bluuebunny/crossref_metadata_2025 huggingface dataset:
The acquired vitamin K-dependent γ-carboxylation deficiency in hepatocellular carcinoma involves not only prothrombin, but also protein C
Authors: Yoshikawa Yuji , Sakata Yoichi , Toda Gotaro , Oka Hiroshi
Published: March 2007

Protein C, one of the vitamin K-dependent plasma proteins synthesized in the liver, was measured immunologically in normal subjects (n = 20), patients with hepatocellular carcinoma (n = 60), liver cirrhosis (n = 60), acute hepatitis (n = 16), chronic hepatitis (n = 19), malignant neoplasms other than hepatocellular carcinoma (n = 35) and patients on warfarin treatment (n = 20). We also assayed γ-carboxyglutamic acid-complete (carboxylated) protein C in these population by using a monoclonal antibody directed against human protein C, JTC-1, which recognizes the γ-carboxyglutamic acid domain-related conformational change induced by metal ions. We demonstrated that the plasma of patients with hepatocellular carcinoma contains considerable amounts of γ-carboxyglutamic acid-incomplete protein C, evidenced by the significantly reduced protein C:γ-carboxyglutamic acid/protein C:antigen ratios in hepatocellular carcinoma as compared to those seen in normal controls, other liver diseases and other malignant neoplasms (p < 0.01). In two patients with hepatocellular carcinoma with the reduced protein C:γ-carboxyglutamic acid/protein C:antigen ratios, successful treatment (transcatheter hepatic arterial embolization or lipiodolization of antitumor agent) led to the very rapid normalization of the ratios. Intravenous administration of vitamin K, however, induced no such effects in three other patients with hepatocellular carcinoma with the abnormality. We conclude that the impaired vitamin K-dependent γ-carboxylation observed in patients with hepatocellular carcinoma involves not only prothrombin, but also protein C, and that the impairment is not due to vitamin K deficiency.
Identification and characterization of a murine receptor for galactose-terminated glycoproteins
Authors: Hong Wanjin , Van Le Anh , Doyle Darrell
Published: March 2007

The asialoglycoprotein receptor, the hepatic binding lectin for galactose-terminated glycoproteins, has been isolated and characterized from human, rabbit and rat liver. Several recent studies have shown the existence of the same receptor in murine liver. However, the biochemical structure of the receptor in murine liver has not been resolved. In this paper, we describe the identification and purification of the receptor for asialoglycoproteins from murine liver. The purified receptor has three polypeptides with apparent molecular weights of 42,000, 45,000 and 51,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthemore, our studies suggest that the receptor from murine liver is very similar to its counterpart in rat liver, although some potential interesting differences have also been observed. Initial studies indicate that this receptor is well conserved in different mouse strains.
Cytokeratin intermediate filaments of rat hepatocytes: Different cytoskeletal domains and their three-dimensional structure
Authors: Katsuma Yoshinori , Marceau Normand , Ohta Masaharu , French Samuel W.
Published: March 2007

A new method of visualizing the three-dimensional architecture of the cytokeratin filaments of the intact rat hepatocyte in situ has been achieved. Frozen sections of liver cut 10 μm thick were serially extracted to remove all elements of the cells except the intermediate filaments. Parallel sections were stained with monoclonal antibodies to the two main cytokeratins found in bile duct and liver cells. Immunofluorescent antibody and immunogold electron microscopy techniques were used to identify the proteins morphologically. Several new observations resulted from these studies. The pericanalicular sheath of intermediate filaments was visualized using steropairs as an uninterrupted branching tubular structure composed of cytokeratins located in the cell cortex of adjacent hepatocytes. Intermediate filaments in the cell cortex formed a distinct sheet of matted filaments which enveloped the entire hepatocyte. The cortical intermediate filaments were in continuity with the pericanalicular sheath and the filaments located within the cytoplasm. The intermediate filaments are attached to the centrioles and appeared to tent the nuclear lamina-pore complex at points of contact. Monoclonal antibodies to rat liver intermediate filament cytokeratins (CK49 and CK55) each stained intermediate filaments located in the cell cortex, within the cytoplasm and at the nucleus. By immunogold staining, some of the intermediate filament filaments were shown to contain both cytokeratins. Filaments which did not stain were thought to be either actin at the cell periphery or nuclear lamins around the nucleus. It is concluded that the cytokeratins form a specialized framework for the cell cortex, canaliculus, centrioles and the nucleus of hepatocytes. The filaments run continuously throughout the cytoplasm without terminating.
Oxidant stress during reperfusion of ischemic liver: No evidence for a role of xanthine oxidase
Authors: Metzger Jacques , Dore Srinath P. , Lauterburg Bernhard H.
Published: March 2007

Oxygen-derived free radicals might play a role in the injury produced by reperfusion of ischemic organs. Since the generation of reactive oxygen species results in an increased formation of glutathione disulfide, we have attempted to document an oxidant stress during reperfusion of ischemic liver by following the hepatic production of glutathione disulfide in vivo and in the perfused rat liver. Following partial hepatic ischemia of 120 min duration, the plasma concentration of glutathione disulfide gradually increased from 0.8 ± 0.1 to 9.1 ± 1.6 μM (mean ± S.E., n = 6) after 1 hr of reperfusion. The plasma concentration of reduced glutathione increased only 2-fold from 21.4 ± 2.4 to 38.1 ± 3.4 μM. The rise in plasma glutathione disulfide was higher with increasing duration of ischemia from 30 to 120 min and was associated with a gradual increase in hepatic glutathione disulfide. The hepatic origin of glutathione disulfide was documented in the perfused rat liver where the release of glutathione disulfide increased gradually during reperfusion following 90 min of warm ischemia from 1.0 ± 0.1 to 2.6 ± 0.5 nmole per min per gm liver at 30 min after onset of reperfusion. The administration of allopurinol, an inhibitor of xanthine oxidase, did not decrease the release of glutathione disulfide (29.9 ± 3.8 nmoles per gm in controls vs. 44.7 ± 10.0 nmoles per gm in 30 min with allopurinol) and ALT (3.3 ± 1.4 units per gm in controls vs. 2.6 ± 0.8 units per gm in 30 min with allopurinol). Our studies document an oxidant stress associated with reperfusion of ischemic liver. The absent effect of allopurinol and the observation that the oxidant stress is not maximal when the available concentration of substrates for xanthine oxidase is highest (i.e., at the onset of reperfusion) indicate that the oxidant stress may not be due to the generation of superoxide anion radicals by xanthine oxidase, but rather may result from the activation of leukocytes and resident macrophages.
Atrial natriuretic factor in cirrhosis with ascites: Plasma levels, cardiac release and splanchnic extraction
Authors: Ginès Pere , Jiménez Wladimiro , Arroyo Vicente , Navasa Miquel , López Clara , Titó Llúcia , Serra Antoni , Bosch Jaime , Sanz Ginès , Rivera Francisca , Rodés Joan
Published: March 2007

The aim of this study was to determine the plasma levels, cardiac release and splanchnic extraction of atrial natriuretic factor in cirrhosis with ascites. The plasma concentration of immunoreactive atrial natriuretic factor in samples obtained from an antecubital vein was measured in 18 healthy volunteers and in 35 cirrhotics with ascites. In 11 of these cirrhotics and in 11 patients admitted to the hospital for the study of a thoracic pain who had no clinical or hemodynamic signs of cardiac failure (control group), the plasma levels of immunoreactive atrial natriuretic factor in samples from the coronary sinus, right atrium, pulmonary artery, hepatic vein and femoral vein were determined and the coronary sinus blood flow measured by thermodilution. Cirrhotic patients showed significantly higher plasma levels of immunoreactive atrial natriuretic factor in each vascular territory studied than did control subjects (coronary sinus: 101.2 ± 10.6 vs. 26.1 ± 4.7 fmoles per ml; right atrium: 32.5 ± 5.8 vs. 9.4 ± 3.5; pulmonary artery: 36.8 ± 10.1 vs. 7.5 ± 2.4; hepatic vein: 10.7 ± 2.0 vs. 2.7 ± 0.8; femoral vein: 18.2 ± 2.4 vs. 3.1 ± 0.9; antecubital vein: 14.7 ± 1.6 vs. 4.0 ± 0.8). The coronary sinus blood flow was also higher in cirrhotics (200 + 22 ml per min) than in controls (105 ± 7 ml per min). Consequently, the estimated cardiac release and cardiac production of immunoreactive atrial natriuretic factor were strikingly increased in cirrhotics (13,334 ± 2,007 and 5,484 ± 1,734 fmoles per min, respectively) as compared to control subjects (1,669 ± 338 and 1,431 ± 350 fmoles per min, respectively; p < 0.01). The splanchnic extraction of immunoreactive atrial natriuretic factor was similar in both groups. These results indicate that the plasma levels of immunoreactive atrial natriuretic factor are increased in patients with cirrhosis and ascites, and that this is due to an increased release and not to an impaired splanchnic degradation of the peptide.
Systemic and splanchnic hemodynamic effects of intravenous hypertonic glucose in patients with cirrhosis
Authors: Pugliese Domenico , Lee Samuel S. , Koshy Abraham , Cerini Raimondo , Ozier Yves , Lebrec Didier
Published: March 2007

In animals, there may exist a hyperemic response in the portal circulation during intravenous administration of hypertonic glucose, but a hemodynamic response of this kind has never been described in man. This study was designed to evaluate if hyperglycemia itself could induce systemic or splanchnic hemodynamic changes in patients with cirrhosis. Sixteen patients with cirrhosis were studied before and during i.v. infusions of hypertonic (900 mOsmoles per liter) glucose (n = 8), mannitol (n = 4) or saline (n = 4) at 2 ml per min. In the group receiving glucose, there were significant increases in hepatic venous pressure gradient (+12%), azygos blood flow (+27%) and pulmonary capillary pressure (+32%), while calf blood flow decreased (-26%). No changes occurred in the mannitol or saline groups. Changes in plasma osmolality, plasma volume, splanchnic oxygen extraction and vasoactive hormones, including vasoactive intestinal polypeptide and glucagon, did not appear to be involved in the mechanism of these vasoactive phenomena. It is suggested that the possible deleterious effects of increase in portal pressure and azygos blood flow should be taken into consideration when administering hypertonic glucose to patients with portal hypertension.
Postprandial hemodynamic responses in patients with cirrhosis
Authors: Lee Samuel S. , Hadengue Antoine , Moreau Richard , Sayegh Raymond , Hillon Patrick , Lebrec Didier
Published: March 2007

The hemodynamic response to a 800 kcal liquid meal was investigated in 24 patients (study group) with cirrhosis and two control groups. One control group of six cirrhotic patients (volume control) had a calorie-free equivolumic electrolyte solution. The second control group (normal control) of six patients with normal hepatic function had the same test meal. Cardiac index, mean arterial pressure, heart rate, hepatic venous pressures, hepatic blood flow and azygos blood flow (only measured in the study group) were measured before and 30 and 60 min after the meal. Systemic circulatory responses were generally absent. Hepatic blood flow and wedged hepatic venous pressure increased significantly in both cirrhotic patients and normal controls; however, cirrhotic patients tended to have a quicker response (peaks at 30 min) than normals (peaks at 60 min). Azygos blood flow showed large postprandial variability, but overall did not change significantly. These results demonstrate that the splanchnic circulation of the patient with cirrhosis reacts differently from normals to the physiologic stimulus of a meal, and in particular appears to have precocious vasoactive reactivity.
Ventricular function in cirrhosis and portasystemic shunt: A two-dimensional echocardiographic study
Authors: Keller Helmut , Bezjak Vera , Stegaru Beatrice , Buss Jan , Holm Eggert , Heene Dieter L.
Published: March 2007

Cardiovascular alterations such as increased heart rate, high cardiac output, reduced systemic vascular resistance, and in most of the cases, increased contractility parameters have been recognized in patients with advanced liver disease.

Some investigators define a cirrhotic cardiomyopathy as a hyperdynamic failure of the heart. Consequently, in patients with cirrhosis, the risk of developing further circulatory deterioration may be increased in situations which stress the cardiovascular system. After opening a portocaval shunt, it is expected that a large amount of blood will be distributed from the splanchnic to the pulmonary circulation and put a strain on the heart.

This two-dimensional echocardiographic study was made in 30 patients with cirrhosis and in 20 patients who were chronically treated (range: 16 to 156 months) with portasystemic shunt for prevention of hemorrhage from esophageal variceal bleeding. Patients with portasystemic shunts revealed a change in hemodynamic pattern. There was a significant increase in the left ventricular end-diastolic volume index and also a slight increase in the left ventricular end-systolic index. Cardiac output remained high despite a significant decrease in heart rate due to an elevated left ventricular stroke volume index.

The parameters of systolic ventricular performance were normal.

In contrast to the acute opening of the portacaval shunt, the chronic shunt volume put no strain of clinical significance on the heart.
The oncogenicity of azathioprine?
Authors: Schaffner Fenton
Published: March 2007

One hundred and fifty-four patients with histologically verified non-alcoholic chronic liver disease were randomized to azathioprine or prednisone treatment. After a median of 91 months observation time, the cause of death was assessed retrospectively. Autopsy was performed in 82% of 71 deaths. In the azathioprine group 33% (13/39) died from malignant neoplasia, and in the prednisone group (13%) (4/32) (p = 0.08). Considering a possible fatal outcome as a consequence of treatment, this finding urges caution in the long-term application of azathioprine at the usual dose level.
Percutaneous portal-systemic shunts: Promise and problems
Authors: Zimmon David S.
Published: March 2007

Original Gianturco expandable stents and their modifications were used to create an experimental intra-hepatic portacaval anastomosis (EIPCA) in 30 young domestic swine without portal hypertension. The study focused on the design of a suitable stent, the technique of its application, the the evaluation of short-term patency of the EIPCA. A stent with a 2.5-cm-long body and wire skirts on both ends was most suitable for EIPCA creation. Well-positioned stents shunted most of the portal blood in the inferior vena cava circulation and remained patent for 4-6 weeks. Ingrowth of liver parenchyma and abundant proliferation of the intima and connective tissue inside the stent lumen in these rapidly growing animals gradually decreased EIPCA patency, and thrombus formation with diminished blood flow closed them completely.
Correlation of hepatocyte HBsAg expression with virus replication and liver pathology
Authors: Hsu Hey-Chi , Lai Ming-Yang , Su Ih-Jen , Chen Ding-Shinn , Chang Mei-Hwei , Yang Pei-Ming , Wu Chieng-Yen , Hsieh Hong-Chung
Published: March 2007

To elucidate the biologic significance of hepatocyte HBsAg, its expression patterns were correlated with virus replication and liver pathology in 578 liver biopsies taken from chronic HBsAg carriers aged 1 to 80 years. Five major patterns of hepatocyte HBsAg were identified: homogeneous [intense and discrete, (Pattern A), faint and discrete, (Pattern B) and faint and grouped (Pattern C)]; globular or spotty (Pattern D), and marginal (Pattern E). Pattern A was always associated with viremia and also very frequently with membrane HBsAg expression, but rarely with active liver disease. It occurred most commonly in HBeAg-positive carrier children and young adults, reflecting an early immune tolerance phase with active virus replication. Pattern B was also usually associated with viremia, but very commonly associated with active disease (70%), reflecting active virus replication with enhanced immune response. Pattern E (marginal HBsAg), which was always in group distribution resembling a clonal expansion, predominated the HBeAg-negative phase and was associated with absence of viremia and occurred mostly in older adults with inactive bipolar disease spectrum (normal liver/mild disease or cirrhosis/hepatocellular carcinoma); this reflects a late phase of inactive virus replication or integration. Patterns C and D did not correlate well with viremia, but also tended to have inactive diseases as did Pattern E. These findings suggest that hepatocyte HBsAg expression is closely related to the natural course of chronic hepatitis B virus infection.
Prevalence of isolated antibody to hepatitis B core antigen in an area endemic for hepatitis B virus infection: Implications in hepatitis B vaccination programs
Authors: Lok Anna S. F. , Lai Ching-Lung , Wu Pui-Chee
Published: March 2007

Of 1,801 Chinese subjects, age 1 to 90 years, screened for hepatitis B surface antigen and antibody (HBsAg, anti-HBs) and antibody to hepatitis B core antigen (anti-HBc), 214 (11.9%) had an isolated, positive anti-HBc result; anti-HBc was reproducibly present in the initial sera in only 66% and persisted after an interval of 2 weeks to 3 months in only 73%. There was a strong correlation between the rates of reproducibility and persistence of isolated anti-HBc and the initial anti-HBc titers. Thirty-two subjects with persistent, isolated anti-HBc received four doses of hepatitis B vaccine (5 μg, HEVAC B) at 0, 1, 2 and 12 months: 56% developed a primary anti-HBs response in response to hepatitis B vaccine, 16% developed an anamnestic or secondary anti-HBs response, and 28% were undetectable for anti-HBs even after four doses of vaccine. The low rates of reproducibility and persistence of anti-HBc together with the high rate of primary anti-HBs response to hepatitis B vaccine in subjects with isolated anti-HBc raise doubts as to the reliability of anti-HBc (Corzyme, Abbott Laboratories, North Chicago, Ill.) as a single screening test for hepatitis B infection prior to vaccination and suggests that subjects with isolated anti-HBc, in particular those with low anti-HBc titers, be included in vaccination programs.
The effect of retinol on ito cell proliferation in vitro
Authors: Davis Bernard H. , Vucic Angelina
Published: March 2007

Hepatic sinusoidal fat-storing Ito cells are felt to represent the primary storage site for hepatic vitamin A and may be important collagen-producing effector cells during hepatic fibrogenesis. The cirrhotic liver generally has a decreased vitamin A content with increased numbers of “transitional” myofibroblasts adjacent to developing fibrous bands. It has been suggested that Ito cells “transform” into these myofibroblasts. The in vivo loss of Ito cell vitamin A can be simulated in vitro as Ito cells spontaneously lose their vitamin A lipid droplets during primary culture. The current study evaluated Ito cell proliferation in vitro with respect to vitamin A content and the extracellular collagen matrix. The cells were grown on a Type I or Type IV collagen matrix to simulate the types of collagens presumed to be present in the space of Disse. Initially it was observed that freshly isolated Ito cells begin to proliferate several days after isolation coincident with the decline of the vitamin A lipid droplets and a decrease in cellular retinyl palmitate. The proliferation rate for passaged Ito cells was similar on either matrix (on Type I collagen: T1/2 = 2.2 ± 1.1 days, n = 16; on Type IV collagen: T1/2 = 3.3 ± 1.4 days, n = 4; p < 0.11). This proliferation rate remained constant through Cell Generation 16 and was similar to the rate for primary Ito cells in culture. To evaluate the possibility that primary Ito cell proliferation is causally related to the loss of vitamin A, Ito cells were re-exposed to an increased concentration of retinol in vitro. Passaged Ito cells (grown on a Type I or Type IV collagen matrix) which lack vitamin A droplets were exposed to media containing 10−5
M retinol. Cell proliferation was markedly inhibited for the 2 weeks of observation without apparent loss of cell viability. The effect was noted to be reversible and dose related with a submaximal effect at 10−6
M retinol. In conclusion, retinol can modulate Ito cell proliferation in vitro and may represent an important controlling factor with respect to Ito cell behavior.
Hepatocytes may produce laminin in fibrotic liver and in primary culture
Authors: Clément Bruno , Rescan Pierre-Yves , Baffet Georges , Loréal Olivier , Lehry Dominique , Campion Jean-Pierre , Guillouzo André
Published: March 2007

Previous studies have shown that laminin is present in basement membranes in normal liver but failed to identify cellular sources. We have investigated the extracellular and intracellular distribution of laminin in normal rat and human liver, in fibrotic human liver and in primary hepatocyte cultures from both species by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver from both species, antibodies to laminin strongly stained basement membranes and formed discontinuous discrete deposits in the wall of the sinusoid. Vascular endothelial and bile duct cells as well as fat-storing cells and sinusoidal endothelial cells strongly stained for this glycoprotein while hepatocytes were negative. In fibrotic human liver, increased amounts of extracellular laminin were usually found. Continuous deposition in the space of Disse was observed in some cases. In addition to fat-storing cells and endothelial cells, hepatocytes were also sometimes positive. Normal rat and human hepatocytes synthesize and secrete laminin in conventional culture, but it remains soluble in the medium. By contrast, in coculture with another rat liver cell type, laminin accumulated around hepatocyte cords. These observations suggest that fat-storing cells and endothelial cells are the major sites of production of laminin in normal liver. However, when their environment is altered (e.g., liver injury, culture), adult hepatocytes are able to synthesize detectable amounts of laminin.
Increased production of collagen in Vivo by hepatocytes and nonparenchymal cells in rats with carbon tetrachloride-induced hepatic fibrosis
Authors: Chojkier Mario , Lyche Kip D. , Filip Michael
Published: March 2007

We have shown, using the proline:ornithine dual label method, that in normal rats, hepatocytes contribute in vivo about 80 to 90% of the newly synthesized hepatic collagen. In order to quantify the contribution of hepatocytes and nonparenchymal cells to collagen synthesis in vivo in hepatic fibrogenesis, rats with CCl4-induced liver fibrosis were given [53H]proline and [14C]ornithine intraperitoneally. About 80% of the 14C in albumin and transferrin was present as arginine, following conversion of [14C]ornithine via the urea cycle. In contrast to hepatocyte proteins, in nonparenchymal cells and serum a negligible percentage of the radioactivity was present as [14C]arginine. These combined findings indicate that, in spite of the hepatocellular damage, the labeling of hepatocyte proteins was efficient and specific, validating the use of the proline:ornithine method in this experimental model of hepatic fibrosis. We calculated the [3H]proline/[14C]arginine ratio in hepatic collagen (after correcting for the relative frequencies of amino acids) as a percentage of the same ratio in either albumin or transferrin, the index hepatocyte proteins. In this experimental model, during active fibrogenesis, both hepatocytes and nonparenchymal cells increase their production of collagen 2-fold when compared to normal animals, and hepatocytes produce the majority of the newly synthesized hepatic collagen.
Modulation of hepatotoxicity by macrophages in the liver
Authors: Shiratori Yasushi , Kawase Tateo , Shiina Shuichiro , Okano Ken’Ichi , Sugimoto Tsuneaki , Teraoka Hitoshi , Matano Sunao , Matsumoto Kazunori , Kamii Kazuo
Published: March 2007

In an attempt to elucidate the role of hepatic macrophages in liver injury, we investigated galactosamine-treated rats (500 mg per kg body weight). The rats received an i.v. injection of latex particles (2 × 109 particles per animal) prior to (latex-galactosamine) or 12 to 16 hr subsequent to the galactosamine treatment (galactosamine-latex). Effect of superoxide dismutase on hepatic injury induced by galactosamine or galactosamine-latex treatment was also examined. Oxygen-derived free radical-generating capacity of isolated hepatic macrophages was measured as chemiluminescence with the stimulation of phorbol myristate acetate or latex particles. As compared with normal rats, chemiluminescence of hepatic macrophages from galactosamine-treated rats was 5- to 10-fold enhanced 12 hr following galactosamine treatment and remained elevated for 48 hr. Chemiluminescence of the latex particle-pretreated macrophages in the liver was markedly suppressed even following the galactosamine treatment (p < 0.01). Compared to galactosamine-treated rats, both lipid peroxide level in the liver tissue and AST and ALT concentration in serum were significantly decreased in the latex-galactosamine-treated rats < p < 0.01) and increased in the galactosamine-latex-treated rats (p < 0.01). Furthermore, superoxide dismutase supplementation protected against liver injury induced by the galactosamine-latex treatment. From these results, pretreatment with latex particles suppressed the free radical-generating capacity of hepatic macrophages and protected against hepatic injury induced by galactosamine. In contrast, injection of latex particles after galactosamine treatment aggravated hepatic injury, which was prevented by superoxide dismutase. These data suggest that liver injury induced by galactosamine is modulated by oxygen-derived free radicals from hepatic macrophages.
Comparative study of aminoglycoside nephrotoxicity in normal rats and rats with experimental cirrhosis
Authors: Camps Jordi , Sola Xavier , Rimola Antoni , Parés Albert , Rives Antoni , Salmeron Juan M. , Arroyo Vicente , Rodés Joan
Published: March 2007

Several authors have suggested that the risk of developing aminoglycoside nephrotoxicity is greater in cirrhotic patients than in the noncirrhotic population. However, this has not been confirmed by other investigators. To compare the intensity and characteristics of aminoglycoside nephrotoxicity in cirrhotic and normal rats, 31 rats with carbon tetrachloride-induced cirrhosis with ascites and 35 control rats were treated with gentamicin. Each group of rats was divided into two subgroups in order to receive 10 or 40 mg per kg per day of gentamicin, and different subsets of animals were killed on Days 4, 8 and 12 of treatment for renal histological examination and determination of renal tissue gentamicin concentration. Urine volume, osmolality, sodium excretion and N-acetyl-β-D-glucosaminidase activity were measured daily throughout the study. Creatinine clearance and trough plasma concentration of gentamicin were determined in each animal immediately before killing. There were no significant differences between cirrhotic and control rats in relation to the magnitude of changes in urine volume, osmolality, sodium excretion and N-acetyl-β-D-glucosaminidase activity and creatinine clearance during gentamicin administration. The values of a histopathological score semiquantitatively assessing the renal morphological changes observed by light microscopy were not significantly different in cirrhotic and control rats. In addition, similar trough plasma and renal cortical tissue concentrations of gentamicin were observed in both groups of animals. These results suggest that, in this experimental model, cirrhosis does not increase the risk for aminoglycoside nephrotoxicity.
Hepatic encephalopathy in cirrhotic and portacaval shunted dogs: Lack of changes in brain GABA uptake, brain GABA levels, brain glutamic acid decarboxylase activity and brain postsynaptic GABA receptors
Authors: Roy Sophie , Pomier-Layrargues Gilles , Butterworth Roger F. , Huet P.-Michel
Published: March 2007

It has been suggested, from studies of a rabbit model of fulminant hepatic failure, that hepatic encephalopathy might be related to an increase in brain γ-aminobutyric acid uptake through a more permeable blood-brain barrier, leading to an overactivity of brain γ-aminobutyric acid-mediated inhibitory neurotrans-mission. Five groups of dogs were studied: normal dogs, dogs with secondary biliary cirrhosis without and with hepatic encephalopathy and portacaval shunted dogs without and with hepatic encephalopathy. Brain γ-aminobutyric acid and sucrose uptake was investigated using the multiple indicator dilution curve technique in unanesthetized dogs. Tracer doses of 99mTc-labeled albumin (extracellular reference substance), 3H-labeled γ-aminobutyric acid and 14C-labeled sucrose prepared in autologous dog plasma were injected in one carotid artery, and dorsal sagittal sinus dilution curves were obtained. Uptake was calculated by comparing the areas under the 99mTc-labeled albumin and the [3H]γ-aminobutyric acid (or [14C]sucrose) curves from appearance to peak height. After killing, brain γ-aminobutyric acid levels were measured in the frontal cortex by high-performance liquid chromatography and glutamic acid decarboxylase activities using a radioenzymatic assay. Brain γ-aminobutyric acid postsynaptic receptors were assessed using [3H]muscimol binding studies. There were no significant changes in cirrhotic and shunted dogs with or without hepatic encephalopathy with regard to brain γ-aminobutyric acid and sucrose uptake, brain γ-aminobutyric acid levels and glutamic acid decarboxylase activities. [3H]Muscimol binding studies did not show any changes in the number nor in the affinity of postsynaptic γ-aminobutyric acid receptors. Moreover, trypan blue injected in four dogs with hepatic encephalopathy did not induce a staining of the brain. These findings support neither a selective increase in brain γ-aminobutyric acid uptake in hepatic encephalopathy nor a physical breakdown of the blood-brain barrier. Moreover, brain studies suggest that impaired central γ-aminobutyric acid-ergic function does not play an important role in the pathogenesis of hepatic encephalopathy in dogs with cirrhosis or portacaval shunts.
Computer simulation of portal venous shunting and other isolated hepatobiliary defects of the enterohepatic circulation of bile acids using a physiological pharmacokinetic model
Authors: Cravetto Cesare , Molino Gianpaolo , Hofmann Alan F. , Belforte Gustavo , Bona Basilio
Published: March 2007

The effect of three isolated defects in the enterohepatic circulation of bile acids on the size and distribution of the bile acid pool, plasma bile acid levels and bile acid secretion into the intestine was simulated using a linear multicompartmental physiological pharmacokinetic model previously used to simulate these aspects of bile acid metabolism in healthy man. Stepwise increases in portal-systemic shunting (with a reciprocal decrease in hepatic blood flow) caused an exponential increase in systemic plasma concentrations of bile acids, but no other major changes in bile acid metabolism. When the effect of varying fractional hepatic extraction was simulated, it was found that the greater the fractional hepatic extraction, the greater the elevation observed for systemic plasma bile acid levels for a given degree of portal-systemic shunting. When total hepatic blood flow was restored to normal by simulating “arterialization,” systemic plasma levels of bile acids decreased strikingly, yet remained elevated. For cholate with a fractional hepatic extraction of 0.9 and 100% portal-systemic shunting, arterialization caused a decrease from a 20-fold elevation to a 5-fold elevation. This simulation thus defined the effect of the presence of the portal venous system per se on plasma bile acid levels and also quantified the circulatory route by which substances reach the liver when portal-systemic shunting is present. An isolated defect in hepatic uptake of bile acids caused little change in overall bile acid metabolism other than modestly increased plasma levels. Loss of bile acid storage by the gallbladder caused the majority of the bile acid pool to move from the gallbladder compartments to the proximal small intestine during fasting but had little effect on the dynamics of the enterohepatic circulation during eating. The results of these novel simulations of isolated defects in bile acid transport should aid in the interpretation of the more complex changes in bile acid metabolism which are likely to occur in hepatic or biliary disease.
Regulation of bile acid synthesis. II. Effect of bile acid feeding on enzymes regulating hepatic cholesterol and bile acid synthesis in the rat
Authors: Heuman Douglas M. , Vlahcevic Reno Z. , Bailey Marsha L. , Hylemon Phillip B.
Published: March 2007

Bile acid synthesis is believed to be regulated by bile salts returning to the liver via the portal vein and suppressing cholesterol 7α-hydroxylase, the rate-limiting enzyme in the bile acid biosynthesis pathway. In order to characterize the relative effectiveness of bile salts in regulating bile acid synthesis, seven different bile acids were administered (1% w/w in chow) to rats over a 14-day period. Biliary bile salt composition was determined from bile samples obtained prior to killing; in all cases, the fed bile acid became the predominant bile salt in bile. The specific activities of microsomal cholesterol 7α-hydroxylase, HMG-CoA reductase and acylcoenzyme A:cholesterol acyltransferase were determined after killing.

Hydrophilic bile salts (ursocholic, hyocholic, ursodeoxycholic and hyodeoxycholic) did not inhibit HMG-CoA reductase or cholesterol 7α-hydroxylase activities. By contrast, more hydrophobic bile salts (cholic, chenodeoxycholic and deoxycholic) inhibited the activities of these two enzymes in order of increasing hydrophobicity. Neither hydrophobic nor hydrophilic bile salts inhibited acylcoenzyme A:cholesterol acyltransferase activity. No consistent effect of bile acid feeding on total microsomal cholesterol was observed.

Based on the results of these studies, we propose that the hydrophilic-hydrophobic balance of the bile acid pool may play an important role in the regulation of bile acid synthesis. We postulate that the activities of cholesterol 7α-hydroxylase and HMG-CoA reductase may be regulated by hydrophobic bile acid-induced changes in the lipid composition and physicochemical properties (fluidity) of the microsomal membranes to which both of these rate-limiting enzymes are attached. Alternatively, hydrophobic bile salts by increasing biliary cholesterol secretion and suppressing HMG-CoA reductase activity may reduce the availability of cholesterol substrate for bile acid synthesis.
Monoconjugated bilirubin is a major component of hemolysis-induced gallstones in mice
Authors: Trotman Bruce W. , Nair Rajagopalan C. , Bernstein Seldon E.
Published: March 2007

The role of bilirubin conjugates in the formation of pigment gallstones is not known. In this study, we completely solubilized and then analyzed by high-performance liquid chromatography specimens of black pigment gallstones from eight nb/nb mice with hereditary hemolytic anemia. Each dried gallstone specimen of about 200 μg was dissolved in 5 ml of dimethyl sulfoxide/0.15 M HCl/50 mM disodium-EDTA (8:1:1 by volume) at room temperature. Stone dissolution was complete by 30 min as monitored by the A456 and direct observation, and no oxidative products of bilirubin were observed in the visible spectrum, 350 to 750 nm. By high-performance liquid chromatography, the intact tetrapyrroles were separated as diconjugated and monoconjugated bilirubins; unconjugated bilirubin was resolved as XIII, IX and III α-isomers. The isocratic solvent system used was 0.1 M di-n-dodecylamine acetate/0.1 M di-n-octylamine acetate (4:1, v/v) in methanol, pH 7.4, at a flow of 1 ml per min. Diconjugated bilirubin accounted for 6.0 ± 2.4 molar % (mean ± S.E.), monoconjugated bilirubin for 37.4 ± 8.4% and unconjugated bilirubin for 56.3 ± 8.9% of the solubilized pigments. The IX α-isomer represented 96 ± 1.9% of the unconjugated bilirubin. The presence of bilirubin conjugates in gallstones was confirmed by ethylanthranilate diazotization: the conjugated azodipyrrole in stone had the same retention time as that of conjugated azodipyrrole from rat and mouse bile. A majority of the bilirubin conjugates was sensitive to β-glucuronidase of liver origin, indicating that the C-1 glucuronide ester was present. We conclude that (i) monoconjugated bilirubins are a significant component of black pigment gallstones from mice with hereditary hemolytic anemia, and (ii) the IX α-isomer is the predominant unconjugated bilirubin isomer in these gallstones. Monoconjugated bilirubin may play a role in black pigment stone formation as a coprecipitant and source of unconjugated bilirubin.
Wilson’s disease: Transplantation when all else has failed
Authors: Sternlieb Irmin
Published: March 2007

Experience with liver transplantation for patients with Wilson’s disease who have major neurological impairment is limited, and this report describes the results obtained in two such patients. The first was a 30-year-old man with a 14-month history of hepatic and neurological impairment. In spite of treatment with d-penicillamine, he developed increasing dysarthria, dysphagia, akinesia and rigidity of all four limbs, and required continuous nursing care. Following transplantation, liver function was almost normal from four weeks onwards, but recovery of neurological function was much slower and was not seen until two to three months after surgery. By four months he was sufficiently mobile to be discharged, and when he returned for assessment at eight months, no abnormal neurological signs were detectable. The second patient was a 27-year-old woman with worsening liver dysfunction for eight years; one year previously she had developed dysarthria, akinesia, a fine tremor and moderate rigidity of all limbs as well as marked psychological impairment. There was no improvement on treatment with d-penicillamine or trientine, but as liver function returned to normal two months after liver grafting, her neurological and psychological function began to improve so that by three months she could be discharged.
In situ cellular analysis of α-fetoprotein gene expression in regenerating rat liver after partial hepatectomy
Authors: Bernuau Dominique , Poliard Anne , Feldmann Gérard
Published: March 2007

Cellular analysis of hepatic α-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few α-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of α-fetoprotein secretion, 10 to 20% α-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2, 6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of α-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in α-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of α-fetoprotein mRNA sequences and a late cytoplasmic accumulation of α-fetoprotein mRNA molecules.
Serum vitamin A deficiency and increased intrahepatic expression of cytokeratin antigen in alcoholic liver disease
Authors: Ray Mukunda B. , Mendenhall Charles L. , French Samuel W. , Gartside Peter S.
Published: March 2007

The clinical and histologic significance of cytokeratin antigen expression in various intrahepatic locations was assessed in 57 patients with alcoholic liver disease as part of a large Veterans Administration Cooperative Study of Alcoholic Hepatitis. Cytokeratin antigen was demonstrated in fixed, paraffin-embedded liver tissue by an avidin-biotin peroxidase method using a mixture of two different monoclonal antibodies, AE1 (acidic; 48, 50 and 56.5 kD) and AE3 (basic; 52, 56, 58 and 65 to 67 kD). In contrast to the normal liver, in which only bile duct epithelium was positive, this antibody mixture stained both bile ducts and hepatocytes in pathologic livers.

Serum levels of vitamin A showed a significant inverse correlation with the amount of cytokeratin antigen (scale: 0 to 3) in hepatocytes without Mallory bodies (p = 0.001), in Mallory body-containing hepatocytes (p < 0.0001) and in bile ducts (p = 0.0074). Increased amount of cytokeratin antigen in each of these locations, in turn, correlated directly with the histologic severity of the liver disease. Histologic severity (fibrosis, parenchymal degeneration/necrosis, hepatocyte regeneration and inflammation) was significantly higher in patients when either Mallory bodies (p < 0.0001) or cytokeratin antigen (p = 0.0021) was present in hepatocytes. Demonstration of cytokeratin antigen in hepatocytes which contained Mallory bodies correlated positively (p = 0.03) with clinical severity of the liver disease as determined by high serum bilirubin and prolonged prothrombin time (Maddrey’s discriminant function). This study suggests that (i) vitamin A deficiency may be a triggering factor in aggregation of cytokeratin filaments, and (ii) the degree of aggregation of cytokeratin may determine the histologic severity and thereby the prognosis of patients with alcoholic liver disease.
Different mechanisms decrease hepatic collagen and albumin production in fasted rats
Authors: Chojkier Mario , Flaherty Michael , Peterkofsky Beverly , Majmudar Gopa H. , Spanheimer Robert G. , Brenner David A.
Published: March 2007

Weight loss is correlated with a specific decrease in collagen synthesis in extrahepatic tissues, mainly through modulation of mRNA levels. Here, we investigated the response to weight loss in the rat liver. Male rats were either fed ad libitum or fasted for 92 hr; fasted animals lost ˜20% of their initial body weight. Following i.p. injection of [5-3H]proline, hepatic collagen was extracted and de novo collagen production was measured. There was a decrease in the specific radioactivities of purified hepatic collagen (−75%) and albumin (−70%) relative to total hepatic protein, indicating that production of both of these proteins was specifically decreased. In fasted animals, the absolute hepatic collagen production was markedly decreased (−60%), while changes in absolute hepatic protein production were small (−15%). Using hybridization with specific DNA probes, we found that fasting causes about a 70% decrease in albumin mRNA, but the quantities of hepatic procollagen α1(I) and α2(I) mRNAs were unchanged. These results are consistent with regulation of albumin production during fasting by modulation of mRNA levels. The inhibition of hepatic collagen production in fasted animals, however, appears to be modulated at a posttranscriptional level or may result from increased degradation. This response differs from the pretranslational regulation of collagen synthesis in extrahepatic tissues during fasting. Furthermore, our results suggest that decreased body weight could be a potentially complicating variable in studies of collagen metabolism and fibrogenesis in the liver.
Activation of liver macrophages following phenobarbital treatment of rats
Authors: Laskin Debra L. , Robertson Fredika M. , Pilaro Anne M. , Laskin Jeffrey D.
Published: March 2007

Phenobarbital is a potent inducer of hepatic cytochrome P-450 and is a tumor promoter in the two-stage model of liver carcinogenesis. In the present studies, we show that phenobarbital also induces an accumulation of activated macrophages in the livers of treated rats. These macrophages are larger and more stellate than resident Kupffer cells and are highly vacuolated. In addition, macrophages isolated from livers of phenobarbital-treated rats display increased phagocytosis of sheep red blood cells, chemotaxis toward the complement fragment C5a and enhanced production of hydrogen peroxide. Biologically active mediators released by activated macrophages have been implicated in tumor promotion as well as in the regulation of cytochrome P-450-mediated drug biotransformation. We propose that the activated macrophages that accumulate in the liver following phenobarbital treatment may contribute, at least in part, to the biological responses to this drug.
Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: Possible role in microvesicular steatosis induced by this antibiotic
Authors: Fréneaux Eric , Labbe Gilles , Letteron Philippe , Dinh The Le , Degott Claude , Genève Jean , Larrey Dominique , Pessayre Dominique
Published: March 2007

Intravenous administration of high doses of tetracycline may produce severe microvesicular steatosis of the liver in man. A similar disease is observed after ingestion of drugs which inhibit hepatic mitochondrial fatty acid β-oxidation and in subjects with various inborn defects in this metabolic pathway. We therefore determined the effects of tetracycline on the mitochondrial oxidation of fatty acids in mice and in man.

In vitro, addition of tetracycline 0.25, 0.5, 1 or 2 mM inhibited by 15, 38, 56 and 65%, respectively, the formation of β-oxidation products during incubation of palmitic acid with mouse liver mitochondria and the various cofactors necessary for β-oxidation. Inhibition was reversible. Inhibition appeared even greater with human liver mitochondria.

Tricarboxylic acid cycle activity, assessed by the in vitro formation of [14C]CO2 from [1-14C]acetylcoenzyme A by mouse liver mitochondria, was inhibited by 25, 32 and 43%, respectively, in the presence of 0.5, 1 or 2 mM of tetracycline.

In vivo, administration of tetracycline, 0.25 or 1 mmole per kg, inhibited by 53 and 84%, respectively, the exhalation of [14C]CO2 during the first 3 hours following the administration of a tracer dose of [U-14C] palmitic acid. Administration of tetracycline, 0.0625, 0.25 or 1 mmole per kg, 6 hr before the measurement, increased hepatic triglycerides by 100, 170 and 250%, respectively. After 1 mmole per kg, accumulation of hepatic triglycerides was maximum at 24 hr, reaching 9-fold the control value; liver histology showed microvesicular steatosis at 6 and 24 hr.

We conclude that tetracycline inhibits the mitochondrial oxidation of fatty acids in mice and in man. This effect may contribute to the development and severity of microvesicular steatosis observed after high doses of this antibiotic in man.