive uptake of vascular glutamate and alpha-ketoglutarate into perivenous glutamine synthetase containing hepatocytes. In addition, evidence is presented suggesting that perivenous uptake of alpha-ketoglutarate may be one determinant for hepatic glutamine synthesis, at least under the experimental conditions used here

[en] Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

[en] A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of ¹²⁵I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens