Molecular aspects of iron absorption and HFE expression.

Hereditary hemochromatosis, a disease of iron overload, occurs in about 1 in 200-400 Caucasians. The gene mutated in this disorder is termed HFE. The product of this gene, HFE protein, is homologous to major histocompatibility complex class I proteins, but HFE does not present peptides to T cells. Based on recent structural, biochemical, and cell biological studies, transferrin receptor (TfR) is a ligand for HFE. This association directly links HFE protein to the TfR-mediated regulation of iron homeostasis. Although evidence is accumulating that binding of HFE to TfR is critical for the effects of HFE, the final pieces in the HFE puzzle have not been established. This review focuses on recent advances in HFE research and presents a hypothetical model of HFE function.

Increased hepatic oxidative DNA damage in patients with hepatocellular carcinoma.

Since oxidative DNA damage plays a role in experimental carcinogen-induced cancers, the purpose of the present study was to determine if hepatic oxidative DNA damage was increased in patients with HCC compared to patients with benign hepatic tumors or hepatic metastases (non-HCC) or to patients with end-stage alcoholic liver disease undergoing liver transplantation. Oxidative DNA damage was assessed by 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Results showed that peritumoral 8-OH-dG was markedly increased in HCC (N= 51) (180 +/- 74 vs 32 +/- 58-OH-dG/10(6)dG for tumor, P < 0.005) in contrast to patients with non-HCC (N = 17), in whom the peritumoral 8-OH-dG did not differ from that in tumor (39 +/- 7 vs. 31 +/- 108-OH-dG/10(6)dG). Oxidative DNA damage can be both mutagenic and carcinogenic; our data suggested it will be important in future studies to determine the chronology of this type of liver injury relative to hepatocarcinogenesis.

Changes in serum lipoprotein profile during interferon therapy in chronic hepatitis C.

OBJECTIVES: Therapy with a interferon is associated with a rise in serum triglyceride levels, although this effect has not been well studied with newer forms of interferon or interferon in combination with ribavirin. METHODS: Review of combined data obtained from several prospective, randomized controlled clinical trials conducted in the clinical studies unit of a tertiary care referral center among patients with chronic hepatitis C undergoing treatment with various forms of a interferon, with or without the addition of ribavirin. Serum levels of triglycerides and cholesterol were measured before and during therapy. Changes in these levels were correlated with baseline characteristics. RESULTS: At baseline, the mean serum triglyceride level among 152 patients studied was 130 mg/dL (range 32-620) and was elevated above normal in three patients (2%). During therapy, triglyceride levels rose significantly early on and began to decline spontaneously after 12 wk, returning to baseline after stopping treatment. Triglyceride levels rose above 500 mg/dL in 18 patients (12%) and above 1000 mg/dL in two patients (1.3%) although none experienced acute complications or clinical symptoms. Serum cholesterol levels did not change significantly during therapy (mean at baseline 172 vs 168 mg/dL at 24 wk). Factors correlated with the rise in triglycerides included baseline triglyceride levels, HCV genotype, and the type of interferon used. CONCLUSIONS: Serum triglyceride levels increase consistently in patients with chronic hepatitis C treated with all forms of a interferon, often to very high levels. These changes do not seem to be associated with clinical signs or complications and triglyceride levels decline while patients are still on therapy and return to normal after stopping.

Naturally variant autosomal and sex-linked loci determine the severity of iron overload in beta 2-microglobulin-deficient mice.

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the beta(2)-microglobulin (beta(2)M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in beta(2)M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F(1) and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a beta(2)M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.

Hepatic alpha-smooth muscle actin expression in hepatitis C patients before and after interferon therapy.

BACKGROUND/AIMS: The mechanism of hepatic fibrogenesis with chronic viral hepatitis is not well understood. Persistent activation of hepatic stellate cells is felt to play a role in the development of fibrogenesis and progression to cirrhosis. METHODOLOGY: We determined the expression of hepatic alpha-smooth muscle actin, a marker of hepatic stellate cell activation, in 29 patients with chronic hepatitis C and varying degrees of liver injury and fibrosis. In addition to a baseline evaluation, we assessed the effect of interferon therapy on alpha-smooth muscle actin expression in 11 patients, including 6 with a sustained response to therapy. Specimens were evaluated by light microscopy for grade of inflammation and stage of fibrosis. Expression of alpha-smooth muscle actin was assessed semiquantitatively by immunohistochemical staining. RESULTS: At baseline, all patients had alpha-smooth muscle actin expressed within the liver without an obvious correlation with the severity of liver injury. However, among sustained responders, a reduction in hepatic necroinflammatory activity was associated with a trend towards a decrease in alpha-smooth muscle actin expression. This however did not reach statistical significance. CONCLUSIONS: Hepatic alpha-smooth muscle actin expression, as a marker of hepatic stellate cell activation appears reversible and tends to correlate with necroinflammatory activity.

Mouse strain differences determine severity of iron accumulation in Hfe knockout model of hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/-), and knockout (Hfe -/-) mice from each strain. Although the Hfe -/- mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe -/- AKR mice that were 2.5 or 3.6 times higher than those of Hfe -/- C3H or Hfe -/- C57BL/6 mice, respectively. The Hfe -/- mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.

Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts.

BACKGROUND/AIMS: Myofibroblasts are the primary cells responsible for increased matrix deposition in hepatic fibrosis. Activation of hepatic stellate cells and portal fibroblasts to myofibroblasts during cholestatic liver injury is accompanied by increased expression of the activation marker, alpha-smooth muscle actin (SMA), and collagen genes. In contrast to our understanding of injury, the cellular mechanisms of liver repair are not well defined. This study was designed to examine the morphological relationship between bile duct hyperplasia, matrix deposition and myofibroblast phenotype in a model of chronic cholestatic liver injury and repair. METHODS: Reversible extrahepatic obstruction was accomplished in rats using a soft vessel loop suspended from the anterior abdominal wall: duct manipulation alone was performed in sham-operated controls. After 7 days, rats were either sacrificed or decompressed by release of the loop and subsequently sacrificed 2-10 days after reversal. Liver sections were obtained for in situ hybridization for procollagen alpha1(I) mRNA, immunohistochemical staining for SMA and cytokeratin 19, and histochemical staining for reticulin. RESULTS: Cholestatic livers demonstrated bile duct hyperplasia, which reversed to normal within 10 days after decompression. Fibrosis was also substantially reduced during this period. SMA-positive myofibroblasts were abundant and localized to regions adjacent to proliferating ducts and excess matrix in the obstructed animals. Decompressed livers showed a dramatic time-dependent reduction in the number of SMA-positive cells and in the expression of procollagen I mRNA. CONCLUSIONS: Our results show that the disappearance of bile duct hyperplasia after biliary decompression is accompanied by a similarly rapid loss of SMA-positive myofibroblasts. Both cellular events may abrogate enhanced matrix synthesis and allow repair to occur.

Histological evaluation of iron in liver biopsies: relationship to HFE mutations.

OBJECTIVE: Hepatic iron overload is observed in many forms of chronic liver disease. Hereditary hemochromatosis (HH) results in hepatic iron overload and is associated with 2 missense mutations in the HFE gene. The aim of this study was to define the usefulness of the histological pattern of iron deposition in determining the probability of an iron-loaded patient having HFE-related iron overload. METHODS: This study assessed liver biopsies containing stainable iron from 103 patients with various liver diseases; clinical information included hepatic iron concentration and HFE genotype (C282Y, H63D). The biopsies were evaluated using a reproducible histological scoring system for iron deposition. Three separate components of histological iron deposition were recorded: 1) pattern (primarily hepatocellular with a zonal gradient, or reticuloendothelial without an obvious zonal gradient), 2) pattern score to denote the extent of iron within the acinus, and 3) quantitation grade of iron granules within affected hepatocytes. RESULTS: The predominantly hepatocellular pattern (HH pattern) was observed in 72 biopsies of which only 42 were from patients homozygous for the C282Y mutation, indicating that this pattern alone cannot be used as a surrogate marker for HH genotype. The predominantly reticuloendothelial pattern (non-HH pattern) was observed in the remaining 31 patients, none of whom was compound heterozygous or homozygous for the C282Y mutation (negative predictive value: 100%). Thus, the non-HH, reticuloendothelial pattern reliably predicts the absence of homozygosity for the C282Y mutation. CONCLUSIONS: The use of histological evaluation for iron deposition is simple, assists in expanding information communicated from histopathologic observations, and may be clinically useful in determining the necessity of further evaluation of HFE genotype in subjects with histological evidence of hepatic iron overload.

Cell surface expression of HFE protein in epithelial cells, macrophages, and monocytes.

BACKGROUND AND OBJECTIVE: Most patients with hereditary hemochromatosis are homozygous for a Cys282AETyr mutation in the HFE gene. This mutation has been shown to impair the association of the HFE gene product with b(2)-microglobulin and to prevent its cell surface presentation in transfected COS-7 and 293 cells. This study was performed to examine the expression of HFE protein in epithelial cells, macrophages, and circulating leukocytes obtained from normal subjects and patients with hereditary hemochromatosis. DESIGN AND METHODS: Antisera against two different peptides of the HFE protein were used to immunostain tissue sections and isolate granulocytes, lymphocytes and monocytes. RESULTS: Immunocytochemical staining showed that the HFE protein is expressed in gastric epithelial cells, tissue macrophages, and circulating monocytes and granulocytes. The cell surface associated signal, which was seen in normal gastric epithelial cells, monocytes and macrophages, was also present in C282Y mutant cells from patients with hereditary hemochromatosis, although at apparently reduced amounts in these cells. INTERPRETATION AND CONCLUSIONS: From these studies, it is clear that the C282Y mutation reduces but does not completely prevent presentation of the HFE protein on the cell surface of human monocytes, tissue macrophages, and gastric epithelial cells.

Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 microg/kg/hr x 6 hr, for 10 weeks. Procollagen alpha1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

Transferrin receptor 2: continued expression in mouse liver in the face of iron overload and in hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite down-regulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the iron-responsive elements found in the 3' untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE -/-). Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not down-regulated with dietary iron loading or in the HFE -/- model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH.

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells.

AIMS/BACKGROUND: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E2 (PGE2), prostacyclin (PGI), and tumour necrosis factor-alpha (TNF) production in this model. METHODS: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. RESULTS: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE2 production, phagocytosis of hepatocytes did not. CONCLUSIONS: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE2 or TNF.

HFE genotype in patients with hemochromatosis and other liver diseases.

BACKGROUND: Hereditary hemochromatosis is a common inherited disorder of iron metabolism. The gene HFE, which contains two missense mutations (C282Y and H63D), was recently identified. OBJECTIVE: To determine how HFE genotyping for the C282Y and H63D mutations contributes to the diagnosis of hemochromatosis and to determine the prevalence of HFE mutations in a group of patients with liver disease. DESIGN: Cross-sectional study. SETTING: Academic medical center. PATIENTS: 66 patients with hereditary hemochromatosis and 132 referred patients with other liver diseases. MEASUREMENTS: At initial diagnosis, fasting transferrin saturation, ferritin level, routine chemistry panel, and complete blood count were determined. Percutaneous liver biopsy was done on all patients for histologic analysis and measurement of hepatic iron concentration and hepatic iron index. HFE genotyping for the C282Y and H63D mutations was done on all patients by using genomic DNA samples. RESULTS: Of the 66 patients with hemochromatosis diagnosed on the basis of serum iron studies and liver biopsy findings, 60 (91%) were C282Y homozygotes, 2 (3%) were compound heterozygotes, 1 (1.5%) was a C282Y heterozygote, 2 (3%) were H63D heterozygotes, and 1 (1.5%) was negative for both mutations. Of the 132 patients with liver disease, 6 (5%) were C282Y homozygotes, 8 (6%) were compound heterozygotes, 6 (5%) were C282Y heterozygotes, 5 (4%) were H63D homozygotes, 20 (15%) were H63D heterozygotes, and 87 (66%) were negative for both mutations. All 66 C282Y homozygotes had an elevated hepatic iron concentration, and 65 of the 66 patients (98%) had a transferrin saturation of at least 45%. Ten of the 66 patients (15% [95% CI, 7.5% to 26%]) had a hepatic iron index less than 1.9 mmol/kg per year; hemochromatosis was not suspected in 6 of the 10 patients before genotyping. Cirrhosis or substantial hepatic fibrosis was not seen in any (0% [CI, 0% to 18%]) of the 19 patients younger than 40 years of age who were homozygous for the C282Y mutation. CONCLUSIONS: All 66 patients homozygous for the C282Y mutation of HFE had an elevated hepatic iron concentration, but approximately 15% of these patients did not meet a previous diagnostic criterion for hemochromatosis (hepatic iron index > 1.9 mmol/kg per year). Determination of HFE genotype is clinically useful in patients with liver disease and suspected iron overload and may lead to identification of otherwise unsuspected C282Y homozygotes.

Intracellular signaling pathways in stellate cell activation.

Pathological fibrogenesis in the liver is mediated by activated stellate cells. These cells have a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. A number of factors have been proposed to initiate and perpetuate the fibrogenic process in stellate cells, including inflammatory cytokines, alterations in the extracellular matrix, growth factors, and oxidative stress. Some recent research has focused on the intracellular signaling pathways that are stimulated by these factors in stellate cells, including mitogen-activated protein kinases, phosphatidylinositol 3-kinase, focal adhesion kinase, and protein kinase C. This paper will summarize the experimental evidence that implicates these pathways in stellate cell activation, focusing on the effects of exposure to platelet-derived growth factor, tumor necrosis factor-alpha, and fibronectin. Implications for alcohol-induced hepatic fibrosis and future directions for research will also be discussed.

Hepatic lipid peroxidation in hereditary hemochromatosis and alcoholic liver injury.

Studies in experimental animals have indicated that enhanced lipid peroxidation may play a role in the hepatic injury produced by iron overload or by excessive alcohol consumption. The aim of this study was to compare the formation of lipid peroxidation-derived aldehydes in the liver of patients with hereditary hemochromatosis (HH) and alcohol abuse. Liver biopsy specimens from 10 nondrinking patients with HH were evaluated. These patients were classified as having HH based on hepatic iron index or human leukocyte antigen identity with a known proband. All patients were homozygous for the Cys282Tyr mutation. In addition, 8 patients with alcoholic liver disease were examined, 2 of whom also had hemochromatosis. For comparison, 17 patients with liver diseases unrelated to iron overload or alcohol abuse were studied. Liver biopsy specimens were immunostained for protein adducts with malondialdehyde and 4-hydroxynonenal. Both malondialdehyde- and 4-hydroxynonenal-protein adducts were found from liver specimens of patients with HH and alcohol abuse in more abundant amounts than from patients in a control group. In alcoholics the adducts were primarily in zone 3, whereas in hemochromatosis staining had an acinar zone 1 predominance, which followed the localization of iron. The most abundant amounts of protein adducts were noted in patients with alcohol abuse plus iron overload. The data support the concept that both chronic alcohol use and iron overload induce hepatic lipid peroxidation. Through formation of reactive aldehydic products, excessive alcohol consumption and iron overload may have additive hepatotoxic effects.

Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by tissue iron deposition secondary to excessive dietary iron absorption. We recently reported that HFE, the protein defective in HH, was physically associated with the transferrin receptor (TfR) in duodenal crypt cells and proposed that mutations in HFE attenuate the uptake of transferrin-bound iron from plasma by duodenal crypt cells, leading to up-regulation of transporters for dietary iron. Here, we tested the hypothesis that HFE-/- mice have increased duodenal expression of the divalent metal transporter (DMT1). By 4 weeks of age, the HFE-/- mice demonstrated iron loading when compared with HFE+/+ littermates, with elevated transferrin saturations (68.4% vs. 49.8%) and elevated liver iron concentrations (985 micrograms vs. 381 micrograms). By using Northern blot analyses, we quantitated duodenal expression of both classes of DMT1 transcripts: one containing an iron responsive element (IRE), called DMT1(IRE), and one containing no IRE, called DMT1(non-IRE). The positive control for DMT1 up-regulation was a murine model of dietary iron deficiency that demonstrated greatly increased levels of duodenal DMT1(IRE) mRNA. HFE-/- mice also demonstrated an increase in duodenal DMT1(IRE) mRNA (average 7.7-fold), despite their elevated transferrin saturation and hepatic iron content. Duodenal expression of DMT1(non-IRE) was not increased, nor was hepatic expression of DMT1 increased. These data support the model for HH in which HFE mutations lead to inappropriately low crypt cell iron, with resultant stabilization of DMT1(IRE) mRNA, up-regulation of DMT1, and increased absorption of dietary iron.

Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum.

In hereditary hemochromatosis (HH), intestinal absorption of dietary iron is increased, leading to excessive iron accumulation in tissues and resultant organ damage. The HFE protein, which is defective in HH, normally is expressed in crypt enterocytes of the duodenum where it has a unique, predominantly intracellular localization. In placenta, the HFE protein colocalizes with and forms a stable association with the transferrin receptor (TfR), providing a link between the HFE protein and iron transport. In the present study, we examined the relationship of the HFE protein to the TfR in enterocytes of the human duodenum and measured the uptake of transferrin-bound iron and ionic iron by isolated crypt and villus enterocytes. Immunocytochemistry showed that the HFE protein and TfR both are expressed in the crypt enterocytes. Western blots showed that, as was the case in human placenta, the HFE protein in crypt enterocytes is physically associated with the TfR and with beta2-microglobulin. The crypt cell fraction exhibited dramatically higher transferrin-bound iron uptake than villus cells. On the other hand, the villus cells showed 2-3 times higher uptake of ionic iron than crypt cells. We propose that the HFE protein modulates the uptake of transferrin-bound iron from plasma by crypt enterocytes and participates in the mechanism by which the crypt enterocytes sense the level of body iron stores. Impairment of this function caused by HFE gene mutations in HH could provide a paradoxical signal in crypt enterocytes that programs the differentiating enterocytes to absorb more dietary iron when they mature into villus enterocytes.

HFE gene knockout produces mouse model of hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 +/- 5% vs. 77 +/- 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 +/- 450 vs. 255 +/- 23 microg/g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.

Hepatic iron concentration in hereditary hemochromatosis does not saturate or accurately predict phlebotomy requirements.

OBJECTIVE: Biochemical measurement of the hepatic iron concentration (HIC) is essential for the diagnosis of hereditary hemochromatosis (HH). The aim of this study was to determine whether the HIC at the time of diagnosis could predict the subsequent phlebotomy requirements and to determine whether saturation of HIC occurred in HH. METHODS: Fifty-four patients (32 male, 22 female) with homozygous HH were evaluated, and HIC was measured in liver biopsies. Patients were subjected to weekly phlebotomy (500 ml) until the transferrin saturation was <50% and/or the serum ferritin concentration was <50 microg/L. The relationship between HIC and total body iron stores (as measured by phlebotomy requirements) was determined using both linear and nonlinear (sigmoidal model) least squares regression. RESULTS: The HIC ranged from 3,742 to 41,040 microg/g dry wt. A linear relationship between HIC and total body iron stores (iron removed, IR, g) best described the data both in male (HIC = 1986 IR - 3494; r = 0.83; p < 0.001) and female HH patients (HIC = 1251 IR + 2690; r = 0.75; p < 0.001). Men required eight more phlebotomies (2 g iron) on average, compared with women, to reach normal iron stores. There was no evidence of saturation of hepatic iron levels at higher total body iron stores. However, accurate prediction of individual phlebotomy requirements based on the HIC or serum ferritin concentration at the time of diagnosis was not possible. CONCLUSION: The phlebotomy requirement for treatment of HH cannot be accurately predicted from the initial HIC or serum ferritin level. Within the range examined, hepatic iron deposition did not saturate in HH.

Ascorbate-stimulated lipid peroxidation and non-heme iron concentrations in Alzheimer disease.

Lipid peroxidation has been suggested to be a potential cause of neuronal damage in neurodegenerative diseases. Changes in several parameters of lipid peroxidation, including basal (unstimulated) lipid peroxidation, stimulated lipid peroxidation, tissue iron concentrations, and the concentrations of some oxygen radical scavengers, have been reported in neurodegenerative diseases. However, the in vitro interaction of oxygen radical scavengers and stimulated lipid peroxidation in neurodegenerative disease has been less well-studied. The purpose of the present study was to determine the effects of oxygen radical scavengers on ascorbate-stimulated lipid peroxidation in Alzheimer disease (AD). We have found that some parameters of ascorbate-stimulated lipid peroxidation are altered in AD and that the effects of superoxide dismutase (SOD) on ascorbate-stimulated lipid peroxidation are significantly different in AD as compared to aged.