Anthony T. Murphy

Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications.

The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and the master regulator hepcidin. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. Many patients, however, present with both ACD and iron deficiency anemia (ACD/IDA), the latter resulting from chronic blood loss. We used a rat model of ACD resulting from chronic arthritis and mimicked ACD/IDA by additional phlebotomy to define differing iron-regulatory pathways. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In rats and humans with ACD, serum hepcidin concentrations are elevated, which is paralleled by reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects, and ACD/IDA persons, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Circulating hepcidin levels affect iron traffic in ACD and ACD/IDA and are more responsive to the erythropoietic demands for iron than to inflammation. Hepcidin determination may aid to differentiate between ACD and ACD/IDA and in selecting appropriate therapy for these patients.

Recent Advances in use of LC/MS/MS for Quantitative High-Throughput Bioanalytical Support of Drug Discovery

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.

Hypoxia induced downregulation of hepcidin is mediated by platelet derived growth factor BB

Objective Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood. Design Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. Results Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. Conclusions Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.

High-fat diet causes iron deficiency via hepcidin-independent reduction of duodenal iron absorption ☆ ☆☆

Obesity is often associated with disorders of iron homeostasis; however, the underlying mechanisms are not fully understood. Hepcidin is a key regulator of iron metabolism and may be responsible for obesity-driven iron deficiency. Herein, we used an animal model of diet-induced obesity to study high-fat-diet-induced changes in iron homeostasis. C57BL/6 mice were fed a standard (SD) or high-fat diet (HFD) for 8 weeks, and in addition, half of the mice received high dietary iron (Fe+) for the last 2 weeks. Surprisingly, HFD led to systemic iron deficiency which was traced back to reduced duodenal iron absorption. The mRNA and protein expressions of the duodenal iron transporters Dmt1 and Tfr1 were significantly higher in HFD- than in SD-fed mice, indicating enterocyte iron deficiency, whereas the mRNA levels of the duodenal iron oxidoreductases Dcytb and hephaestin were lower in HFD-fed mice. Neither hepatic and adipose tissue nor serum hepcidin concentrations differed significantly between SD- and HFD-fed mice, whereas dietary iron supplementation resulted in increased hepatic hepcidin mRNA expression and serum hepcidin levels in SD as compared to HFD mice. Our study suggests that HFD results in iron deficiency which is neither due to intake of energy-dense nutrient poor food nor due to increased sequestration in the reticulo-endothelial system but is the consequence of diminished intestinal iron uptake. We found that impaired iron absorption is independent of hepcidin but rather results from reduced metal uptake into the mucosa and discordant oxidoreductases expressions despite enterocyte iron deficiency.

Behavioral, biochemical, and genetic analysis of iron metabolism in high‐intensity blood donors

BACKGROUND: Individuals donating whole blood 13 times in a 2‐year period without development of iron deficiency anemia (superdonors) are a self‐selected population that is deferred for low hematocrit (Hct) level less frequently than other donors.

A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25

BACKGROUND Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%-75% range 23.2-93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%-75% range 5.9-18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%-75% range 0.42-3.07 μg/L, n = 100). CONCLUSIONS The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

Identification of a common variant in the TFR2 gene implicated in the physiological regulation of serum iron levels

The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.

Growth differentiation factor 15 in anaemia of chronic disease, iron deficiency anaemia and mixed type anaemia.

Recently, the iron and erythropoiesis‐controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β‐thalassaemia patients, thereby increasing iron absorption despite iron overload. To access the diagnostic and pathogenic impact of GDF15 in inflammatory anaemia the association of GDF15 expression with serum iron parameters and hepcidin was studied in patients suffering from iron deficiency anaemia (IDA), anaemia of chronic disease (ACD) and ACD subjects with true iron deficiency (ACD/IDA). GDF15 was significantly increased in both ACD and ACD/IDA, but not in IDA subjects as compared to controls. In contrast, hepcidin levels were significantly lower in IDA and ACD/IDA subjects than in ACD patients. IDA and ACD/IDA, but not ACD, showed an association between GDF15 and soluble transferrin receptor, an indicator of iron requirement for erythropoiesis. However, GDF15 did not correlate to hepcidin in either patient group. While GDF15 levels were linked to the needs for erythropoiesis and iron homeostasis in IDA, the immunity‐driven increase of GDF15 may not primarily affect iron homeostasis and hepcidin expression. This indicates that other ACD‐related factors may overcome the regulatory effects of GDF15 on hepcidin expression during inflammation.

Regulation of iron metabolism through GDF15 and hepcidin in pyruvate kinase deficiency.

Iron absorption is inadequately increased in patients with chronic haemolytic anaemia, which is commonly complicated by iron overload. Growth differentiation factor 15 (GDF15) has been identified as a bone marrow‐derived factor that abrogates hepcidin‐mediated protection from iron overload under conditions of increased erythropoiesis. Increased concentrations of GDF15 have been reported in β‐thalassaemia patients and GDF15 has been found to suppress hepcidin expression in vitro. To further study the interdependencies of iron metabolism and erythropoiesis in vivo, the concentrations of hepcidin and GDF15 were determined in sera from 22 patients with pyruvate kinase deficiency (PKD) and 21 healthy control subjects. In PKD patients, serum hepcidin levels were 13‐fold lower than in controls (2·0 ng/ml vs. 26·2 ng/ml) and GDF15 was significantly higher (859 pg/ml vs. 528 pg/ml). Serum hepcidin concentrations correlated positively with haemoglobin and negatively with serum GDF15. These results suggest that GDF15 contributes to low hepcidin expression and iron loading in PKD.

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.