The Effect of Ethanol Metabolism on Ferritin Uptake by Freshly Isolated Rat Hepatocytes: Is Acetaldehyde Responsible for This Alteration?

Alcohol abuse is associated with disturbances to iron metabolism in man, ranging from anemia to siderosis. Also seen in these patients are increased serum ferritin levels. Since the liver not only stores iron in cytosolic ferritin, but has also been shown to take up this molecule from the plasma by an active transport mechanism, it has been suggested that the iron in this circulating ferritin may contribute to the increased incidence of siderosis seen in alcoholics. As part of an ongoing study of these disturbances, using a rat model, we have examined the uptake of ferritin by freshly isolated hepatocyte suspension to test the hypothesis that increased hepatocyte uptake of ferritin iron contributes to the siderosis seen in some alcoholics. Incubation of hepatocytes in the presence of ethanol resulted in a progressive reduction in uptake with increasing alcohol concentration, from 1.23 +/- 0.05 ng of ferritin/10(6) cells/min to 0.65 +/- 0.02 ng/10(6) cells/min (mean +/- SD) at an ethanol concentration of 100 mM. 4-Methylpyrazole (0.1 mM) restored 70% of this activity, but higher concentrations also decreased ferritin uptake in the absence of ethanol. The addition of 5 microM cyanamide decreased ferritin uptake slightly in the presence of ethanol (0.82 +/- 0.04 ng of ferritin/10(6) hepatocytes/min vs. 0.86 +/- 0.03 ng/10(6) cells/min for ethanol alone), while having no effect in the absence of ethanol (1.01 +/- 0.04 vs. 1.12 +/- 0.05 ng/10(6) cells/min). Preincubation of the hepatocytes with acetaldehyde resulted in a dose-dependent reduction to a maximum reduction of approximately 25% at 300 microM acetaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron Uptake from Transferrin and Asialotransferrin by Hepatocytes from Chronically Alcohol-Fed Rats

Chronic alcohol intake is often associated with alterations to iron homeostasis and an increase in the serum levels of carbohydrate-deficient transferrin. As the liver is a major iron storage site and also synthesizes transferrin, the normal serum iron transport protein, the aim of this study was to test the hypothesis that these disturbances in iron homeostasis were caused by altered hepatocyte iron uptake from the abnormal transferrin. To achieve this, we have investigated iron uptake from both transferrin and asialotransferrin by hepatocytes from male Sprague-Dawley rats fed the De Carli and Lieber alcohol diet. Iron uptake from transferrin by hepatocytes from alcoholic rats was less than 60% that of control values, and in the presence of 50 mM ethanol decreased still further to 35% of the uptake by the corresponding control cells. Iron uptake from rat asialotransferrin was reduced in both groups when compared to that observed from normal transferrin; 13% by control cells and 39% by hepatocytes from alcohol-fed rats. Alcohol, however, had no further effect on asialotransferrin uptake by either hepatocytes from alcohol-fed rats, or their pair-fed controls. Transferrin binding to hepatocytes was also influenced by the alcohol diet. Although there was no difference in binding at 37 degrees C, cells from alcohol-fed rats bound 85% of this total at 4 degrees C, compared to 44% by control hepatocytes. Similar values were also obtained for hepatocyte binding of asialotransferrin; alcohol feeding resulted in an increase in binding at 4 degrees C to 73% from 58% with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in Glycosylated and Nonglycosylated Serum Ferritin in Chronic Alcoholism and Their Evolution during Alcohol Withdrawal

Increase in serum ferritin, which occurs in 40 to 70% of chronic alcoholics, remains poorly understood. We tested the hypothesis which links hyperferritinemia in chronic alcoholism not only to ferritin release from damaged liver cells, but also to increased ferritin secretion. Fifty-eight chronic alcoholic patients hospitalized for alcohol withdrawal were subdivided into three groups according to liver damage. Their serum levels of ferritin and ferritin bound to concanavalin A (ferritin Con A, which represents glycosylated, i.e., secreted ferritin) were measured serially on days 1, 7, and 11 of withdrawal and compared with a control group. The results were: (1) Total serum ferritin increased in alcoholics. Both free and Con A ferritins increased in equal proportions, the ferritin Con A to total ferritin ratio remaining unchanged. The increase was dependent on liver disease, as both free and Con A ferritins increased significantly with the severity of liver illness. Serum ferritin levels were related to iron status: it correlated with hepatic iron concentration (obtained in 19 patients); however, high ferritin values were not related to the degree of iron overload, which remained low. Finally, there was no correlation between serum ferritin and the average of alcohol consumption. (2) Both free and Con A ferritin decreased by about 40% during alcohol withdrawal. In conclusion, we have demonstrated that (1) total serum ferritin is increased in chronic alcoholism and (2) that this ferritin increase is due in part to an increase in ferritin Con A, proof of the induction of ferritin secretion by alcohol in humans.     

Estimating Iron Overload in Patients with Suspected Liver Disease and Elevated Serum Ferritin

BackgroundIron status evaluation in patients with suspected liver disease and elevated serum ferritin is often challenging because hyperferritinemia does not always indicate iron overload. A reliable approach to estimate iron overload without exposing the patient to unnecessary investigations would help the clinician to identify patients who may take advantage of iron-removal therapy.MethodsWe analyzed all liver biopsies, including measurement of hepatic iron concentration, performed at the University Hospital Zurich from 1997 to 2010 to identify clinical and laboratory predictors of iron overload in patients with elevated serum ferritin (n = 147).ResultsHyperferritinemia was predictive of iron overload only in patients with a high level of serum ferritin (>2000 μg/L). In patients with moderate hyperferritinemia, liver transaminases inversely correlated with hepatic iron concentration. A combination of both parameters expressed as ferritin/aspartate transaminase ratio was highly predictive of tissue iron overload (sensitivity 83.3%, specificity 78.6%). Receiver operating characteristic analysis resulted in an area under the curve of 0.83.ConclusionsWe established a simple and reliable method to correctly estimate iron overload in patients with suspected liver disease and elevated serum ferritin.     

Distribution of Alcohol Dehydrogenase and the Low Km Form of Aldehyde Dehydrogenase in Isolated Perivenous and Periportal Hepatocytes in Rats

Hepatic damage induced by chronic alcohol abuse starts in the perivenous (PV) zone of the hepatic lobule. To explain this vulnerability in the PV zone, periportal (PP) and PV hepatocytes were isolated by digitonin-collagenase perfusion and the distributions of class I alcohol dehydrogenase (ADH) and the low-Km mitochondrial aldehyde dehydrogenase (ALDH) were studied. ADH was measured by three approaches i.e., specific activity, immunoreactive enzyme content and ADH mRNA level. ALDH was determined by specific activity and immunoreactive enzyme content. When compared with PV hepatocytes, isolated PP cells exhibited higher lactate dehydrogenase (PP/PV = 1.3-1.5), higher alanine aminotransferase (PP/PV = 1.7-1.9), but lower glutamine synthase (PP/PV less than 0.01). By prelabeling the PP zone with acridine orange before digitonin-collagenase digestion, flow cytometry indicated that mainly the isolated PP hepatocytes exhibited fluorescence. ADH activities and ADH mRNA levels did not differ in PP and PV cells. With a polyclonal antibody directed specifically against class I ADH, ADH immunoreactive protein also did not differ in PP vs PV cells. By activity assay, the low Km ALDH activities were found to be lower in the PV hepatocytes (PP/PV = 1.3). This was confirmed by immunotransblot with anti-ALDH IgG (PP/PV = 1.6). In conclusion: the preferential damage of the PV zone produced by ethanol is not caused by differences of ADH distribution in liver but could be related partly to a decrease in the low-Km ALDH in the PV zone.     

Alcohol Mediates Increases in Hepatic and Serum Nonheme Iron Stores in a Rat Model for Alcohol-Induced Liver Injury

The notion that prolonged ethanol consumption promotes hepatocellular damage through interactions with iron was evaluated in rats fed ethanol with or without supplemental dietary carbonyl iron. The individual and combined pro-oxidant potential of these agents was evaluated in terms of their ability to perturb iron homeostasis and initiate hepatocellular injury. Sprague-Dawely rats received a high fat liquid diet for 8 weeks supplemented with 35% ethanol-derived calories (Alcohol group), 0.02 to 0.04% (w/v) carbonyl iron (Iron group), ethanol plus carbonyl iron (Alcohol + Iron group), or a diet containing carbohydrate-derived isocaloric calories (Control group). Hepatic and serum nonheme iron stores were significantly elevated (p < 0.05) in all treatment groups, compared with the Controls. Catalytically active low-molecular weight iron was detected in rats consuming alcohol and was markedly elevated (p < 0.05) in rats ingesting iron alone or iron in combination with alcohol. Elevations in serum ALT indicated significant hepatocellular injury in rats ingesting only alcohol, but was most prominent in the rats consuming ethanol in combination with iron (p < 0.05). Significant hepatic fatty infiltration, increased hydroxyproline content, and perturbations in reduced glutathione were also observed in the Alcohol and Iron treatment groups. Histochemical assessment of hepatic iron sequestration revealed that alcohol feeding resulted in deposition of ferric iron in the centrilobular area of the liver lobule. This unique alcohol-mediated iron deposition was histologically graded above Control group and was observed in both hepatocytes and Kupffer cells. Data presented herein suggest that alcohol alone or in combination with iron results in rather specific lobular patterns of hepatic iron deposition relevant to iron overload observed in human alcoholics. Furthermore, data suggest that alcohol- and iron-initiated prefibrotic events occur before extensive hepatocellular necrosis.     

Effects of Chronic Ethanol Administration on the Endocytosis of Cytokines by Rat Hepatocytes

The effects of chronic ethanol administration on the endocytosis of three representative cytokines were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 12 to 13 weeks, these cells exhibited a decreased ability to internalize and degrade transforming growth factor-α, tumor necrosis factor-α and interleukin-6, compared with hepatocytes from the pair-fed controls. This impaired endocytosis of all three cytokines was accompanied by significant decreases in the amount of hepatocyte surface-bound cytokine. Changes in cytokine binding to surface receptors and reduced rates of receptor-cytokine complex internalization into the cells seem to be major contributors to defective endocytosis in hepatocytes from the ethanol-fed rats. Impaired hepatocyte endocytosis could lead to altered steady-state levels of cytokines in the liver and modified physiological responses to cytokines. These changes could affect homeostasis among the various cell types in the liver and could contribute to liver dysfunction and injury.     

Chronic Alcohol Feeding in Liquid Diet or in Drinking Water Has Similar Effects on Electron Microscopic Appearance of the Hepatic Sinusoid in the Rat

Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronically in a complete liquid diet or in sucrose-containing drinking water. The animals were kept on liquid diet (±alcohol) for 14 weeks or on sucrose-containing drinking water (±alcohol) for 12.5 weeks and sacrificed thereafter. To rule out possible artifact induced by fixation procedure, livers were fixed by immersion (no perfusion), immersion preceded by perfusion, and by perfusion with glutaraldehyde and examined with both scanning and transmission electron microscopy. Regardless of the mode of its administration, and of the fixation procedure used, alcohol induced similar changes in liver sinusoid ultrastructure. Such changes included disruption of the sieve-plate pattern of the sinusoidal endothelial cell fenestrations with the appearance of large gaps and resulting in a meshwork lining, wherein large areas of the sinusoid communicated freely with the underlying hepatocytes. Transmission electron microscopy complemented these findings. The results reported in this study demonstrate that alcohol-induced structural changes of the liver sinusoid in the rat are similar whether alcohol is fed via a liquid diet or in drinking water. Therefore, alcohol administration in drinking water may provide a simple, inexpensive, and convenient method of inducing structural changes in the rat liver sinusoid.     

Ferritin in bone marrow and serum in iron deficiency and iron overload

Summary Nonheme iron and ferritin in the bone marrow and serum ferritin was investigated in patients with iron deficiency anaemia or iron overload. As controls served patients without any disturbance of the iron metabolism.There is a precise correlation between the nonheme iron and ferritin in the bone marrow of patients with and without disturbance of iron metabolism. A correlation was also found between the ferritin in the bone marrow and the serum. Nonheme iron and ferritin in the bone marrow and serum ferritin was decreased in patients with iron deficiency anaemia. Conversely, the same parameters were increased in patients with iron overload.     

Effects of Alcohol, Carbon Tetrachloride, and Choline Deficiency on Iron Metabolism in the Rat

The effects of alcohol on hepatic iron uptake and intestinal iron transport were studied in rats fed a nutritionally replete liquid diet containing varying quantities of ethanol. Results were compared with those from animals exposed to carbon tetrachloride (CCI₄) to produce hepatocellular necrosis or a choline-deficient diet to produce steatosis and cirrhosis. A high ethanol intake for 4 or 10 weeks produced hepatic steatosis. CCU produced hepatocellular necrosis. Choline deficiency was associated with steatosis ± cirrhosis. Intestinal iron transport was unaffected by ethanol, CCU, or choline deficiency. Hepatic iron uptake was significantly depressed in rats consuming 11.7 g/kg/day ethanol (p < 0.01) for 4 weeks. Choline-deficient animals studied at 14 weeks also had significantly decreased hepatic iron uptake (p < 0.01); results were similar in the cirrhotic and noncirrhotic animals. Conversely, CCI₄ exposure produced a significant 5-fold increase in hepatic iron uptake (p < 0.001). Results suggest that ethanol consumption, fatty liver, and cirrhosis are not responsible for any increase in iron absorption or of hepatic iron uptake in the rat model. Acute hepatocellular injury is followed by increased hepatic iron uptake.     

Efficacy for Anemia and Changes in Serum Ferritin Levels by Long‐Term Oral Iron Administration in Hemodialysis Patients

Oral iron preparations are useful for the treatment of anemia in hemodialysis patients; however, long‐term changes in the iron dynamics and effects of anemia are unknown. Serum ferritin levels and erythropoietin‐stimulating agent (ESA)/hemoglobin (Hb) ratios were investigated for 750 days in the following four groups: patients treated with sodium ferrous citrate (SF) (de novo 50 mg/day and 150 mg/day switched from 50 mg/day) and patients treated with 1500 mg/day of ferric citrate (FC) (de novo and switched from 50 mg/day of SF). Compared with the other groups, serum ferritin levels increased less apparently with de novo 50 mg/day SF. ESA/Hb ratios did not change in groups switched from 50 mg/day SF. Conversely, in groups with de novo iron, ESA/Hb ratios decreased and ultimately reached the same levels in all groups. Although more iron results in higher serum ferritin levels, 50 mg/day SF has an equivalent effect for anemia treatment.     

Non-Transferrin-Bound Iron in Alcohol Abusers

BACKGROUND: Non-transferrin-bound iron, a low-molecular-weight iron complex capable of initiating free radical formation and lipid peroxidation, has been detected in the serum of animals experimentally fed with alcohol, but no data have been reported in alcohol abusers. The purpose of this study was to evaluate whether non-transferrin-bound iron is present in chronic alcohol abusers with liver involvement and whether alcohol plays any part in its appearance. METHODS: We measured non-transferrin-bound iron in a cohort of chronic alcohol abusers with and without liver cirrhosis at presentation, when 43 were active abusers and 33 were abstainers, and in a smaller group during a follow-up period. RESULTS: At presentation, non-transferrin-bound iron was detectable in 83.7% of active abusers but only in 21.2% of abstainers, and within the group of abusers, patients with cirrhosis had significantly higher non-transferrin-bound iron than patients without. Non-transferrin-bound iron was present not only in patients with transferrin saturation >45% but also in those with transferrin saturation < or =45%. Multiple regression analyses revealed that only alcohol intake and total bilirubin were associated independently with non-transferrin-bound iron values. Longitudinal study confirmed the data of the cross-sectional study. CONCLUSIONS: Non-transferrin-bound iron could have a role in initiating or promoting alcohol-induced liver damage.     

Expression and Cytoskeletal Association of Integrin Subunits Is Selectively Increased in Rat Perivenous Hepatocytes After Chronic Ethanol Administration

BACKGROUND: For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. METHODS: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. RESULTS: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. CONCLUSIONS: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.     

Iron storage in macrophages and endothelial cells. histochemistry,ultrastructure, and clinical significance

Summary l hour after i. v. infusion of colloidal iron in iron deficient subjects uniform phagosomal iron granules were observed in macrophages and endothelial cells of several organs. 7 to 10 days later transformation into ferritin could be visualized in macrophages only. Now, these cells showed diffuse iron staining of the cytoplasm due to dispersed ferritin molecules. Polymorphous lysosomes contained densely packed particles from still unchanged ferric hydroxide to paracristalline ferritin. The macrophageal iron was mobilizable in few days to several weeks. The uniform lysosomal iron granules of endothelial cells disappeared after 1 to 2 years. Endothelial iron siderosis without previous i.v. iron application was a frequent finding in pernicious anaemia and iron overload of diverse origin.

---

Zusammenfassung l Stunde nach i.v. Infusion kolloidaler EisenprÄparate wurden gleichförmige phagosomale Eisengranula in Makrophagen und Endothelzellen verschiedener Organe gefunden. 7 bis 10 Tage spÄter konnte eine Umwandlung in Ferritin nur in Makrophagen sichtbar gemacht werden. Diese Zellen zeigten nun eine diffuse, durch fein verteiltes Ferritin bedingte EisenfÄrbung des Zytoplasma. Die polymorphen Lysosomen enthielten dicht gelagerte Eisenteilchen von z.T. noch unverÄndertem Ferrihydroxyd bis zu parakristallinem Ferritin. Das Makrophageneisen war in wenigen Tagen bis zu mehreren Wochen mobilisierbar. Die uniformen lysosomalen Eisengranula der Endothelzellen schwanden nach 1 bis 2 Jahren. Eine endotheliale Siderose ohne vorherige i.v. Eisengabe war ein hÄufiger Befund bei perniziöser AnÄmie und Eisenüberladung verschiedener Ätiologie.

     

Effect of Chronic Alcohol Ingestion on the Binding of High Density Lipoproteins to Rat Hepatic Membranes: Involvement of Apolipoprotein E

Feeding alcohol to rats produces high density lipoproteins (HDL) particles that exhibit lowered apolipoprotein (apo) E:apo A1 ratio. In this study, we have carried out experiments to compare the abilities of apo E-deficient HDL particles of the alcohol-fed rat and apo E-sufficient HDL particles of the control rat to bind to hepatic membranes. When rat hepatic membranes were incubated with rat serum HDL of physiological concentrations (< or = 200 micrograms HDL-apo A1/ml), binding of HDL to hepatic membranes showed concentration dependent on HDL-apo A1. Polyclonal antibodies that specifically recognize apo A1 and apo E inhibited HDL binding to hepatic membrane while the antibody against apo AIV did not. The binding of 125I-apo A1-HDL was diminished by adding excess amount of unlabeled HDL to the incubation mixture. Apo E-deficient serum HDL obtained from alcohol-fed rats competed less efficiently against radiolabeled HDL for binding to rat hepatic membrane than normal HDL from control animals. The defect in apo E-deficient serum HDL obtained from alcohol-fed rats can be corrected by preincubation with added purified apo E. We hypothesize that this weaker binding may result in slower degradation of apo E-deficient HDL particles by the liver and in part explains the higher plasma HDL levels found in alcohol-drinking animals.     

Ferritin in erythrocytes and plasma of patients with iron overload

Summary Erythrocyte and plasma ferritin was followed in 13 patients with iron overload undergoing phlebotomies for at least 6 months in comparison with untreated patients and normal males. Plasma ferritin was widely scattered with an average of only twice the normal, whereas erythrocyte ferritin was highly elevated to about twelve times the normal (p<0.0001). — The time course of plasma and erythrocyte ferritin during phlebotomy therapy was analyzed in 3 patients with idiopathic hemochromatosis. Three stages were established: 1. plasma ferritin dropped gradually into the normal range while erythrocyte ferritin remained high, 2. appropriate phlebotomies maintained normal plasma ferritin and high erythrocyte ferritin, and indicated a monthly uptake of dietary iron of 150–200 mg at a steady state, 3. at low plasma ferritin levels, erythrocyte ferritin was rapidly decreased by further intensive phlebotomy therapy. Based on the presumed net removal of iron, 1 g/l plasma ferritin was equivalent to 3–6 mg of body iron and 1 g/l erythrocyte ferritin to somewhat less than 1 mg of body iron. — An elevated erythrocyte ferritin during phlebotomy therapy in iron overload not only depends on body iron stores like plasma ferritin but may also be regulated by the activity of erythropoiesis.     

Alcohol Induction of Ferritin Expression in a Human Hepatoblastoma Cell Line (HEP G2)

Hyperferritinemia, an unclear mechanism, is frequently observed in chronic alcoholics. The aim of this work was to study the effect of alcohol on ferritin expression in a human hepatoblastoma cell line, HepG2. This cell line proved to be sensitive to alcohol, since alcohol increased gamma-GT activity both in cells and media. The most striking result was the increase of ferritin in cells and media by alcohol. Moreover, this effect was specific, since it contrasted with a decrease in total protein synthesis and secretion, a decrease in transferrin excretion and a lack of effect on orosomucoid. In our model, alcohol was able to induce, in a specific manner, ferritin expression.     

The Uptake of Ferric Iron by Rat Liver Ferritin in Vivo and in Vitro

S ummary . The incorporation of ferric iron labelled with ⁵⁹Fe into rat liver ferritin has been studied in whole animals, into liver homogenate and into purified protein. Uptake of Fe³⁺ into purified rat liver ferritin followed a pattern similar to that with horse spleen ferritin given Fe²⁺ and an oxidant. The distributions of iron incorporated as a function of molecular iron content obtained in vivo resembled the in vitro patterns for iron contents above 500 Fe atoms/molecule and times after injection of up to 12 h. At larger intervals a maximum label moved to molecules of highest iron content as the molecules accumulated more iron. The apparently reduced uptake of injected ⁵⁹Fe into molecules of low iron content might be due either to the chase of cold iron through an existing iron pool or the presence of functionally different ferritins at more than one anatomic site within the cell.