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Iron overload in paediatrics undergoing cardiopulmonary bypass.

Pathological changes in iron status are known to occur during bypass and will be superimposed upon physiological abnormalities in iron distribution, characteristic of the neonatal period. We have sought to define the severity of iron overload in these patients. Plasma samples from 65 paediatric patients undergoing cardiopulmonary bypass (CPB) were analysed for non-haem iron, total iron binding capacity, transferrin and bleomycin-detectable iron. Patients were divided into four age groups for analysis. Within each age group, patients who were in iron overload at any time point were statistically compared to those who were not. The most significant changes in iron chemistry were seen in the plasma of neonates, with 25% in a state of plasma iron overload. 18.5% of infants and 14.3% of children at 1-5 years were also in iron overload at some time point during CPB. No children over 5 years, however, went into iron overload. Increased iron saturation of transferrin eliminates its ability to bind reactive forms of iron and to act as an antioxidant. When transferrin is fully saturated with iron, reactive forms of iron are present in the plasma which can stimulate iron-driven oxidative reactions. Our data suggest that paediatric patients are at greater risk of iron overload during CPB, and that some form of iron chelation therapy may be advantageous to decrease oxidative stress.

Bleomycin↗

Diagnosis and clinical evaluation of iron overload.

Diagnostic evaluation of the various forms of iron overload requires information about the total amount and distribution of iron stores. Direct information on the quantity of storage iron can be obtained only by its mobilization in response to repeated phlebotomy or after dilution of a labelled iron test dose in the total body iron pool. Both approaches are cumbersome and time-consuming and are suitable only for research purposes. Detailed information on the amount and distribution of tissue iron in iron overload can be obtained from biopsy specimens of the major iron storage organs such as the liver and bone marrow. However, the invasive nature of these procedures limits their clinical usefulness. Indirect measures, such as serum iron concentration, TIBC saturation, serum ferritin, chelate-induced urinary iron excretion or intestinal iron absorption, and ferrokinetic measurements may provide useful information on the amount of total body iron reserve. However, they all have important limitations in their diagnostic use for evaluating iron overload. The most suitable indirect storage iron index among these methods is the ferritin assay, which has a well established place in the diagnosis of iron overload and monitoring of the effect of therapy. Recent developments in physical methods such as CT, SQUID and NMR have significantly improved the applicability of these techniques for non-invasive measurement of liver iron. It is expected that quantitative measurement of hepatic iron stores will soon be integrated into the diagnostic procedures available by imaging techniques such as CT and NMR. In combination with screening parameters such as serum ferritin and TIBC saturation these new but expensive diagnostic tools may simplify and shorten the diagnostic process and may also be useful for monitoring the treatment of iron overload by phlebotomy or chelating drugs.

Biopsy↗

Molecular pathogenesis of iron overload.

Our current understanding of iron absorption under normal conditions is presented, together with an overview of the clinical disorders of iron overload and the molecular processes that contribute to increased iron deposition in iron overload. Recently, a number of new genes involved in iron metabolism have been identified which is allowing the molecular mechanisms of iron absorption to be elucidated.

Enterocytes↗

Hepatocyte apoptosis in hepatic iron overload diseases.

In this retrospective study, we systematically analyzed hepatocyte apoptosis in three situations of hepatic iron overload (hereditary hemochromatosis; hepatic iron overload of unknown reason; iron overload due to hyperhemolysis or exogenous iron administration). Apoptosis was assessed by use of DNA nick end-labelling. The results suggest that hepatic iron overload is associated with an increased apoptotic rate of hepatocytes, and that iron-laden hepatocytes in hemochromatosis behave, with respect to apoptosis, differently from those in other states of iron overload. The hepatocyte apoptotic rate tended to increase as a function of the degree of iron storage. As in other pathological liver changes studied so far, an elevated apoptotic rate of hepatocytes predominated in the pericentral parts of liver acini in hemochromatosis, but not in the two other groups of hepatic iron overload. Possible mechanisms for this difference are discussed, particularly with respect to a participation of the Kupffer cell system.

Adult↗

Iron overload enhances epithelial cell proliferation in endometriotic lesions induced in a murine model.

BACKGROUND: Iron deposits are characteristic of endometriotic lesions, and pelvic iron concentrations are higher in endometriosis patients than in women without endometriosis. In this study, the effect of iron overload and iron chelation on the development of endometriosis in a murine model was investigated. METHODS: Human menstrual endometrium was injected i.p. into nude mice, either alone (controls) or supplemented with erythrocytes or desferrioxamine (DFO), an iron chelator. After 5 days, the iron load of endometriosis-like lesions and peritoneal macrophages and fluid was evaluated. Lesions were quantified by immunohistochemical morphometry, and their proliferative activity was assessed. RESULTS: Injection of erythrocytes into the pelvic cavity caused iron overload in lesions (P < 0.025) and peritoneal macrophages (P < 0.01) and fluid (P < 0.05), whereas DFO effectively reduced iron status in lesions (P < 0.05) and macrophages (P < 0.01) compared with controls. No difference was observed in the number or surface area of lesions between the three groups. Erythrocytes increased (P < 0.05) and DFO significantly decreased (P < 0.01) the proliferative activity of lesions. CONCLUSIONS: Iron overload does not appear to affect lesion establishment but may contribute to the further growth of endometriosis by promoting cell proliferation of lesions. Iron chelator treatment could therefore be beneficial in endometriosis to prevent iron overload in the pelvic cavity and decrease cellular proliferation of lesions.

Animals↗

Safety and efficacy of subcutaneous bolus injection of deferoxamine in adult patients with iron overload.

We compared 48-hour urinary iron excretion after a twice-daily subcutaneous bolus injection of deferoxamine and after 12 hours of subcutaneous continuous infusion of the drug in 27 patients with iron overload (mean age, 55.7 years). In most patients, the iron overload was due to multiple transfusions administered during chemotherapy or as part of supportive care for a hematologic or oncologic disorder. One patient had sickle cell anemia and 1 had hereditary hemochromatosis and spherocytosis. Similar urinary iron excretion was observed with the 2 methods of administration; mean +/- SD values were 6935.3 +/- 3832.3 microg/48 hours with subcutaneous bolus injection and 6630.4 +/- 3606.9 microg/48 hours with subcutaneous continuous infusion (P =.3). Twenty-six patients (96.3%) chose to continue therapy with bolus injection. The long-term efficacy of bolus injection was evaluated by measuring the serum ferritin concentration at regular intervals for a follow-up time of 20.1 +/- 4.5 months. Ferritin concentration decreased to below 1000 microg/L in 73% of the patients and to below 500 microg/L in 42% and became normal in 26%. Best results were obtained in patients who were no longer receiving blood transfusions when chelation therapy was initiated. Three of 26 patients (11.5%) had mild, transient side effects after bolus injection. Larger prospective, randomized studies must be conducted before deferoxamine bolus injection can be routinely recommended for patients with iron overload. (Blood. 2000;95:2776-2779)

Adult↗

Risk of iron overload among middle-aged women.

Iron overload, expressed as increased body iron stores, has been recognized as a potential hazard because it promotes the generation of oxygen radicals. We analyzed factors associated with serum ferritin levels (an indicator of body iron stores) among middle-aged women with a high prevalence of nutrient supplement use. Serum ferritin concentrations were determined on automated immunoassay for 487 healthy women with the mean age of 57 years who participated in the New York University Women's Health Study. The mean serum ferritin concentration in postmenopausal women was more than twice that in premenopausal women. Serum ferritin concentrations progressively increased with advancing age, but adjustment for menopausal status considerably weakened this association. Among non-dietary factors, nonwhite ethnicity, obesity and cigarette smoking were positively associated with serum ferritin concentrations. After adjustment for these factors and for menopausal status, serum ferritin levels were positively associated with meat intake and multivitamin use and inversely associated with breakfast cereal consumption. However, none of these lifestyle factors positively associated with serum ferritin levels had a significant impact on serum ferritin levels above 100 ng/ml (approximately equal to median concentration). Our results suggest that iron overload seems unlikely among middle aged women through their diet and nutritional supplements.

Adult↗

Use of the ferritin/alanine aspartate transaminase ratio as an iron overload marker independent of liver cell damage.

To define an iron overload index independent of liver cell damage, the mean annual levels of alanine aspartate transaminase (ALAT) and serum ferritin and their ratios were determined. Ferritin/ALAT ratio values were compared between two groups of patients with acute or chronic hepatitis without iron overload, and one group of thalassaemic patients with iron overload. The two groups without iron overload exhibited ferritin/ALAT ratio values of 2 and 1.2 respectively; a ratio value higher than 10 was always observed in those patients with iron overload. The ferritin/ALAT ratio is correlated with the degree of iron overload. This ratio increases in regularly-transfused patients without chelation treatment. It generally remains stable or decreases after initiation of iron chelation therapy. The ferritin/ALAT ratio thus appears useful in the follow-up of patients subjected to a long-term transfusional treatment particularly when acute or chronic liver cell damage may interfere with iron overload by increasing serum ferritin values.

Alanine Transaminase↗

Iron overload augments the development of atherosclerotic lesions in rabbits.

Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n = 8), iron-overloaded (group B, n = 6), hypercholesterolemic (group C, n = 6), and untreated (group D, n = 6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P < .05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits.

Animals↗

Hepatic iron overload: direct HFE (HLA-H) mutation analysis vs quantitative iron assays for the diagnosis of hereditary hemochromatosis.

Among patients with hepatic iron overload, the distinction between hereditary hemochromatosis (HH), a common yet treatable genetic disease, and other causes of siderosis remains problematic. The recent discovery of a specific homozygous mutation (C282Y) in a novel major histocompatibility complex class I-like gene (named HLA-H or HFE) in 80% to 100% of well-characterized cases of HH suggests that direct DNA-based mutation analysis may help resolve this dilemma. To assess the clinical utility of direct HLA-H mutation analysis in a typical diagnostic setting, we measured genotypic and phenotypic parameters of iron overload in 37 subjects with biopsy-proven hepatic siderosis (2+ or greater) and in 127 healthy control subjects. The prevalence of C282Y homozygotes was significantly greater in the hepatic siderosis group (32%) than in the control group (0%), confirming the association between this homozygous mutation and hepatic iron overload. In the hepatic siderosis group, C282Y homozygotes had significantly higher hepatic iron and ferritin levels, a significantly lower prevalence of hepatitis C virus or alcoholic liver disease, but no significant difference in the saturation of serum transferrin. Of the 20 subjects with a hepatic iron index (HII) in the previously defined "hemochromatosis range" (>1.9), 9 (45%) were C282Y homozygotes. Of the 11 nonhomozygous subjects with an HII greater than 1.9 (presumed false-positive HIIs), 10 (91%) had hepatic cirrhosis compared with 3 of 9 (33%) homozygotes with an HII greater than 1.9 who had cirrhosis (P<.02). The HII thus has poor diagnostic specificity for predicting genotypic HH in patients with cirrhosis. We conclude that direct determination of the HLA-H C282Y genotype may be the single best diagnostic test for HH, particularly in patients with cirrhosis, for whom the HII is quite nonspecific.

Adult↗

Antioxidant activity of silybin in vivo during long-term iron overload in rats.

BACKGROUND & AIMS: Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. METHODS: Rats were fed a 2.5% carbonyl-iron diet and 100 mg.kg body wt-1.day-1 silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. RESULTS: Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. CONCLUSIONS: Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.

Adenosine Triphosphate↗

H63D homozygotes with hyperferritinaemia: Is this genotype, the primary cause of iron overload?

OBJECTIVES: Hereditary haemochromatosis is a disease that affects iron metabolism and leads to iron overload. Homozygosity for the H63D mutation is associated with increased transferrin saturation (TS) and ferritin levels. Our objective was to find out if the homozygosity of H63D mutation was the primary cause of iron overload. PATIENTS AND METHODS: We studied 45 H63D homozygotes (31 males and 14 females) with biochemical iron overload and/or clinical features of haemochromatosis. The simultaneous detection of 18 known HFE, TFR2 and FPN1 mutations and sequencing of the HAMP gene were performed to rule out the possible existence of genetic modifier factors related with iron overload. RESULTS: Values of biochemical iron overload, measured as percentage TS and serum ferritin concentration (SF), in our H63D homozygotes were significantly higher in patients than in controls: TS 55 +/- 15% vs. 35 +/- 15% and SF 764 (645-883) microg/L vs. 115 (108-123) microg/L for patients and controls, respectively. These H63D homozygotes presented extreme hyperferritinaemia and no additional mutations in HFE, TFR2, FPN1 and HAMP genes were detected. CONCLUSIONS: The lack of additional mutations in our H63D homozygotes suggests that this genotype could be the primary cause of iron overload in these patients. Despite our results, we cannot entirely discount the possibility that one or more genetic modifier factor exists, simply because we were unable to find it, although there was a precedent in the HFE gene. Genetic modifier factors have been described for C282Y mutations in the HFE gene, but at the present time they have never been reported in H63D homozygotes.

Adolescent↗

Evidence that iron overload plus croton oil induce skin tumours in mice.

Iron overload is known to occur due to different factors including genetic disorders. It has been shown that iron overload predisposes humans to an increased risk of cancer. However, the mechanism by which iron overload enhances chemically induced carcinogenesis is not known. In this report, for the first time it is shown that iron overload acts as a tumour initiator. Female albino Swiss mice were given iron dextran 1 mg/mouse per day intramuscularly for 15 days and croton oil 0.5 mg in 200 microL acetone/mouse topically twice a week for 30 weeks. During this period, the animals were observed for tumour incidence. There were significantly higher yields of tumours in those animals receiving both iron and croton oil. However, the group of animals treated only with acetone, iron, croton oil and 7,12-dimethylbenz(a)anthracene (DMBA) alone did not develop any tumours during 30 weeks of observation. Further, croton oil-mediated induction in cutaneous lipid peroxidation (LPO) level was higher in the iron-overload group. The results of this study suggest that oxidative stress generated by iron overload is responsible for croton oil-mediated skin carcinogenesis.

Animals↗

Low dose intravenous ascorbic acid for erythropoietin-hyporesponsive anemia in diabetic hemodialysis patients with iron overload.

BACKGROUND: Recent report demonstrates that inadequate iron mobilization and defective iron utilization may cause recombinant erythropoieitin (rEPO) hyporesponsiveness in hemodialysis (HD) patients with iron overload. The effect of intravenous ascorbic acid (IVAA) in HD patients selected on the basis of iron overload and EPO resistance also has been proven. However, it is uncertain whether IVAA still works in diabetic ESRD patients with hyperferritinemia. Therefore, the aim of this study focusing on diabetic ESRD patients was to analyze the potential effect of low dose IVAA on improvement of anemia and erythropoiesis-related parameters when compared with control period. PATIENTS AND METHOD: This study consisted of 22 chronic hemodialysis patients with type II diabetes in a single dialysis unit. In studies of this type, all eligible patients are followed up, but the primary comparison is still between different sequentially treatment including control period and post-IVAA period in same patients. IVAA patients received ascorbic acid, 100 mg each administered intravenously three times per week for eight weeks of treatment and four months of post-treatment follow-up. RESULTS: The demographic characteristics of 22 diabetic uremic patients show that mean age is 63.6 +/- 10.2 years old. The ratio of sex (M/F) = 10/12. Mean duration of HD is 46.7 +/- 33.2 months. As for the urea kinetic study between these two periods including KT/V, nPCR, and URR, there is no significantly different. As for anemia-related parameters, Hb and Hct increased significantly in post-IVAA period after 3 months compared with control period, while MCV did not increase significantly. Serum ferritin significantly decreased at study completion. The same situation is for iron. As for TS, it significantly increased at one month and further markedly increased at subsequent three months. CONCLUSION: This study has demonstrated that short-term low dose IVAA therapy can facilitate iron release from reticuloendothelial system but also increase iron utilization in diabetic hemodialysis patients with iron overload. Therefore, IVAA is a potential adjuvant therapy to treat erythropoeitin-hyporesponsive anemia in iron-overloaded patients.

Aged↗

Functional characterization of the E399D DMT1/NRAMP2/SLC11A2 protein produced by an exon 12 mutation in a patient with microcytic anemia and iron overload.

DMT1 (Nramp2, Slc11a2) mediates iron uptake at the intestinal brush border and across the membrane of acidified endosomes. A single patient with severe microcytic anemia and iron overload was recently reported to carry a mutation in exon 12 of DMT1 (1285G>C). The mutation has two effects: it severely impairs splicing causing skipping of exon 12 and introduces an amino acid polymorphism (E399D) in the protein encoded by the remaining properly spliced transcript found in the patient. The functional properties and possible contribution to disease of the DMT1 E399D mutation are unknown and have been studied in independent mutants at that position (E399D, E399Q, E399A) expressed in LLC-PK1 kidney cells. The 3 mutants are shown to be fully functional with respect to stability, targeting and trafficking to the membrane, and are transport-competent. This indicates that DMT1G1285C is not a complete loss of function but rather that a modest amount of active DMT1 is produced in this patient. This activity may explain the distinguishing iron overload seen in this patient in addition to microcytic anemia that is absent in parallel rodent models of DMT1 deficiency.

Adult↗

Dietary supplementation of baicalin and quercetin attenuates iron overload induced mouse liver injury.

The introduction of new iron chelating drugs may ultimately improve iron-chelation therapy for patients with iron overload diseases such as thalassaemia and other disorders. In this paper, the in vivo effects of baicalin and quercetin on iron overload induced liver injury were studied on mice. It was found that when iron-dextran induced iron overload mice were fed baicalin or quercetin containing diet (1% w/w) for 45 days, both flavonoids significantly inhibited iron overload induced lipid peroxidation and protein oxidation of liver, decreased hepatic iron and hepatic collagen content, increased the serum non-heme iron level but not serum ferritin level. Flavonoids supplementation also increased the excretion of iron through feces. In vitro study demonstrated that both flavonoids could release iron from ferritin. These results indicate that besides acting as antioxidants, both flavonoids can also release iron from liver and finally excrete it through feces. The present study provides further support for flavonoids to be medicines for iron overload diseases.

Animals↗

Analysis of genes implicated in iron regulation in individuals presenting with primary iron overload.

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C-->G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.

Adult↗

Hepatic mitochondrial oxidative metabolism in rats with chronic dietary iron overload.

We examined the effects of chronic dietary iron overload on hepatic mitochondrial oxidative metabolism. Experimental iron overload was produced by feeding rats a chow diet supplemented with carbonyl iron over a 7-week period. Biochemical and histologic evaluations of liver tissue confirmed moderate degrees of hepatic parenchymal iron overload. Electron microscopy showed no abnormalities in hepatic mitochondrial ultrastructure in blocks of tissue or in mitochondrial fractions from iron-loaded liver. Studies of mitochondrial oxidative metabolism revealed a consistent and progressive decrease in state 3 (ADP-stimulated) respiration and in respiratory control ratios at hepatic iron concentrations above 1,000 micrograms per gm for all three substrates studied, glutamate, beta-hydroxybutyrate and succinate. Changes in state 4 (ADP-limited) respiration and ADP/O ratios were not progressive with increasing hepatic iron concentrations. At hepatic iron concentrations at which there were decreases in state 3 respiration and respiratory control ratios, there was also evidence of lipid-conjugated diene formation, indicative of mitochondrial lipid peroxidation. There were no changes in mitochondrial function when iron as either ferritin or hemosiderin or as a combination of ferritin, hemosiderin and ferric nitrilotriacetate was added in vitro to normal liver homogenates. Use of density gradient centrifugation to reduce iron and lysosomal contamination of mitochondrial fractions failed to prevent the reduction in mitochondrial function. We conclude that moderate degrees of chronic hepatic iron overload in vivo result in an inhibitory defect in the mitochondrial electron transport chain as evidenced by a decrease in state 3 respiration and respiratory control ratios.

Acetylglucosaminidase↗