The cellular labile iron pool and intracellular ferritin in K562 cells.

The labile iron pool (LIP) harbors the metabolically active and regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels. Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 micromol/L, as monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator-free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to reappear was relatively small and occurred within 1 hour, whereas the second was larger and relatively slow to occur, paralleling the decline in intracellular ferritin level (t1/2= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation. The changes in the LIP were also inversely reflected in the activity of iron regulatory protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively similarly in response to long-term treatments with the iron chelator deferoxamine, although L-ferritin declined more rapidly, resulting in a 4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not H-ferritin, was partially attenuated by the lysosomotrophic agent, chloroquine; on the other hand, antiproteases inhibited the degradation of both subunits to the same extent. These findings indicate that, after acute LIP depletion with fast-acting chelators, iron can be mobilized into the LIP from intracellular sources. The underlying mechanisms can be kinetically analyzed into components associated with fast release from accessible cellular sources and slow release from cytosolic ferritin via proteolysis. Because these iron forms are known to be redox-active, our studies are important for understanding the biological effects of cellular iron chelation.     

Hepatic ferritin uptake and hepatic iron

The effect of hepatic iron on the uptake of ferritin was studied by perfusing livers from normal, iron-deficient and iron-loaded rats with 125I-labeled ferritin. Unlabeled ferritin with tracer doses of labeled ferritin in concentrations of 0.02 to 2,700 nmol/L were studied. Rats were made iron deficient by feeding an established iron-deficient diet for 3 wk. Rats were iron loaded by injection of iron dextran (50 mg/wk) for 3 wk. The mean percentage of uptake of ferritin was similar for doses ranging from 0.22 to 22.2 nmol/L of 125I-labeled ferritin. Uptake of ferritin in the normal animal was saturable, with an apparent maximal velocity of uptake of approximately 9.1 pmol/gm/min and a Michaelis-Menten constant of approximately 5 nmol/L at 37 degrees C. Uptake was minimal at 4 degrees C. The mean uptake of ferritin was 78% +/- 10% in the iron-deficient rats (mean hepatic iron = 1.5 mumol/gm), 79% +/- 10% in the normal animals (mean hepatic iron = 9.2 mumol/gm) and 78% +/- 8% in the iron-loaded animals (mean hepatic iron = 192 mumol/gm). In this experimental system, modulation of hepatic iron did not affect uptake of ferritin, suggesting that regulation of the hepatic ferritin receptor may not depend on hepatic iron content. The rapid uptake of ferritin by the liver despite iron overload is consistent with other observations of the nonregulation of non-transferrin-bound iron by hepatic iron and may play a role in the progressive iron overload seen in hemochromatosis.     

The Relationship between Body Iron Stores and Ferritin Turnover in Rat Liver and Intestinal Mucosa

A single dose of oral or parenteral iron is known to stimulate ferritin synthesis in the liver and small intestine in experimental animals. A study has been carried out on the ferritin content and ferritin turnover in rat liver and intestinal mucosa in iron deficient, normal and iron loaded animals. [I-¹⁴C]DL-leucine incorporation into ferritin was used as a measure of its synthesis.
A significantly greater ferritin accumulation was found in the intestinal mucosa of the group of iron loaded animals compared with normal or iron deficient rats ( P < 0.001). In the liver the ferritin protein, ferritin iron and labelled ferritin content was significantly different between all three groups ( P < 0.001), the amounts being higher in the iron loaded animals and lower in the iron deficient animals when compared with the normal group. These results may explain how the absorption of iron in the small intestine is controlled by body iron stores.     

Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein

Like the rat peritoneal macrophage, the isolated Kupffer cell is capable of processing and releasing iron acquired by phagocytosis of immunosensitized homologous red blood cells. When erythrophagocytosis is restrained to levels which do not affect cell viability, about one red cell per macrophage, close to 50% of iron acquired from red cells is released within 24 hr in the form of ferritin. Immunoradiometric assay of the extracellular medium indicates that 160 ng ferritin are released by 10(6) Kupffer cells after 24-hr incubation at 37 degrees C. Iron release is temperature-dependent, the rate at 37 degrees C being nearly 5-fold greater than at 4 degrees C. As estimated by sucrose-gradient ultracentrifugation, ferritin released by the erythrophagocytosing Kupffer cell averages 2,400 iron atoms per molecule. When reincubated with isolated hepatocytes, this released ferritin is rapidly taken up by the cells. Via this process, hepatocytes may accumulate more than 160,000 iron atoms per cell per min. Such accumulation is not impeded by the presence of iron-loaded transferrin in the culture medium, but is markedly depressed by rat liver ferritin. In contrast to the conservation of transferrin during its interaction with hepatocytes, the protein shell of the ferritin molecule is rapidly degraded into trichloroacetic acid-soluble fragments. Ferritin-mediated transfer of iron from Kupffer cells to hepatocytes may help explain the resistance of the liver to iron deficiency as well as the liver's susceptibility to iron overload.     

Differential response of non-transferrin bound iron uptake in rat liver cells on long-term and short-term treatment with iron.

BACKGROUND: Uptake of non-transferrin-bound iron by the liver is important as a clearance mechanism in iron overload. In contrast to physiological uptake via receptor-mediated endocytosis of transferrin, no regulatory mechanisms for this process are known. This study compares the influence of long-term and short-term depletion and loading of hepatocytes with iron on the uptake of non-transferrin bound iron, its affinity, specificity and the interaction with the transferrin-mediated pathways. METHODS: Rats were fed iron-deficient, normal and 3,5,5-trimethylhexanoyl-ferrocene-containing diets to obtain livers with the corresponding desired status and the hepatocytes from these livers were used for transport studies. Hepatocytes from normal rats were depleted or loaded with iron by short-term treatment with desferrioxamine or ferric ammonium citrate, respectively. Uptake of non-transferrin bound iron was assayed from ferric citrate and from ferric diethylene triammine pentaacetate. RESULTS: Uptake of non-transferrin-bound iron in hepatocytes could be seen as consisting of a high-affinity (Km=600 nM) and a low-affinity component. Whereas in normal and in iron-starved rats the high-affinity component was more prominent, it disappeared altogether in hepatocytes from rats with iron overload resulting from prolonged feeding with TMH-ferrocene-enriched diet. Overloading also led to loss of inhibition by diferric transferrin, which occured in starved as well as normal cells. In contrast, short-term iron-depletion of isolated hepatocytes with desferrioxamine had only a weak stimulatory effect, whereas treatment with ferric ammonium citrate strongly increased the uptake rates. However, the inhibition by diferric transferrin also disappeared. In both cases, uptake of non-transferrin bound iron was inhibited by apotransferrin. CONCLUSIONS: Non-transferrin bound iron uptake in liver cells is apparently regulated by the iron status of the liver. The mode of response to iron loading depends on the method of loading in terms of time course and the form of iron used. It cannot be explained by the behavior of the iron regulatory protein, and it is complex, seeming to involve more than one transport system.     

Regulation of intracellular iron metabolism in human erythroid precursors by internalized extracellular ferritin.

Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with (59)Fe- and/or (125)I-labeled acid isoferritin and chased. The (125)I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, (59)Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.     

Effect of Chronic Alcohol Feeding on Hepatic Iron Status and Ferritin Uptake by Rat Hepatocytes

Alcohol abuse is known to cause disturbances to iron homeostasis in man and is associated with elevated serum ferritin levels. We have previously shown that ethanol metabolism in the rat hepatocyte is associated with an immediate reduction in ferritin uptake by this cell. In this study we have examined the effect of pair-feeding the Lieber-DeCarli liquid alcohol diet on ferritin uptake by rat hepatocytes. Rat liver ferritin was radiolabeled with ⁵⁹Fe in vivo and isolated by conventional techniques. Rats were pair-fed the Lieber-DeCarli liquid alcoholic diet for 4–6 weeks. Hepatocytes, isolated from their livers by collagenase perfusion, were incubated with [⁵⁹Fe]ferritin in L-15 medium at 37°C and 4° to measure ferritin uptake and binding. The in vitro effect of ethanol on these hepatocytes was also studied. Ferritin and iron parameters were measured in the sera and hepatocytes of these animals and a comparable group of normal chowfed rats. The rate of ferritin uptake by hepatocytes from alcohol-fed rats was significantly faster than that of their pair-fed controls (0.743 ± 0.061 vs. 0.540 ± 0.042 ng/min/10⁶ cells, p < 0.05). However, the rats on Lieber-DeCarli control diet exhibited a lower hepatocyte ferritin uptake rate than chow-fed animals (79.3 ± 8.1% of the control values, p < 0.01). In vitro incubation of cells in 100 mm ethanol resulted in less inhibition of ferritin uptake by hepatocytes from alcoholic rats than from their pair-fed controls (11 ± 7.1% inhibition vs. 43.6 ± 10.7% for controls, p < 0.05). Receptor-mediated binding of ferritin to hepatocytes showed a 61% increase in saturable binding capacity for alcoholic rats (15,820 ± 4950 molecules/cell vs. 9798 ± 3622, p= 0.05). The presence of ethanol in the medium did not affect ferritin binding significantly. Although there was no significant difference in the serum iron values between all three groups, transferrin concentrations were markedly elevated in the alcohol-fed rats, resulting in a much lower transferrin iron saturation than for the control animals. Because the corresponding serum values for the diet controls were intermediate between those for the alcohol-fed rats and the chow-fed animals, these findings may reflect dietary restriction by the liquid diet, which is exacerbated by the addition of alcohol. These findings suggest that there is increased iron uptake by the hepatocyte following chronic alcohol administration, which may be due to the increased ferritin receptors. This is supported by the observation that this alcohol treatment also causes a depletion of serum ferritin. However, the decreased iron content in the alcohol-fed rats indicate that this may be due to a response to changes in iron homeostasis by the hepatocyte and/or redistribution in the body.     

Hepatocyte iron kinetics in the rat explored with an iron chelator

S ummary . The hepatocyte metabolism of ⁵⁹Fe-labelled ferritin, haemoglobin-haptoglobin and transferrin has been examined in rats. All three forms of ⁵⁹Fe became transiently available to desferrioxamine (DF) at the time they would otherwise have entered storage or alternative pathways of iron metabolism. However, differences in both the patterns of spontaneous ⁵⁹Fe reutilization by normal and iron deficient rats and the partition of chelate iron excretion between bile and urine, suggested that iron in transit within hepatocytes did not behave as a single common pool. Ferritin ⁵⁹Fe, entering a pool of non-radioactive iron the size of which is determined by liver iron stores, was chelated predominantly into the bile. Transferrin ⁵⁹Fe was distinguished by a greater reflux to the erythron in iron deficient rats, and by excretion of a larger proportion of ⁵⁹Fe chelated by DF in the urine. Haemoglobin-haptoglobin ⁵⁹Fe followed a metabolic pathway which was relatively independent of both the iron stores and DF. If the heterogeneous behaviour of rat hepatocyte transit iron has a parallel in man, alterations in the size of similar chelatable iron pools could explain the dependence of DF-induced urine and faecal iron excretion on both liver iron stores and the level of erythropoiesis.     

Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool

Evaluation of myocardial iron during iron chelation therapy is not feasible by repeated endomyocardial biopsies owing to the heterogeneity of iron distribution and the risk of complications. Recently, we described a noninvasive method based on magnetic resonance imaging. Here, the method was used for repeated estimation of the myocardial iron content during iron chelation with deferrioxamine in 14 adult nonthalassemic patients with transfusional iron overload. We investigated the repeatability of the method and the relationship between the myocardial iron estimates and iron status. The repeatability coefficient (2sD) was 2.8 micromol/g in the controls (day-to-day) and 4.0 micromol/g in the patients (within-day). Myocardial iron estimates were elevated in 10 of all 14 patients at first examination, but normalized in 6 patients after 6 to 18 months of treatment. If liver iron declined below 350 micromol/g all but one of the myocardial iron estimates were normal or nearly normal. At start (R2 = 0.69, P =.0014) and still after 6 months of iron chelation (R2 = 0.76, P =.001), the estimates were significantly and more closely related to the urinary iron excretion than to liver iron or serum ferritin levels. In conclusion, our preliminary data, which may only pertain to patients with acquired anemias, suggest the existence of a critical liver iron concentration, above which elevated myocardial iron is present, but its extent seems related to the size of the chelatable iron pool, as reflected by the urinary iron excretion. This further supports the concept of the labile iron pool as the compartment directly involved in transfusional iron toxicity.     

Serum Ferritin in Ascorbic Acid Deficiency

S ummary Serum ferritin levels were monitored in guinea-pigs rendered scorbutic by dietary deprivation of ascorbic acid (AA). Hepatic and splenic concentrations of AA fell rapidly during the first 8 d of deprivation, and then declined more slowly to reach approximately 10% of control values after 14 d. Normal levels of serum ferritin (geometric mean, 324 μg/1) were observed for 20 d but a significant fall occurred between days 20 and 23 of deficiency (143 γg/1; P <0·05).
Intramuscular administration of iron dextran to scorbutic animals (75 mg Fe per kg body weight) resulted in a non-significant elevation of serum ferritin levels (geometric mean, 470 μg/1; P >0·1). In control animals, the same dose of iron dextran caused a 10-fold rise in serum ferritin levels (3400 μg/1). A single subcutaneous dose of AA (75 mg/kg weight) given to scorbutic animals caused a significant rise in serum ferritin levels to 599 μg/1 ( P <0·001) but had no effect in control animals. The same dose of AA given to iron loaded, scorbutic animals elevated serum ferritin levels from 554 μg/1 to 1297 μg/1 ( P <0·025).
Thus, AA deficiency in guinea-pigs caused serum ferritin levels to fall and blocked the rise in serum ferritin levels observed in response to acute iron loading in control animals. Adequate tissue concentrations of AA appeared to be necessary for the maintenance of a quantitative correlation between tissue iron stores and serum ferritin levels. The mechanisms by which AA influences serum ferritin levels are at present unknown.     

The labile iron pool in human erythroid cells

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5·8-fold and 8·8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2·5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3·9-fold. We also show that in β-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.     

Myocardial iron loading by magnetic resonance imaging T2* in good prognostic myelodysplastic syndrome patients on long-term blood transfusions

Magnetic resonance imaging (MRI) was used to quantify myocardial iron loading by T2* in 11 transfusion-dependent good prognostic myelodysplastic syndrome (MDS) patients. Myocardial T2*, left ventricular function and hepatic T2* were measured simultaneously. Patients had been on transfusion therapy for 13-123 months and had serum ferritin levels of 1109-6148 microg/l at the time of study. Five patients had not commenced iron chelation and had been transfused with a median of 63 red cell units and had a median serum ferritin level of 1490 microg/l. Six patients were on iron chelation and had been transfused with a median of 112 red cell units and had a median serum ferritin level of 4809 mug/l. Hepatic iron overload was mild in two, moderate in seven and severe in two patients. The median liver iron concentration was 5.9 mg/g dry weight in chelated patients and 9.5 mg/g in non-chelated patients (P = 0.17; not significant). Myocardial T2* indicated absent iron loading in 10/11 patients (91%; 95% confidence interval 62-98%) and borderline-normal in one patient. Left ventricular function was normal in all patients. No correlation was observed between increasing serum ferritin levels, hepatic iron overload and myocardial T2*. A long latent period relative to hepatic iron loading appears to predate the development of myocardial iron loading in transfusion-dependent MDS patients.     

Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells.

Acute administration of iron to rats has been previously shown to induce liver ferritin synthesis by increasing the translation of inactive cytoplasmic ferritin mRNAs for both heavy (H) and light (L) subunits by mobilizing them onto polyribosomes. In this report rat hepatoma cells in culture are used to explore the relationship of this response to intracellular iron levels. After adding iron as ferric ammonium citrate to the medium, latent ferritin H- and L-mRNAs were extensively transferred to polyribosomes, accompanied by increased uptake of [35S]methionine into ferritin protein. Because total cellular levels of L- and H-mRNA were not significantly changed by exposure to iron, the increased ferritin mRNAs on polyribosomes most probably come from an inactive cytoplasmic pool, consistent with the inability of actinomycin-D and of cordycepin to inhibit iron-induced ferritin synthesis. When deferoxamine mesylate, an intracellular iron chelator, was added after the addition of iron to the medium, ferritin mRNA on the polyribosomes was reduced, while the free messenger pool increased, and ferritin synthesis diminished. In contrast, the extracellular iron chelator diethylenetriaminepentaacetic acid failed to inhibit the induction of ferritin protein synthesis. Addition of iron in the form of hemin also caused translocation of mRNA to polyribosomes, a response that could be similarly quenched by deferoxamine. Because hemin does not release chelatable iron extracellularly, we conclude that the level of chelatable iron within the cell has a regulatory role in ferritin synthesis through redistribution of the messenger RNAs between the free mRNA pool and the polyribosomes.     

Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload

After a single subcutaneous dose of iron-dextran (600 mg of iron/kg), iron overload developed in C57BL/10ScSn mice. At 4, 24 and 78 wk liver nonheme iron concentrations were 67-, 42- and 21-fold higher than controls, respectively. Much of the iron was in macrophages, but hepatocytes were also strongly positive for Perls' stainable iron. One feature was the development of iron-positive nuclear inclusions in hepatocytes. After a delay of at least 8 wk when no stainable iron was evident, a maximum of 37% of periportal hepatocytes contained inclusions by 24 wk. Although this proportion remained constant for the remainder of the study, the size of the inclusions (which were not membrane-limited) increased to greater than 3 microns in diameter, occupying greater than 25% of the nuclear volume. The presence of iron in the inclusions was confirmed by energy dispersive x-ray microanalysis. Immunocytochemical studies showed that the iron was present as aggregates of ferritin. Quantitation of nonaggregated ferritin molecules by image analyses after electron microscopy demonstrated that within 4 wk ferritin levels in cytoplasm and nucleoplasm had greatly increased but that there was a concentration gradient of approximately one order of magnitude across the nuclear envelope. These findings are consistent with the hypothesis that in iron-loaded mouse hepatocytes there is a slow passage of ferritin-molecules through the nuclear pores; the gradient is maintained by the continual aggregation of ferritin within the nucleus. Intranuclear ferritin may provide a source of iron for catalyzing hydroxyl radical formation in nuclei during some toxic, carcinogenic and aging processes.     

Cellular expression and regulation of iron transport and storage proteins in genetic haemochromatosis

: Genetic haemochromatosis is a common iron overload disorder of unknown aetiology. To characterize the defect of iron metabolism responsible for this disease, this study localized and semiquantified the mRNA and protein expression of transferrin, transferrin receptor and ferritin in the liver and duodenum of patients with genetic haemochromatosis. Biopsies were obtained from iron-loaded non-cirrhotic patients with genetic haemochromatotic and control patients with normal iron stores. Additional duodenal biopsies were obtained from patients with iron deficiency. Immunohistochemical and in situ hybridization analysis for transferrin, transferrin receptor and ferritin was performed. Hepatic transferrin, transferrin receptor and ferritin protein expression was localized predominantly to hepatocytes and was increased in patients with genetic haemochromatosis when compared with normal controls. Interestingly, hepatic ferritin mRNA expression was not increased in these same patients. In the genetic haemochromatotic duodenum, ferritin mRNA and protein was localized mainly to crypt and villus epithelial cells and the level of expression was decreased compared with normal controls, but similar to iron deficiency. Duodenal transferrin receptor mRNA and protein levels colocalized to epithelial cells of the crypt and villus were similar to normal controls. Early in the course of genetic haemochromatosis and before the onset of hepatic fibrosis, transferrin receptor-mediated iron uptake by hepatocytes contributes to hepatic iron overload. Increased hepatic ferritin expression suggests this is the major iron storage protein. While persisting duodenal transferrin receptor expression may be a normal response to increased body iron stores in patients with genetic haemochromatosis, decreased duodenal ferritin levels suggest that duodenal mucosa is regulated as if the patient were iron deficient.     

Erythrocytapheresis limits iron accumulation in chronically transfused sickle cell patients

Cerebrovascular accidents (CVA) as a complication of sickle cell disease occur most frequently in childhood. Life-long transfusion prevents recurrent stroke, but inevitably leads to iron overload. Although effective chelation exists, many patients are not compliant. Erythrocytapheresis, an automated method of red blood cell exchange, was evaluated as an alternative to control transfusion-related iron load. Eleven patients with sickle cell anemia and a history of stroke were converted from simple transfusion to pheresis. Total time on pheresis for the group averaged 19 months (range 4–36 months). No significant complications occurred with a mean pre-pheresis hemoglobin S (Hb S) level of 44%. Blood utilization increased by an average of 50%. The effect of pheresis on serum ferritin depended on the patient's pre-pheresis ferritin level and chelation regimen. Ferritin levels remained stable for chelated patients with ferritin levels ⩾5,000 ng/ml, but decreased in a chelated patient with a pre-pheresis ferritin level of 4,000 ng/ml. For non-chelated patients with significant pre-pheresis iron load, ferritin levels remained stable. No patient on chelation prior to pheresis was able to discontinue deferoxamine. However, one patient with pre-pheresis ferritin of 500 ng/ml maintained serum ferritin levels <200 ng/ml for 36 months of pheresis without chelation. Pheresis is more expensive than simple transfusion unless the cost of chelation and organ damage from iron overload are considered. Erythrocytapheresis is a safe method of controlling Hb S levels and limiting or preventing iron load in chronically transfused sickle cell patients. Am. J. Hematol. 59:28–35, 1998. © 1998 Wiley-Liss, Inc.     

Effects of iron loading on pathogenicity in hepatitis C virus-infected chimpanzees.

Elevated iron levels have been associated with raised serum alanine transaminase (ALT) levels in hepatitis C virus (HCV)-infected humans. However, it is not clear if HCV infection causes increased iron accumulation by the liver or if the severity of HCV infection is actually worsened by higher iron levels in the host. To better understand the relationship between iron and persistent HCV infections, we examined the effect of excess dietary iron on disease severity in HCV-infected chimpanzees. Iron was supplemented in the diets of four HCV-infected and two uninfected chimpanzees for 29 weeks to achieve iron loading. Iron loading was confirmed by increases in serum iron levels, percentages of transferrin saturation, ferritin levels, elevations in hepatic iron concentration (HIC), and by histological examination. The majority of HCV-infected chimpanzees had higher iron levels before iron feeding than the uninfected animals. Although various degrees of iron loading occurred in all chimpanzees, HCV-infected animals exhibited increased loading in comparison with uninfected animals. The effects of iron loading on HCV disease expression was determined by comparing disease parameters during an extended baseline period before iron loading with the period during iron loading and immediately following iron loading. Iron loading did not influence the viral load, but did exacerbate liver injury in HCV-infected chimpanzees, as evidenced by elevated ALT and histological changes. Because all chimpanzees on high iron diets experienced iron loading, but pathological effects were only observed in HCV-infected chimpanzees, HCV infection appears to increase the susceptibility of the liver to injury following iron loading. These results confirm and extend previous observations made in human populations and serve to further validate the chimpanzee model of chronic hepatitis C.