Body iron metabolism and pathophysiology of iron overload

Iron is an essential metal for the body, while excess iron accumulation causes organ dysfunction through the production of reactive oxygen species. There is a sophisticated balance of body iron metabolism of storage and transport, which is regulated by several factors including the newly identified peptide hepcidin. As there is no passive excretory mechanism of iron, iron is easily accumulated when exogenous iron is loaded by hereditary factors, repeated transfusions, and other diseased conditions. The free irons, non-transferrin-bound iron, and labile plasma iron in the circulation, and the labile iron pool within the cells, are responsible for iron toxicity. The characteristic features of advanced iron overload are failure of vital organs such as liver and heart in addition to endocrine dysfunctions. For the estimation of body iron, there are direct and indirect methods available. Serum ferritin is the most convenient and widely available modality, even though its specificity is sometimes problematic. Recently, new physical detection methods using magnetic resonance imaging and superconducting quantum interference devices have become available to estimate iron concentration in liver and myocardium. The widely used application of iron chelators with high compliance will resolve the problems of organ dysfunction by excess iron and improve patient outcomes.     

Identification of new mutations of hepcidin and hemojuvelin in patients with HFE C282Y allele

HFE-hemochromatosis is the most common form of hereditary hemochromatosis. The disorder is associated with the homozygous C282Y mutation and has variable phenotype, being modulated by environmental and genetic factors. Candidate modifier genes are hemojuvelin and hepcidin, which are responsible for juvenile hemochromatosis. We used DHPLC to scan mutations in these genes in a cohort of unrelated patients with C282Y mutation. They consisted of 136 C282Y homozygous, 43 heterozygous, and 42 C282Y/H63D compound heterozygous, plus 62 controls subjects. Mutations and polymorphisms were found in 16 patients and 4 controls. Abnormally high indices of iron status were found in subjects C282Y/H63D heterozygous for the N196K hemojuvelin mutation and the -72C > T hepcidin substitution. The already described G71D mutation of hepcidin did not induce evident modification of the C282Y/H63D phenotype. The data show that heterozygous mutations of the hemojuvelin gene contribute like those of hepcidin to the phenotypic heterogeneity of hemochromatosis. However, they are rare and explain only a minor portion of the variable penetrance of the disorder.     

Optimizing the diagnosis and the treatment of iron overload diseases

A number of human disorders are related to chronic iron overload, either of genetic or acquired origin. The multi-organ damage produced by iron excess leads, in adults and in children, to severe clinical consequences, affecting both quality of life and life expectancy. The diagnosis is increasingly based on a non-invasive strategy, resorting to clinical, biological and imaging data. The treatment rests on either venesection or chelation therapy, depending on the etiology. Major advances in the fields of molecular biology, pharmacology, and biotechnology pave the road for key improvements in the diagnostic and therapeutic management of the patients.     

慢性乙型肝炎患者血清铁调素和铁代谢指标及体外HBV转染Huh7细胞铁调素水平变化*

目的 探讨慢性乙型肝炎(CHB)患者血清铁调素(Hepc)和铁代谢指标的变化,并在体外观察HBV转染肿瘤细胞Hepc表达的变化。方法 在71例CHB患者和24例健康体检者,采用ELISA法检测血清Hepc水平,使用全自动蛋白分析仪检测血清铁(SI)、铁蛋白(Ferr)和转铁蛋白(Tf)水平。使用电化学发光全自动免疫分析仪检测血清IL-6水平。构建HBV感染Huh7细胞模型,分别采用RT-qPCR和Western blot法检测细胞Hepc表达情况。结果 与健康人比较,CHB组血清SI、Ferr、IL-6、丙氨酸氨基转移酶 (ALT)、天门冬氨酸氨基转移酶(AST)、血清总胆红素 (TBIL)水平均显著升高,Hepc、Tf和可溶性转铁蛋白受体(sTfR)、白蛋白(ALB)显著降低(P<0.05);转染细胞24 h,Hepc mRNA相对水平为(5.21±0.43),空白对照细胞的(0.73±0.14)显著升高(P<0.05),在转染48 h,细胞Hepc mRNA水平为(8.45±0.61),较空白对照的(1.16±0.17)显著升高(P<0.05);在质粒转染48 h后,细胞Hepc蛋白相对表达量为(0.78±0.08),较空白对照的(0.41±0.02)显著升高(P<0.05)。结论 检测铁调素及其铁代谢相关指标有助于评估慢性乙型肝炎病情进展,适当予以铁调素或者其相应内源性激活剂针对性地治疗铁超载CHB患者,是否可应用于临床,值得期待。     

Mutations in HAMP and HJV genes and their impact on expression of clinical hemochromatosis in a cohort of 100 Spanish patients homozygous for the C282Y mutation of HFE gene

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype–genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.     

The role of iron in the pathophysiology and treatment of chronic hepatitis C

Increased hepatic iron content may be observed in patients with chronic hepatitis C infection, and may contribute to disease severity. The presence of hemochromatosis gene mutations is associated with increased hepatic iron accumulation and may lead to accelerated disease progression. Hepatic iron depletion has been postulated to decrease the risk of hepatocellular carcinoma in patients with cirrhosis due to chronic hepatitis C. It is possible that iron depletion stabilizes or improves liver histology and slows disease progression in these individuals. The present article reviews the prevalence and risk factors for hepatic iron overload in chronic hepatitis C, with emphasis on the available data regarding the efficacy of iron depletion in the treatment of this common liver disease.     

Screening for iron overload: Lessons from the HEmochromatosis and IRon Overload Screening (HEIRS) Study

BACKGROUND:

The HEmochromatosis and IRon Overload Screening (HEIRS) Study provided data on a racially, ethnically and geographically diverse cohort of participants in North America screened from primary care populations.

METHODS:

A total of 101,168 participants were screened by testing for HFE C282Y and H63D mutations, and measuring serum ferritin concentration and transferrin saturation. In the present review, lessons from the HEIRS Study are highlighted in the context of the principles of screening for a medical disease as previously outlined by the World Health Organization.

RESULTS:

Genetic testing is well accepted, with minimal risk of discrimination. Transferrin saturation has high biological variability and relatively low sensitivity to detect HFE C282Y homozygotes, which limits its role as a screening test. Symptoms attributable to HFE C282Y homozygosity are no more common in individuals identified by population screening than in control subjects.

CONCLUSIONS:

Generalized population screening in a primary care population as performed in the HEIRS Study is not recommended. There may be a role for focused screening in Caucasian men, with some debate regarding genotyping followed by phenotyping, or phenotyping followed by genotyping.     

A novel N491S mutation in the human SLC11A2 gene impairs protein trafficking and in association with the G212V mutation leads to microcytic anemia and liver iron overload

Background

DMT1 is a transmembrane iron transporter involved in iron duodenal absorption and cellular iron uptake. Mutations in the human SLC11A2 gene coding DMT1 lead to microcytic anemia and hepatic iron overload, with unexpectedly low levels of plasma ferritin in the presence of iron stores.

Design and methods

We report a patient with a similar phenotype due to two mutations in the SLC11A2 gene, the known p.Gly212Val (G212V) mutation and a novel one, p.Asn491Ser (N491S). To assess the expression of DMT1 in human liver, we studied the expression of the four DMT1 mRNA isoforms by real-time quantitative PCR in control human liver samples. We also studied the effect of G212V and N491S DMT1 mutations on RNA splicing in blood leukocytes and cellular trafficking of dsRed2-tagged-DMT1 protein in the human hepatic cell line HuH7.

Results

Our results showed that i) only the isoforms 1B-IRE and 1B-nonIRE were significantly expressed in human liver; ii) the G212V mutation did not seem to affect mRNA splicing and the N491S mutation induced a splicing alteration leading to a truncated protein, which seemed quantitatively of low relevance; and iii) the N491S mutation, in contrast to the G212V mutation, led to abnormal protein trafficking.

Conclusions

Our data confirm the major role of DMT1 in the maintenance of iron homeostasis in humans and demonstrate that the N491S mutation, through its deleterious effect on protein trafficking, contributes together with the G212V mutation to the development of anemia and hepatic iron overload.     

Serum hepcidin levels are related to serum markers for iron metabolism and fibrosis stage in patients with chronic hepatitis B: A cross-sectional study

Background and study aimsThe clinical significance of serum parameters of iron metabolism and hepcidin in liver disease remains unknown. Therefore, this study aimed to evaluate the association of serum hepcidin levels with fibrosis stage and serum iron parameters in patients with chronic hepatitis B (CHB).Patients and MethodsThis cross-sectional study included 126 treatment-naïve patients with CHB (median age, 39.0 years; 64.3% males) who were positive for hepatitis B surface antigen and 23 healthy controls (median age, 33.0 years; 52.2% males). Data on patient demographics, serum hepcidin levels, liver function tests and serum iron parameters and liver biopsy findings including fibrosis grade, histological activity index (HAI) and liver iron level were recorded.ResultsThe median (minimum–maximum) serum hepcidin levels were significantly lower in the CHB group than in the control group [71.2 (13.3–672.7) vs. 657.5 (201.7–2714.2) pg/mL, p < 0.001]. Higher fibrosis stage was associated with higher transferrin saturation (p = 0.029), serum ferritin level (p < 0.001) and viral load (p < 0.001). Fibrosis stage and HAI were positively correlated with ferritin (r = 0.407, p < 0.001 and r = 0.415, p < 0.001, respectively) and transferrin saturation (r = 0.219, p = 0.026 and r = 0.290, p = 0.003, respectively) levels, whereas hepcidin level was negatively correlated with fibrosis stage (r = −0.175, p = 0.051), viral load (r = −0.209, p = 0.020) and ferritin level (r = −0.244, p = 0.006) level. There were no significant differences in serum iron level, total iron binding capacity and liver iron level among patients with different stages of fibrosis.ConclusionReduced hepcidin levels and elevated transferrin saturation and ferritin levels are linked to fibrosis severity and HAI in patients with CHB.     

Hemochromatosis: pathophysiology,evaluation, and management of hepatic iron overload with a focus on MRI

Introduction: Hereditary hemochromatosis (HH) is an autosomal recessive disorder that occurs in approximately 1 in 200–250 individuals. Mutations in the HFE gene lead to excess iron absorption. Excess iron in the form of non-transferrin-bound iron (NTBI) causes injury and is readily uptaken by cardiomyocytes, pancreatic islet cells, and hepatocytes. Symptoms greatly vary among patients and include fatigue, abdominal pain, arthralgias, impotence, decreased libido, diabetes, and heart failure. Untreated hemochromatosis can lead to chronic liver disease, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Many invasive and noninvasive diagnostic tests are available to aid in diagnosis and treatment. MRI has emerged as the reference standard imaging modality for the detection and quantification of hepatic iron deposition, as ultrasound (US) is unable to detect iron overload and computed tomography (CT) findings are nonspecific and influenced by multiple confounding variables. If caught and treated early, HH disease progression can significantly be altered.

Area covered: The data on Hemochromatosis, iron overload, and MRI were gathered by searching PubMed.

Expert commentary: MRI is a great tool for diagnosis and management of iron overload. It is safe, effective, and a standard protocol should be included in diagnostic algorithms of future treatment guidelines.     

Furin-mediated processing in the early secretory pathway: sequential cleavage and degradation of misfolded insulin receptors

Improperly folded membrane proteins are retained in the endoplasmic reticulum and then diverted to a degradative pathway by a network of molecular chaperones and intracellular proteases. Here we report that mutant insulin proreceptors (Pro(62)) retained in the early secretory pathway undergo proteolytic cleavage at a tetrabasic concensus site for the subtilisin-like protease furin (SPC 1), generating two unstable proteolytic intermediates of 80/120 kDa corresponding to alpha (135 kDa) and beta (90 kDa) subunits. These are degraded more rapidly than the uncleaved proreceptor protein. Site-directed mutagenesis of the normal RKRR processing site prevented cleavage. Use of inhibitors and furin-deficient cell lines confirmed that furin is responsible for proreceptor cleavage; furin overexpression increased the degradation of mutant but not wild-type receptors. Together, these results suggest that processing and degradation occur sequentially for mutant proreceptors.     

Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to thwart the host innate immune response. One of the effectors, YopT, causes the disruption of the actin cytoskeleton and contributes to the inhibition of phagocytosis of the pathogen. YopT functions as a cysteine protease to cleave Rho family GTPases. We have analyzed the YopT cleavage products of Rho GTPases by TLC and determined their chemical structure by MS. Amino acid labeling experiments were performed to locate the exact site in RhoA where the YopT cleavage occurs. Our data unambiguously demonstrate that YopT cleaves N-terminal to the prenylated cysteine in RhoA, Rac, and Cdc42 and that the cleavage product of the GTPases is geranylgeranyl cysteine methyl ester. YopT cleaves GTP- and GDP-bound forms of RhoA equally, suggesting that the cleavage does not depend upon the conformation status of the GTPases. YopT also cleaves both farnesylated and geranylgeranylated forms of RhoA. The polybasic sequence in the C terminus of RhoA is essential for YopT substrate recognition and cleavage.     

Biochemical and morphological characterization of parathyroid hormone receptor binding to the rat osteosarcoma cell line UMR-106

We have used both biochemical and morphological techniques to characterize PTH receptors on the clonal osteosarcoma cell line UMR-106, a widely used model of the osteoblast phenotype. 125I-labeled rat (r) PTH-(1-34) bound to a single class of specific saturable receptors on both whole cells and membranes prepared from UMR-106 cells in a time- and temperature-dependent manner. A decrease in PTH receptor affinity seen in the presence of guanine nucleotides demonstrated that PTH receptors on the UMR-106 cells are coupled to guanyl nucleotide-binding proteins. Although PTH is a potent stimulator of adenylate cyclase in the UMR-106 cells, comparison of PTH-stimulated adenylate cyclase and PTH binding curves indicated the presence of receptors that are not linked to the adenylate cyclase system. Our studies also demonstrated that 125I-labeled rPTH-(1-34) bound to UMR-106 cells is rapidly internalized at 22 C, whereas PTH bound at 4 C remains intact and on the cell surface. Internalization of 125I-labeled rPTH-(1-34) was associated with degradation and release of the hormone at 22 C. Three morphologically distinct cell types were identified in subconfluent cultures of UMR-106 cells. Autoradiographic analysis of 125I-labeled rPTH-(1-34) binding demonstrated differential PTH receptor expression in these cell types. The most abundant PTH binding was observed over a cell type with long cytoplasmic extensions. This cell was reminiscent of the predominant PTH target cell previously identified in the rat metaphysis in vivo, suggesting that the UMR-106 cell line may represent neoplastic transformation of the PTH target cell.     

Hepatic iron concentration, fibrosis and response to venesection associated with the A77D and V162del "loss of function" mutations in ferroportin disease

Ferroportin disease is an autosomal dominant form of hemochromatosis associated with siderosis in cells of the mononuclear phagocyte system and, to varying degrees, in hepatocytes. Ferroportin was investigated as a candidate gene in two pedigrees with hyperferritinaemia and siderosis in mononuclear phagocytes. The entire ferroportin coding region was sequenced and hepatic iron concentration, histology and response to treatment were determined. The results were compared with previously reported cases. The A77D mutation was detected in patient 1, his father (patient 2) and his brother (patient 3), who had portal fibrosis. The V162del mutation was detected in patient 4, who developed anemia after the third weekly venesection. While the disease is rare, A77D and V162del are the most common ferroportin mutations in Caucasians. The spectrum of clinical expression of these two mutations was reviewed in all cases described to date. These mutations were associated with fibrosis in about a third of cases. For A77D and V162del, this analysis confirms that the threshold hepatic iron concentration for development of fibrosis may be higher than for classical hemochromatosis. These two mutations, which both decreased iron export in cell culture studies, give rise to similar patterns of clinical expression and morbidity, although the highest hepatic iron concentrations have been observed with A77D. It is important for clinicians to consider ferroportin disease in cases where there are features of iron overload unrelated to HFE, autosomal dominant inheritance and/or iron deposition in mononuclear phagocytes.     

Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C

BACKGROUND & AIMS: Iron overload may cause or contribute to hepatic injury and fibrosis. Mutations in the HFE gene may influence development or progression of chronic liver disease by increasing iron stores or modulating immune responses. The aim of this work was to assess the influence of HFE mutations and serum and hepatic measures of iron status on baseline features and response to lead-in therapy in subjects with advanced chronic hepatitis C enrolled in the Hepatitis C Anti-viral Long-term Treatment to prevent Cirrhosis (HALT-C) Trial. METHODS: Entry criteria included an Ishak fibrosis score >2 and lack of iron overload (Scheuer iron grade <3+) according to local study pathologists. All baseline biopsy specimens were rescored by consensus of study pathologists, and detailed assessment of stainable iron was performed. Hepatic iron concentrations were measured on portions of 144 liver biopsy specimens. A total of 1051 out of 1145 subjects agreed to HFE mutational testing (C282Y, H63D, S65C). RESULTS: Thirty-five percent carried at least one HFE gene mutation. There were no significant differences in the prevalence of HFE gene mutations among subjects with fibrosis (35.5%) versus cirrhosis (32.9%). Thirty-three percent of subjects had end-of-treatment and 16% sustained virologic responses. Presence of HFE mutations, in particular the H63D variation, was associated with increased end-of-treatment (40% vs 29%, P = .0078) and sustained virologic responses (20% with HFE mutation vs 14% sustained virologic response without HFE mutation; P = .009). CONCLUSIONS: Although HFE mutations (especially the most frequent H63D mutation) are associated with increased iron loading, they are also associated with increased sustained virologic responses in US patients with advanced chronic hepatitis C.     

Underestimation of the coexistence of iron deficiencies and thalassemia minors: a single institution experience in Taiwan

Some physicians neglect the possible coexistence of an iron deficiency with a thalassemia minor and do not treat the iron deficiency accordingly. This motivated us to conduct this study. We retrospectively reviewed the records of 3892 patients who visited our clinics and had hemoglobin (Hb) electrophoreses performed in our hematologic laboratory from August 1, 2007 to December 31, 2012. The thalassemia minors were identified by characteristic complete blood count (CBC) parameters obtained from an autoanalyzer and Hb electrophoresis, and some cases were confirmed with molecular tests. Then, we checked iron studies [ferritin and/or serum iron with total iron-binding capacity (TIBC)] to determine the coexistence of an iron deficiency with a thalassemia minor and a response to iron, if such treatments were given. We found 792 cases with thalassemia minors, and excluded those without iron studies, with 661 cases as our sample. A total of 202/661 cases (31%) also had iron deficiencies. They had lower red blood cell (RBC) counts, Hb, and ferritin levels as compared to those thalassemia minor cases without coexistence of iron deficiencies. We concluded that the thalassemia minor patients did not have iron overload complications in our population. On the contrary, iron deficiencies commonly coexist in the clinical visits. We propose that if Hb < 11.5 g/dL in a case of thalassemia minor, one should screen for iron deficiency simultaneously. The sensitivity is 79.8% and the specificity is 82.6%. Therefore, physicians should be aware of this coexisting condition, and know how to recognize and treat it accordingly.     

Iron overload thalassemic cardiomyopathy: Iron status assessment and mechanisms of mechanical and electrical disturbance due to iron toxicity

Patients with thalassemia major have inevitably suffered from complications of the disease, due to iron overload. Among such complications, cardiomyopathy is the leading cause of morbidity and mortality (63.6% to 71%). The major causes of death in this group of patients are congestive heart failure and fatal cardiac tachyarrhythmias leading to sudden cardiac death. The free radical-mediated pathway is the principal mechanism of iron toxicity. The consequent series of events caused by iron overload lead to catastrophic cardiac effects. The authors review the electrophysiological and molecular mechanisms, pathophysiology and correlated clinical insight of heart failure and arrhythmias in iron overload thalassemic cardiomyopathy.     

Metabolism of amino- and carboxyl-sequence immunoreactive parathyroid hormone in the bovine: evidence for peripheral cleavage of hormone.

Radioimmunoassays that detect specifically peptide sequences within either the biologically active amino region (N-assay) or inactive carboxyl region (C-assay) of parathyroid hormone (PTH) were used to evaluate the metabolism of PTH during and after infusion and injection of homogeneous (containing less than 0.1% hormonal fragments) intact bovine PTH (bPTH) into calves. During continuous infusions of hormone, when constant blood levels of immunoreactive PTH were reached, a dissociation between the concentrations of amino versus carboxyl immunoreactivity was observed; concentrations of hormone measured by the C-assay rose to a level of approximately three times higher than that measured by the N-assay. Analysis by gel filtration of immunoreactive PTH in plasma samples from calves after injection of hormone showed the rapid disappearance of intact hormone (N- and C-assays) and the appearance of a large fragment detected by the C-assay but not by the N-assay. The hormonal fragment lacked antigenic determinants within the amino peptide sequence required for biologic activity. No additional fragments of PTH were detected by gel filtration using the N- and C-assays. No detectable conversion of intact PTH to hormonal fragments occurred during incubation in vitro in bovine serum. The results are consistent with the concept that PTH is metabolized after entry into the circulation at peripheral sites located outside the vascular space, resulting in the rapid disappearance from blood of intact hormone and the appearance of a biologically inactive hormonal fragment(s). These studies done in calves agree with earlier studies done in dogs and man and point to the existence in mammals of common pathways for the peripheral metabolism of PTH.