Reticuloendothelial Iron Research Articles

Ferritin Reference Ranges in an Adult Population of Working-Age Adults without Anemia

Introduction Considerable controversy has surrounded the creation of reference intervals for ferritin within tested populations. This is particularly true for women and has been attributed to a high prevalence of nonanemic iron deficiency due to physiologic blood loss during menses and physiologic iron transfer during pregnancy. The Quest Blueprint for Wellness (BFW) program is an annual health screening that provides a range of testing to Quest employees and families. Using deidentified data on 24,812 individuals (55% female; age range 18-80 years old) collected as part of this program, we studied the relationship between serum ferritin and characteristics of the study population, red blood cell parameters, and iron saturation in order to better define normal reference ranges for serum ferritin. Methods Test results of participants in the annual BFW screening program conducted between January 2022 and May 2023 were identified (26,535 [53% female]); tests included hematocrit, hemoglobin, RBC indices (including RDW), total serum iron and total iron binding capacity and CRP. Individuals with abnormalities of hemoglobin, hematocrit or RBC indices (including RDW); serum iron or serum TIBC; or elevated CRP were successively excluded, leaving 24,812 participants (55% female). None of these remaining individuals had a personal history of anemia or an identified ICD-10 code associated with anemia or other RBC disorder. This study population was used in subsequent analyses. Multimodal decomposition (MMD) in R (version 4.2.2) was used to calculate reference intervals based on the distribution of results associated with the healthy study subpopulation that was defined. To address the possibility that the TIBC saturation initially employed (20%) was too low, reference intervals were also determined when varying TIBC saturations from 20%-25%. Results As shown in Table 1, the lower limits of the ranges identified did not vary significantly as the TIBC saturation percent was increased from 20% to 25%. The lower limit for women below 50 years of age ranged from 11-13 ng/mL, which was substantially lower than the lower limits for men of the same age. Over the age of 50, the lower limit of the ferritin reference ranges for women increased, approaching but not equaling or exceeding the values observed for men until ages greater than 70. This may reflect the end of menses and the gradual repletion of iron stores. In contrast, the lower limits of the reference ranges for serum ferritin for men increased at ages 30-40 and then declined and fell below that of women in the eighth decade. Conclusions Comparing the lower range of ferritin values between men and women, these data suggest that iron deficiency may be present in a large proportion of working age women in the absence of anemia or abnormal RBC indices. These data also suggest that normal ferritin values are age dependent for both men and women. These conclusions are tempered by the unavailability of a physiologic reference standard able to demonstrate that iron deficiency is in fact present. The lower limit of the ferritin range for women under the age of 60 and for men over the age of 70 approximates the ferritin values associated with the absence of stainable bone marrow iron. While this test is the traditional “gold standard” it is rarely performed for the evaluation of iron status. The evaluation of iron deficiency at present lacks a physiologic indicator, analogous to the relationship between parathyroid hormone and calcium levels which permitted the definition of blood levels of 25 hydroxyvitamin D that represented sufficiency, or methylmalonic acid concentration in B12 deficiency. The broader availability of measures to assess the functional status of reticuloendothelial iron stores available to support erythropoiesis, such as measurements of hepcidin, would facilitate such an assessment.

Nitric Oxide Alters the Expression of γ-Globin, H-Ferritin, and Transferrin Receptor in Human K562 Cells at the Posttranscriptional Level

Cellular iron metabolism is altered during chronic inflammatory states, leading to reticuloendothelial iron sequestration and an associated anemia. To study the effects of nitric oxide (NO) on the expression of three genes involved in erythroid cell iron metabolism (gamma-globin, H-ferritin, and transferrin receptor [TfR]), we developed a series of human K562 erythroleukemic cell clones retrovirally transduced with inducible nitric oxide synthase (NOS-2) and producing different steady-state levels of NO. gamma-Globin and H-ferritin protein expression was reduced in NO-producing cells in relation to the amount of NO produced. Conversely, cell surface TfR expression increased in NO-producing clones. Both the inhibitory effects of NO on gamma-globin and H-ferritin expression and the stimulatory effect on TfR were reversed by the NOS inhibitor NG-methyl-L-arginine (NGMMA). gamma-Globin and H-ferritin mRNA levels were unaffected by NO production. In the case of TfR, NO appeared to stabilize mRNA in that the half life of TfR mRNA decreased from approximately 15 hours to less than 3 hours when NO production by NOS-transduced clones was inhibited. Thus, NO can regulate expression of these genes at the posttranscriptional level, an effect that is likely mediated by the known effect of NO on the RNA binding activity of iron regulatory protein-1 (Pantopoulos and Hentze, Proc Natl Acad Sci USA 92:1267, 1995). Furthermore, our findings suggest a mechanism for the observed relationship between NO production and the pathophysiology of the anemia of chronic disease.

Mesenchymal iron deposition is associated with adverse long-term outcome in non-alcoholic fatty liver disease.

Background & AimsApproximately one‐third of patients with non‐alcoholic fatty liver disease (NAFLD) show signs of mild‐to‐moderate iron overload. The impact of histological iron deposition on the clinical course of patients with NAFLD has not been established.Methods & ResultsFor this retrospective study, 299 consecutive patients with biopsy‐proven NAFLD and a mean follow‐up of 8.4 (±4.1; range: 0.3‐18.0) years were allocated to one of four groups according to presence of hepatic iron in the reticuloendothelial system (RES) and/or hepatocytes (HC): 156 subjects (52%) showed no stainable iron (NONE), 58 (19%) exclusively reticuloendothelial (xRES), 19 (6%) exclusively hepatocellular (xHC) and 66 (22%) showed a mixed (HC/RES) pattern of iron deposition. A long‐term analysis for overall survival, hepatic, cardiovascular or extrahepatic‐malignant events was conducted. Based on multivariate Cox proportional hazards models any reticuloendothelial iron was associated with fatal and non‐fatal hepatic events. Specifically, xRES showed a cause‐specific hazard ratio (csHR) of 2.4 (95%‐CI, 1.0‐5.8; P = .048) for hepatic as well as cardiovascular fatal and non‐fatal events combined (csHR 3.2; 95%‐CI, 1.2‐8.2; P = .015). Furthermore, the mixed HC/RES iron pattern showed a higher rate of combined hepatic fatal and non‐fatal events (csHR 3.6; 95%‐CI, 1.4‐9.5; P = .010), while xHC iron deposition was not associated with any defined events.ConclusionsThe presence of reticuloendothelial‐accentuated hepatic iron distribution patterns is associated with detrimental long‐term outcomes reflected in a higher rate of both liver‐related and cardiovascular fatal and non‐fatal events.

The diagnostic potential of the iron-regulatory hormone hepcidin.

The diagnostic potential of the iron-regulatory hormone hepcidin.

Association of Vitamin D and secondary hyperparathyroidism with anemia in diabetic kidney disease.

Introduction:Anemia is common in Chronic Kidney Disease (CKD) and diabetes is a major leading risk factor for it. In Diabetic Kidney Disease (DKD), it worsens more, which further increases cardiovascular morbidity and mortality. Despite of adequate iron stores anemia persist, which may be due to impaired iron release from body stores that is unable to meet the demand for erythropoiesis (also called reticuloendothelial cell iron blockade). High parathyroid hormone (PTH) along with vitamin D, may be attributable for anemia.Methods:A cross-sectional study of 150 advanced (Stage 4 & 5) pre dialyzed DKD patients (GFR <30ml/min/1.73 m2), aged 40-70 years were included over a period of 1 year. Any other concomitant illness/ drugs leading to anemia were excluded. Serum samples were collected and urea, creatinine, hemoglobin, iron profile, vitamin D, iPTH, uric acid, calcium, phosphorous and albumin levels were measured. A data base was constructed on Microsoft Excel 2007 and statistical analyses were performed using the SPSS software version 20.0 (IBM, NY, USA).Results:Stage 5 DKD had more pronounced anemia compared to stage 4 DKD (P < 0.001). Hemoglobin (Hb) was inversely correlated with iPTH (r = -0.74, P < 0.001) and was associated with vitamin D deficiency (r = 0.51, P < 0.001) but not with serum ferritin. DKD patients with low eGFR (r = -0.6, P < 0.001), vitamin D (r = -0.43, P < 0.001) and serum calcium (r = -0.37, P < 0.001) had higher iPTH. Secondary hyperparathyroidism (beta=-0.005; P < 0.001) and Vitamin D (beta=0.053; P < 0.01) were strong predictor for Hb while parameters of iron profile was not statistically significant.Conclusion:An efficient control of PTH hypersecretion is therefore required to achieve a better management of anemia as well as mineral metabolism in DKD patients.

Association between metabolic syndrome and liver histology among NAFLD patients without diabetes

BackgroundMetabolic syndrome (MetS) and obesity are associated with non-alcoholic fatty liver disease (NAFLD). The aim of this observational study was to examine the relationship of MetS and a diagnosis of...

Quantitative analysis of dietary iron utilization for erythropoiesis in response to body iron status.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis. There are two sources of iron for erythropoiesis, dietary and stored iron; however, their relative contributions to erythropoiesis remain unknown. In this study, we used the stable iron isotope (57)Fe to quantify synthesis of hemoglobin derived from dietary iron. Using this method, we investigated the activities of dietary iron absorption and the utilization of dietary iron for erythropoiesis in responses to stimulated erythropoiesis and to interventions to alter body iron status. Under iron-loaded conditions, the activity of dietary iron absorption was clearly lowered in response to up-regulation of hepcidin, although the estimated activity of iron release from stored iron was not compared with that under control conditions. This result was supported by the observation that two duodenal iron transporters, divalent metal transporter 1 (DMT1) and ferroportin, were downregulated by iron loading, although the levels of expression of ferroportin in iron storage tissues were not changed by iron loading under erythropoietic stimulation by epoetin-β pegol (C.E.R.A., a long-acting erythropoiesis-stimulating agent). These results indicate that the dietary iron absorption system is more sensitive to body iron status than are reticuloendothelial iron- release mechanisms. Our data indicated that there could be a regulatory mechanism favoring use of stored iron over dietary iron under iron-loaded conditions.

Hepcidin as a predictive factor and therapeutic target in erythropoiesis-stimulating agent treatment for anemia of chronic disease in rats.

Anemia of chronic disease is a multifactorial disorder, resulting mainly from inflammation-driven reticuloendothelial iron retention, impaired erythropoiesis, and reduced biological activity of erythropoietin. Erythropoiesis-stimulating agents have been used for the treatment of anemia of chronic disease, although with varying response rates and potential adverse effects. Serum concentrations of hepcidin, a key regulator of iron homeostasis, are increased in patients with anemia of chronic disease and linked to the pathogenesis of this disease, because hepcidin blocks cellular iron egress, thus limiting availability of iron for erythropoiesis. We tested whether serum hepcidin levels can predict and affect the therapeutic efficacy of erythropoiesis-stimulating agent treatment using a well-established rat model of anemia of chronic disease. We found that high pre-treatment hepcidin levels correlated with an impaired hematologic response to an erythropoiesis-stimulating agent in rats with anemia of chronic disease. Combined treatment with an erythropoiesis-stimulating agent and an inhibitor of hepcidin expression, LDN-193189, significantly reduced serum hepcidin levels, mobilized iron from tissue stores, increased serum iron levels and improved hemoglobin levels more effectively than did the erythropoiesis-stimulating agent or LDN-193189 monotherapy. In parallel, both the erythropoiesis-stimulating agent and erythropoiesis-stimulating agent/LDN-193189 combined reduced the expression of cytokines known to inhibit erythropoiesis. We conclude that serum hepcidin levels can predict the hematologic responsiveness to erythropoiesis-stimulating agent therapy in anemia of chronic disease. Pharmacological inhibition of hepcidin formation improves the erythropoiesis-stimulating agent's therapeutic efficacy, which may favor a reduction of erythropoiesis-stimulating agent dosages, costs and side effects.

A structural model of human ferroportin and of its iron binding site.

A structural model of human ferroportin has been built using two Escherichia coli proteins belonging to the major facilitator superfamily of transporters. A potential iron binding site was identified in the inward-open conformation of the model, and its relevance was tested through measurement of iron export of HEK293T cells expressing wild-type or mutated ferroportin. Aspartates 39 and 181 were found to be essential for the transport ability of the protein. Noteworthy, the D181V mutation is naturally found in type 4 hemochromatosis with reticuloendothelial system iron retention phenotype. The outward-open conformation of ferroportin was also predicted, and showed that significant conformational changes must occur in the inward- to outward-open transition of ferroportin. In particular, putative iron ligands move several ångströms away from each other, leading to the logical conclusion that the iron binding site is not occupied by the metal in the outward-open conformation of ferroportin.

Iron‐mediated upregulation of M1 and downregulation of M2 programs in macrophages suggests a role for iron in the pathogenesis of non‐alcoholic steatohepatitis (1116.13)

Background and Aim: Hepatic reticuloendothelial (RES) iron deposition is associated with nonalcoholic steatohepatitis (NASH) and advanced fibrosis in nonalcoholic fatty liver diseases. We hypothesized that iron loading of macrophages could influence their activation status and contribute to necroinflammation. Methods: Mouse bone marrow derived macrophages (BMDM) were treated with either ferric ammonium citrate (FAC) alone, or cotreated with IL‐4 to determine the effect of iron on proinflammatory or alternative (M2) activation programs. Post treatment, gene expression analysis was performed to assess the status of M1 and M2 markers by qRT‐PCR. Results: BMDMs treated with ferric ammonium citrate (a physiological form of iron) showed increased M1 activation (INOS, TNF‐α, IL‐6, CD14 gene expression) and reduced gene expression of M2 markers (Arginase1, TGF, Mgl1). Further, while cells treated with IL‐4 showed enhanced expression of M2 markers such as Arginase1, Mgl1, Ym1, IL10 and PGC1β, cotreatment with iron abrogated the expression of these M2 markers. Also interestingly, while IL‐4 treatment upregulated the gene expression level of IL‐4 receptor, FAC treatment reduced its expression. Desferrioxamine, an iron chelator, blocked the downregulatory effect of FAC on M2 gene expression. Furthermore, treatment of macrophages with iron led to an elevated expression of hepcidin (p&lt;0.05), the anti‐inflammatory peptide regulating iron homeostasis. Addition of hepcidin to LPS‐treated macrophages resulted in downregulation of TNF‐α (p&lt;0.01), and upregulation of SOCS3 (p=0.05), an anti‐inflammatory mediator. Conclusions: Iron administration causes upregulation of M1 and downregulation of M2 gene expression. We speculate that iron‐induced inflammatory activation may contribute to the necroinflammation, while iron‐mediated downregulation M2 pathway would impact fibrogenesis accompanying NASH. These data provide support for a mechanistic role for iron in NASH.

Hepatic reticuloendothelial system cell iron deposition is associated with increased apoptosis in nonalcoholic fatty liver disease

The aim of this study was to examine the relationship between the presence of hepatic iron deposition, apoptosis, histologic features, and serum markers of oxidative stress (OS) and cell death in nonalcoholic fatty liver disease (NAFLD). Clinical, biochemical, metabolic, and independent histopathologic assessment was conducted in 83 unselected patients with biopsy-proven NAFLD from a single center. Apoptosis and necrosis in serum was quantified using serum cytokeratin 18 (CK18) M30 and M65 enzyme-linked immunosorbent assays and in liver by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in situ. Serum malondialdehyde (MDA) and thioredoxin-1 (Trx1) levels were measured to evaluate OS. Presence of reticuloendothelial system (RES) cell iron in the liver was associated with nonalcoholic steatohepatitis (P < 0.05) and increased hepatic TUNEL staining (P = 0.02), as well as increased serum levels of apoptosis-specific (M30; P = 0.013) and total (M65; P = 0.006) CK18 fragments, higher MDA (P = 0.002) and lower antioxidant Trx1 levels (P = 0.012), compared to patients without stainable hepatic iron. NAFLD patients with a hepatocellular (HC) iron staining pattern also had increased serum MDA (P = 0.006), but not M30 CK18 levels or TUNEL staining, compared to subjects without stainable hepatic iron. Patients with iron deposition limited to hepatocytes had a lower proportion of apoptosis-specific M30 fragments relative to total M65 CK18 levels (37% versus ≤25%; P < 0.05). Presence of iron in liver RES cells is associated with NASH, increased apoptosis, and increased OS. HC iron deposition in NAFLD is also associated with OS and may promote hepatocyte necrosis in this disease.

Endocrinopathies in transfusion‐associated iron overload

Transfusional iron overload primarily results in reticuloendothelial iron accumulation, which is considered to be less harmful than parenchymal iron accumulation. However, systematic and comprehensive data on endocrine function in transfusion-associated haemochromatosis are limited. We examined 25 aplastic anaemia patients (11 men and 14 women) diagnosed with transfusion-associated haemochromatosis at a single institution. Pituitary function was determined with a combined pituitary function test. On a different day, a 75-g oral glucose tolerance test was performed. The bone mineral density (BMD) of the lumbar spine and total hip was assessed with dual-energy X-ray absorptiometry. Twenty-two (88%) of these 25 patients had at least one endocrine abnormality, and 12 had more than one abnormality. The most common pituitary hormonal deficiency involved the pituitary-gonadal axis; 54% of the total subjects had hypogonadotropic hypogonadism. Two patients had an insufficient cortisol response to corticotrophin-releasing hormone stimulation. No patient had a deficiency of growth hormone or thyroid-stimulating hormone. Twelve (48%) had diabetes mellitus, and these patients tended to have higher concentrations of ferritin, alanine aminotransferase and γ-glutamyl transferase. Osteoporosis (T-score <-2·5 SD) was observed in 48% of patients. The reduction in BMD was more pronounced in the lumbar spine than in the total hip. The patients with osteoporosis were accompanied by hypogonadism, which predominantly affected the trabecular bone. Our observations suggest that endocrinopathies are common in transfusion-associated haemochromatosis.

Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats

AbstractAnemia of chronic inflammation (ACI) is the most frequent anemia in hospitalized patients and is associated with significant morbidity. A major underlying mechanism of ACI is the retention of iron within cells of the reticuloendothelial system (RES), thus making the metal unavailable for efficient erythropoiesis. This reticuloendothelial iron sequestration is primarily mediated by excess levels of the iron regulatory peptide hepcidin down-regulating the functional expression of the only known cellular iron export protein ferroportin resulting in blockade of iron egress from these cells. Using a well-established rat model of ACI, we herein provide novel evidence for effective treatment of ACI by blocking endogenous hepcidin production using the small molecule dorsomorphin derivative LDN-193189 or the protein soluble hemojuvelin-Fc (HJV.Fc) to inhibit bone morphogenetic protein-Smad mediated signaling required for effective hepcidin transcription. Pharmacologic inhibition of hepcidin expression results in mobilization of iron from the RES, stimulation of erythropoiesis and correction of anemia. Thus, hepcidin lowering agents are a promising new class of pharmacologic drugs to effectively combat ACI.

The Wide Spectrum of Hepatic Iron Overload σ

We would like to thank Manco and colleagues for their interest in our article.1 We agree that stratification of our study cohort according the presence of diabetes and HFE (hereditary hemochromatosis) genotype status could be of interest. We have conducted additional analyses with regard to diabetes status in our cohort. HFE genotyping was not routinely performed as part of the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database study; this is currently in progress in an ancillary study. Among the 849 adult subjects in our study, 221 were enrolled in the PIVENS (Pioglitazone or Vitamin E for NASH Study) clinical trial,2 which excluded diabetic patients; therefore, to prevent any potential bias, we report here the relationship of diabetes and iron deposition in the remaining 628 subjects. As shown in Table 1, diabetic NASH CRN subjects were less likely to have hepatic iron deposition. In particular, diabetic patients had significantly less hepatocellular but not reticuloendothelial system iron deposition. There are several possible explanations for these findings. The iron regulatory hormone hepcidin is expressed in adipose tissue and has been shown to correlate to body mass index.3 It is possible that increased visceral fat in our diabetic subjects could lead to greater circulating hepcidin levels, reduced iron absorption, and less hepatocellular iron deposition. Recently, alcohol has been shown to down-regulate hepcidin gene expression.4 Because diabetic subjects in our study consumed less alcohol than subjects without diabetes, this could also contribute to higher hepcidin levels in these patients. Diabetic subjects were also more likely to be women, and physiologic blood loss through menstruation and childbirth may have resulted in decreased body iron stores. Additional studies are warranted to define the mechanism for decreased hepatocellular iron content in diabetic patients with NAFLD. In contrast to Manco et al.,5 who have reported hepatic iron deposition in 29% of their Italian pediatric subjects, which was not associated with measurements of insulin resistance, the prevalence of iron deposition in the NASH CRN pediatric subjects was only 4%. It is likely that, as in adults, differences in the Italian and U.S. cohorts, including body mass index (25.2 in Manco et al. versus 33.0 in the NASH CRN pediatric subjects) account for the observed differences.6 James Nelson Ph.D.*, Kris Kowdley M.D.*, * Benaroya Research Institute, Virginia Mason Medical Center, Seattle, WA.

The Effect of Supplemental Ascorbate on Defersirox Iron Chelation In the Iron-Loaded Gerbil

AbstractAbstract 2059 Introduction:Iron dextran injections are often used to induce iron overload in rodents, for the purposes of assessing iron chelation therapy. In gerbils, we have previously described that deferasirox therapy preferentially clears hepatocellular iron when compared with reticuloendothelial stores. Ascorbate deficiency, which is common in humans with iron overload, produces similar profound disparities between reticuloendothelial and parenchymal iron stores. We postulated that iron-induced ascorbate deficiency might be exaggerating reticuloendothelial iron retention in gerbils receiving deferasirox therapy. This study examined the effect of supplemental ascorbate on spontaneous iron loss and deferasirox chelation efficiency in the iron-dextran loaded gerbil. Methods:48 female gerbils underwent iron dextran loading at 200 mg/kg/week for 10 weeks. Sixteen animals were sacrificed at 11 weeks to characterize iron loading; eight were on standard rodent chow and eight had chow supplemented with 2250 ppm of ascorbate. 32 additional animals that were not ascorbate supplemented during iron loading transitioned into the chelation phase. Half were subsequently placed on ascorbate supplemented chow and both groups were assigned to receive either deferasirox 100 mg/kg/day five days per week or sham chelation. Animals received iron chelation for twelve weeks. Liver histology was assessed using H & E and Prussian blue stains. Iron loading was ranked and graded on a five-point scale by an experienced pathologist screened to the treatment arm. Iron quantitation in liver and heart was performed by atomic absorption. Results:Table 1 one summarizes the findings. During iron dextran loading, ascorbate supplementation lowered wet weight liver iron concentration but not liver iron content suggesting primarily changes in tissue water content. Spontaneous iron losses were insignificant, regardless of ascorbate therapy. Deferasirox lowered liver iron content 56% (4.7% per week) in animals without ascorbate supplementation and 48.3% (4.0% per week) with ascorbate supplementation (p=NS).Cardiac iron loading, unloading and redistribution were completely unaffected by ascorbate supplementation. Spontaneous iron redistribution was large (1.9% – 2.3% per week). Deferasirox chelation did not lower cardiac iron to a greater degree than spontaneous cardiac iron redistribution.Histologic grading paralleled tissue wet weight iron concentrations. Ascorbate treatment lowered the rank and absolute iron score in liver during iron loading (p=0.003) and there was a trend toward lower iron scoring in sham treated animals (p=0.13). Ascorbate had no effect on histological score or relative compartment distributions of iron in deferasirox chelated animals (p=0.5).Ascorbate supplementation was sufficient to increase total plasma ascorbate levels from 25 ± 12.2 uM to 38.4 ± 11 uM at 10 weeks (p=0.03). In the liver, ascorbate increased from 1203 ± 212 nmol/g of tissue to 1515 ± 194 nmol/g of tissue (p=0.01) with supplementation. No significant change in total ascorbate was observed in the heart. Discussion:We hypothesized that ascorbate supplementation might improve reticuloendothelial iron accessibility to deferasirox by facilitating redox cycling. Although gerbils synthesize their own ascorbate, supplementation was able to raise both serum and hepatic total ascorbate levels. However, increasing ascorbate did not change either the amount or distribution of tissue iron in deferasirox-treated animals. Disclosures:Nick:Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.

Effect of Hepcidin on Hypoxia-Induced Erythropoietin Production

AbstractAbstract 3219The anemia of chronic disease (ACD) is the major etiology of the anemia observed in in chronically ill patients. ACD typically manifests itself as a hypoproliferative anemia accompanied by a low serum iron concentration despite adequate reticuloendothelial iron stores. In ACD, a slight shortening of red cell survival creates a demand for a small increase in red cell production by the bone marrow. The marrow cannot respond adequately to this demand due to impaired erythropoiesis and impaired mobilization of reticuloendothelial system iron stores. Increased production of the iron regulatory peptide hepcidin has been proposed as the primary factor resulting in ACD. Hepcidin has also been reported to decrease erythroid colony formation in vitro under conditions of restrictive Epo concentration. A blunted erythropoietin (Epo) response to anemia is a characteristic feature of ACD. If hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired Epo production observed in this syndrome.The effect of hepcidin on hypoxia-induced Epo production was evaluated in HepG2 cells exposed to 5% oxygen for 24 hr. 24 hr exposure to hepcidin during hypoxia at the concentrations studied (up to 100 ng/mL) had no adverse effect on HepG2 cell viability compared to controls as evaluated by cell number and Trypan blue exclusion or on cellular synthetic function as measured by alpha fetoprotein. Epo production (whether measured by Western blot or by ELISA) was increased by hypoxia; however, this increase was blunted by the addition of hepcidin to the incubation medium. Impairment of hypoxia-induced Epo production by hepcidin showed a dose-response relationship. The addition of iron-replete transferrin to the incubation mixture did not significantly alter hepcidin effects, suggesting that these effects do not primarily reflect changes in iron availability.In order to evaluate mechanisms by which hepcidin might decrease Epo production, effects of hepcidin 0–100 ng/mL on hypoxia-inducible factor (HIF)-1α protein expression were evaluated in HepG2 cells (HIF expression was normalized to actin expression). The increment in HIF-1α caused by hypoxia was decreased by hepcidin in a dose-dependent manner. The ratio of Epo to HIF was not altered by hepcidin, suggesting that hepcidin effects on HIF may be the mechanism of its effects on Epo production.In conclusion, hepcidin appears to blunt the increment in Epo production induced by hypoxia in vitro. This mechanism does not appear to be reversible by exposure to increased quantities of transferrin-bound iron. This finding is consistent with a role for hepcidin in the impaired Epo production of ACD independent of its effects on iron flux. Disclosures:No relevant conflicts of interest to declare.

Comparison of Two Animal Models for Evaluation of Defersirox Iron Chelation

AbstractAbstract 4244 Introduction:Long-term storage iron (hemosiderin) is insoluble and not directly accessible to iron chelation. Ascorbate is a potent facilitator of redox cycling and facilitates the mobilization of cellular iron stores in patients chelated with deferoxamine. Ascorbate deficiency is quite common in thalassemia major and may create a phenotype of relative chelator refractoriness. However, the synergistic role of ascorbate supplementation has never been demonstrated for other chelators, including the oral chelator deferasirox. Ascorbate can be freely synthesized in many animals, making them unsuitable models to address this question. The osteodystrophic syndrome (ODS) rat is a spontaneous mutant lacking L-gulono-gamma-lactone oxidase, the initial enzyme in ascorbate synthesis. Iron loading and chelation have never been performed in ODS rats, so we report a pilot study aimed at optimizing organ iron loading and clearance. Methods:Twenty-six 8-week old male ODS rats were maintained vitamin C replete by supplying ascorbate in the chow or drinking water. Fourteen animals were iron loaded using a combination of 0.4% TMH ferrocene and 3% carbonyl iron in standard rodent chow. Twelve animals were loaded with iron dextran (200 mg/kg/week) by subcutaneous injection. To characterize loading kinetics, two animals were analyzed at five, ten, and twelve weeks of oral iron loading as well as five and ten weeks of iron dextran injections. Sixteen animals were iron loaded for ten weeks (half enterally, half parenterally) and transitioned into the chelation arm. Iron chelation was performed with oral deferasirox at 75 mg/kg/dose, once daily five times per week. Chelation was administered for six and twelve weeks in six animals, divided equally between parenteral and enteral iron loading; sham chelation was given to the remaining four animals. Following euthanasia, H&E and iron staining were performed and tissue iron was quantified by atomic absorption. Results:Parenteral iron loading was well tolerated and yielded liver iron concentration (LIC) values of approximately 3 and 4.5 mg/g wet weight (∼11-16.8 mg/g dry weight) at five and ten weeks, respectively. Iron loading was predominantly reticuloendothelial, with little parenchymal redistribution. Spontaneous iron loss after 12 weeks of iron chelation was modest at 1.3% per week. In contrast, oral iron loading with the combination of TMH-ferrocene and carbonyl iron was poorly tolerated. Animals developed severe diarrhea and required fluid replacement and frequent dose reductions. These GI disturbances gradually lessened over a period of four weeks and animals received approximately 80% of the targeted iron dose. LIC values were lower at five weeks (2.2 mg/g ww) but nearly equivalent by 10 weeks (4.2 mg/g ww). Iron was entirely parenchymal, with little reticuloendothelial deposition. Spontaneous iron losses after 12 weeks of iron chelation were strikingly higher than for parenteral iron loading, measuring 4.7% per week.Iron chelation with deferasirox was well tolerated in both groups. Iron chelation was less efficient in iron dextran loaded animals. LIC was 3.1 ± 0.7 mg/g ww compared with 3.8 ± 0.2 mg/g ww in sham chelated animals, p=0.25. In contrast, deferasirox treatment nearly completely cleared liver iron in TMH treated animals 0.4 ± 0.1 mg/g ww vs 1.5 ± 0.04 mg/g ww, p = 0.001, with most of the residual iron located primarily in reticuloendothelial store on histologic analysis. Discussion:Iron loading in the ODS rat can be performed with either iron dextran or TMH-ferrocene but the characteristics of iron staining, spontaneous iron loss, and chelator accessibility are completely different. Iron dextran loads the reticuloendothelial system. Iron redistribution to parenchymal tissues is sluggish, based upon the low spontaneous iron elimination rate and modest response to deferasirox therapy (1.5% per week). In contrast, TMH produced exclusively parenchymal loading, high spontaneous iron losses (4.7% per week) and more vigorous response to chelation (6.3% per week). Subsequent studies will determine whether the degree of ascorbate sufficiency modulates deferasirox efficacy in animals with primarily reticuloendothelial iron loading. Disclosures:Nick:Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.

Unexplained Aspects of Anemia of Inflammation

Anemia of inflammation (AI), also known as anemia of chronic inflammation or anemia of chronic disease was described over 50 years ago as anemia in association with clinically overt inflammatory disease, and the findings of low plasma iron, decreased bone marrow sideroblasts and increased reticuloendothelial iron. Pathogenic features underlying AI include a mild shortening of red cell survival, impaired erythropoietin production, blunted responsiveness of the marrow to erythropoietin, and impaired iron metabolism mediated by inflammatory cytokines and the iron regulatory peptide, hepcidin. Despite marked recent advances in understanding AI, gaps remain, including understanding of the pathogenesis of AI associated with “noninflammatory” or mildly inflammatory diseases, the challenge of excluding iron deficiency anemia in the context of concomitant inflammation, and understanding more precisely the contributory role of hepcidin in the development of AI in human inflammatory diseases.

Hepcidin Regulation by the BMP Pathway.

Abstract SCI-25Systemic iron balance is regulated by the key iron regulatory hormone hepcidin. Secreted by the liver, hepcidin inhibits iron absorption from the diet and iron mobilization from body stores by decreasing cell surface expression of the iron export protein ferroportin. Iron administration increases hepcidin expression, thereby providing a feedback mechanism to limit further iron absorption, while anemia and hypoxia inhibit hepcidin expression, thereby increasing iron availability for erythropoiesis. Hepcidin excess is thought to have a role in the anemia of inflammation, while hepcidin deficiency is thought to be the common pathogenic mechanism underlying the iron overload disorder hereditary hemochromatosis, due to mutations in the genes encoding hepcidin itself (HAMP), HFE, transferrin receptor 2 (TFR2), or hemojuvelin (HFE2). Notably the precise molecular mechanisms by which iron levels are “sensed” and how this iron “signal” is transduced to modulate hepcidin expression have remained elusive. We have recently discovered that hemojuvelin is a co-receptor for the bone morphogenetic protein (BMP) signaling pathway, and that hemojuvelin-mediated BMP signals increase hepcidin expression at the transcriptional level. In addition to patients with HFE2 mutations and Hfe2 knockout mice, other genetic mouse models associated with impaired hepatic BMP signaling through a global knockout of the ligand Bmp6, or selective hepatic knockout of an intracellular mediator of BMP signaling, Smad4, also cause inappropriately low hepcidin expression and iron overload. Exogenous BMP6 administration in mice increases hepatic hepcidin expression and reduces serum iron, while BMP6 antagonists inhibit hepatic hepcidin expression, mobilize reticuloendothelial cell iron stores and increase serum iron. Not only does the BMP6-hemojuvelin-SMAD pathway regulate hepcidin expression and thereby systemic iron homeostasis, but also the BMP6-SMAD pathway itself is regulated by iron. Acute iron administration in mice increases phosphorylation of Smad proteins in the liver, and chronic changes in dietary iron modulate hepatic Bmp6 mRNA expression and phosphorylation of Smad proteins concordantly with Hamp mRNA expression. Together, these data support the paramount role of the BMP6-hemojuvelin-SMAD signaling pathway in the iron-mediated regulation of hepcidin expression and systemic iron homeostasis, and suggest that modulators of this pathway may be an alternative therapeutic strategy for treating disorders of iron homeostasis. Recent work elucidating the role of the BMP signaling pathway in hepcidin regulation and systemic iron homeostasis will be presented. DisclosuresBabitt:Ferrumax Pharmaceuticals, Inc.: Equity Ownership.

Magnetic Resonance Imaging of Experimental Inflammatory Bowel Disease

To quantitatively and qualitatively characterize the MR findings of inflammatory bowel disease in a rat model after i.v. injection of the reticuloendothelial system cell specific ultrasmall iron oxide SHU 555 C. Colitis was induced in 15 rats using dinitrobenzene sulfonic acid instillation. Five rats served as controls. T1- and T2-weighted spin-echo- and T2*-weighted gradient-echo-sequences were acquired at 2.4 Tesla before and immediately, 15, 45, 60, and 90 minutes, and 24 hours after i.v.-injection of SHU 555 C (0.1 mmol Fe/kg). MR images were evaluated quantitatively regarding thickness and signal-to-noise ratio (SNR) of the bowel wall and qualitatively regarding overall bowel wall signal intensity and the occurrence of bowel wall ulcerations. MR findings were correlated to histology. The inflamed bowel wall was significantly thicker than the noninflamed bowel wall and 90 minutes after contrast injection it showed a significant reduction of SNR in T1- (94 +/- 27 vs. 61 +/- 29; P < 0.01), T2- (67 +/- 26 vs. 28 +/- 17; P < 0.05), and T2*- (92 +/- 57 vs. 10 +/- 7; P < 0.05) weighted images as compared with unenhanced images. At 24 hours, the respective SNR values remained significantly reduced. The signal loss was homogeneous in 12 and focal in 3 of the 15 rats with colitis. Nine rats showed colonic wall ulcerations. In all but one animal (missed focal ulceration) MR findings correlated to the histologic findings. SHU 555 C leads to a significant signal intensity loss of the inflamed bowel wall in T1-, T2- and T2*-weighted images. SHU 555 C enhanced MRI findings correlate well with histologic findings.