Impact of treating iron deficiency, diagnosed according to hepcidin quantification, on outcomes after a prolonged ICU stay compared to standard care: a multicenter, randomized, single-blinded trial.

BACKGROUND: Anemia is a significant problem in patients on ICU. Its commonest cause, iron deficiency (ID), is difficult to diagnose in the context of inflammation. Hepcidin is a new marker of ID. We aimed to assess whether hepcidin levels would accurately guide treatment of ID in critically ill anemic patients after a prolonged ICU stay and affect the post-ICU outcomes. METHODS: In a controlled, single-blinded, multicenter study, anemic (WHO definition) critically ill patients with an ICU stay ≥ 5 days were randomized when discharge was expected to either intervention by hepcidin treatment protocol or control. In the intervention arm, patients were treated with intravenous iron (1 g of ferric carboxymaltose) when hepcidin was < 20 µg/l and with intravenous iron and erythropoietin for 20 ≤ hepcidin < 41 µg/l. Control patients were treated according to standard care (hepcidin quantification remained blinded). Primary endpoint was the number of days spent in hospital 90 days after ICU discharge (post-ICU LOS). Secondary endpoints were day 15 anemia, day 30 fatigue, day 90 mortality and 1-year survival. RESULTS: Of 405 randomized patients, 399 were analyzed (201 in intervention and 198 in control arm). A total of 220 patients (55%) had ID at discharge (i.e., a hepcidin < 41 µg/l). Primary endpoint was not different (medians (IQR) post-ICU LOS 33(13;90) vs. 33(11;90) days for intervention and control, respectively, median difference - 1(- 3;1) days, p = 0.78). D90 mortality was significantly lower in intervention arm (16(8%) vs 33(16.6%) deaths, absolute risk difference - 8.7 (- 15.1 to - 2.3)%, p = 0.008, OR 95% IC, 0.46, 0.22-0.94, p = 0.035), and one-year survival was improved (p = 0.04). CONCLUSION: Treatment of ID diagnosed according to hepcidin levels did not reduce the post-ICU LOS, but was associated with a significant reduction in D90 mortality and with improved 1-year survival in critically ill patients about to be discharged after a prolonged stay. TRIAL REGISTRATION: www.clinicaltrial.gov NCT02276690 (October 28, 2014; retrospectively registered).

Systemic iron homeostasis.

The iron hormone hepcidin and its receptor and cellular iron exporter ferroportin control the major fluxes of iron into blood plasma: intestinal iron absorption, the delivery of recycled iron from macrophages, and the release of stored iron from hepatocytes. Because iron losses are comparatively very small, iron absorption and its regulation by hepcidin and ferroportin determine total body iron content. Hepcidin is in turn feedback-regulated by plasma iron concentration and iron stores, and negatively regulated by the activity of erythrocyte precursors, the dominant consumers of iron. Hepcidin and ferroportin also play a role in host defense and inflammation, and hepcidin synthesis is induced by inflammatory signals including interleukin-6 and activin B. This review summarizes and discusses recent progress in molecular characterization of systemic iron homeostasis and its disorders, and identifies areas for further investigation.

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains.

Iron plays essential roles in the central nervous system. However, how the iron level is regulated in brain cells including glia and neurons remains to be fully clarified. In this study, the localizations of hepcidin, ferroportin, and hephaestin, which are known to be involved in iron efflux, were immunohistochemically examined in autopsied human brains. Immunoreactivities for hepcidin and ferroportin were observed in granular structures within the cytoplasm of reactive astrocytes and epithelial cells of the choroid plexus. Granular structures showing immunoreactivities for hepcidin and ferroportin were also stained with antibodies for early endosome antigen 1 (EEA1). In addition, immunoreactivity for hephaestin was observed in the cytoplasm of epithelial cells of the choroid plexus as well as reactive astrocytes. Immunoreactivity for hephaestin in the cytoplasm of reactive astrocytes was occasionally colocalized with immunoreactivity for EEA1, while that of hephaestin was frequently observed in the cytoplasm showing no immunoreactivity for EEA1. These findings suggest that immunoreactivities for hepcidin and ferroportin are localized in close proximity to granular structures showing immunoreactivity for EEA1 in the cytoplasm of human brain astrocytes. They also suggest that immunoreactivity of hephaestin is localized in the cytoplasm of the choroid plexus epithelium as well as reactive astrocytes of human brains.

Bacteria-Carried Iron Oxide Nanoparticles for Treatment of Anemia.

The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia.

Hepatic iron is the major determinant of serum ferritin in NAFLD patients.

BACKGROUND AND AIMS: Elevated serum ferritin is common in NAFLD, and is associated with more advanced disease and increased mortality. Hyperferritinaemia in NAFLD is often attributed to inflammation, while in other conditions ferritin closely reflects body iron stores. The aim of this study was to clarify the underlying cause of hyperferritinaemia in NAFLD. METHODS: Ferritin levels were examined with markers of iron status, inflammation and liver injury across the clinical spectrum of NAFLD using blood, tissue and magnetic resonance (MR) imaging. A separate larger group of NAFLD patients with hepatic iron staining and quantification were used for validation. RESULTS: Serum ferritin correlated closely with the iron regulatory hormone hepcidin, and liver iron levels determined by MR. Furthermore, ferritin levels reflected lower serum adiponectin, a marker of insulin resistance, and liver fat, but not cytokine or CRP levels. Ferritin levels differed according to fibrosis stage, increasing from early to moderate disease, and declining in cirrhosis. A similar pattern was found in the validation cohort of NAFLD patients, where ferritin levels were highest in those with macrophage iron deposition. Multivariate analysis revealed liver iron and hepcidin levels as the major determinants of serum ferritin. CONCLUSIONS: While hyperferritinaemia is associated with markers of liver injury and insulin resistance, serum hepcidin and hepatic iron are the strongest predictors of ferritin levels. These findings highlight the role of disordered iron homeostasis in the pathogenesis of NAFLD, suggesting that therapies aimed at correcting iron metabolism may be beneficial.

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement.

BACKGROUND: Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. METHODS: An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). RESULTS: By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). CONCLUSIONS: Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 µg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

Iron deficiency diagnosed using hepcidin on critical care discharge is an independent risk factor for death and poor quality of life at one year: an observational prospective study on 1161 patients.

BACKGROUND: Iron deficiency is difficult to diagnose in critically ill patients, but may be frequent and may impair recovery. Measurement of hepcidin could help in the diagnosis of iron deficiency. We aim to assess if iron deficiency diagnosed using hepcidin is associated with poorer outcome one year after an intensive care unit stay. METHODS: We used the prospective FROG-ICU, multicentre (n = 28 ICUs), observational cohort study of critically ill survivors followed up one year after intensive care unit discharge. Iron deficiency was defined as hepcidin < 20 ng/l, ferritin < 100 ng/l or soluble transferrin receptor (sTfR)/log(ferritin) > 0.8, measured in blood drawn at intensive care unit discharge. Main outcomes were one-year all-cause mortality and poor quality of life (defined as a Short Form 36 (SF-36) score below the median). RESULTS: Among the 2087 patients in the FROG-ICU cohort, 1570 were discharged alive and 1161 had a blood sample available at intensive care unit discharge and were included in the analysis. Using hepcidin, 429 (37%) patients had iron deficiency, compared to 72 (6%) using ferritin alone and 151 (13%) using the sTfR/log(ferritin) ratio. Iron deficiency diagnosed according to low hepcidin was an independent predictor of one-year mortality (OR 1.51 (1.10-2.08)) as was high sTfR/log ferritin ratio (OR = 1.95 (1.27-3.00)), but low ferritin was not. Severe ID, defined as hepcidin < 10 ng/l, was also an independent predictor of poor one-year physical recovery (1.58 (1.01-2.49)). CONCLUSIONS: Iron deficiency, diagnosed using hepcidin, is very frequent at intensive care unit discharge and is associated with increased one-year mortality and poorer physical recovery. Whether iron treatment may improve these outcomes remains to be investigated.

Serum and salivary ferritin and Hepcidin levels in patients with chronic periodontitis and type 2 diabetes mellitus.

BACKGROUND: Iron disorder and abnormal expression of hepcidin play important roles in many diseases, but it is still unclear in chronic periodontitis (CP) and type 2 diabetes mellitus (T2DM). We aimed to assess ferritin and hepcidin levels in serum and saliva of CP patients with or without T2DM. METHODS: Serum and unstimulated whole saliva samples were collected from 88 participants, who were categorized into 4 groups based on the presence or absence of CP or T2DM. Demographics and general health parameters were recorded. Full-mouth clinical periodontal parameters including probing pocket depth, clinical attachment loss, bleeding index, and plaque index were recorded. Chemiluminescence microparticle immunoassay and enzyme-linked immunosorbent assay were used to detect ferritin and hepcidin concentrations, respectively, in serum and saliva. RESULTS: Serum ferritin and hepcidin levels in the CP and CP with T2DM groups were higher than in the control group (P < 0.05). Serum hepcidin and serum ferritin are linear correlated (P < 0.001). Serum hepcidin/ferritin values in the CP with T2DM group were significantly lower than those in the T2DM and control groups. Moreover, salivary ferritin levels in the CP and T2DM groups were higher than those in the control group (P < 0.05). There was positively correlation between salivary ferritin and serum ferritin (P = 0.017). Hepcidin concentrations were relatively low in saliva. CONCLUSIONS: These results suggest that iron overload and hepcidin inadequacy existed in CP with T2DM patients. Salivary ferritin might provide a reference for body iron load. TRIAL REGISTRATION: ChiCTR-ROC-17012780.

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators.

In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.

[Siwu decoction improves iron deficiency anemia in infant rats and regulates iron metabolism].

To investigate the effect of Siwu decoction on improving iron deficiency anemia in infant rats and observe its regulatory effects on iron metabolism. SD rats were fed with low iron fodder for 2 weeks, and then the rats with hemoglobin level less than 75 g•L ⁻¹ were screened out and randomly divided into model group, Ferrous succinate 50 mg•kg ⁻¹ group, Siwu decoction 4 g•kg ⁻¹, 8 g•kg ⁻¹ and 16 g•kg ⁻¹ groups. After 4 weeks' gavage administration, Wright-Giemsa's staining of blood smear and HE staining of the livers were conducted, and all rats were tested for blood routine, serum iron, total iron binding capacity, serum ferritin, serum hepcidin and liver hepcidin. The expression levels of liver ferritin, transferrin and transferrin receptor 1 were also detected. The results showed that as compared with normal group, the activity level of model group was decreased, and the color and lustre of auricles and toes were pale white; the number of red blood cells was decreased; the volume was smaller, with an increased zone of central pallor; the body weight and blood routine parameters were decreased significantly; the livers were pale red, and the hepatic cords around thecentral veins were unclear and misaligned; the serum iron, serum ferritin, liver iron levels and the expression of liver ferritin were decreased significantly; the total iron binding capacity, serum hepcidin, liver hepcidin, the expression levels of liver transferrin and transferrin receptor 1 were significantly increased, indicating successful establishment of models. As compared with the model group, activity was increased in Siwu decoction group; the color and lustre of auricles and toes were ruddy; the number of red blood cells was increased; the volume was larger, with a decreased zone of central pallor; the body weight and blood routine parameters were increased significantly; the livers were red, hepatic cords around the central veins were clear and aligned;the serum iron, serum ferritin, liver iron levels and the expression of liver ferritin were significantly increased, the total iron binding capacity, serum hepcidin, liver hepcidin, the expression of liver transferrin and transferrin receptor 1 were decreased significantly. The results demonstrated that Siwu decoction had a certain effect on improving iron deficiency anemia in infant rats, and the mechanism may be associated with the regulatory effect of hepcidin iron metabolism.

Recombinant human erythropoietin-induced erythropoiesis regulates hepcidin expression over iron status in the rat.

The crosstalk between several factors controlling hepcidin synthesis is poorly clarified for different physiological and pathological conditions. Our aim was to study the impact of increasing recombinant human erythropoietin (rHuEPO) doses on erythropoiesis, iron metabolism and hepcidin, using a rat model. Male Wistar rats were divided in 5 groups: control (vehicle) and rHuEPO-treated groups (100, 200, 400 and 600IU/kgbody weight/week), 3 times per week, during 3weeks. Hematological and iron data were evaluated. The expression of several genes involved in iron metabolism was analyzed by qPCR. Liver hepcidin protein was evaluated by Western Blot. The rHuEPO treatment induced erythropoiesis and increased transferrin saturation (TSAT) in a dose dependent manner. Tf receptor 2 (TfR2), hemojuvelin (HJV) and bone morphogenetic protein 6 (BMP6) were up-regulated in rHuEPO200 group. Matriptase-2 was down-regulated in rHuEPO200 group, and up-regulated in the other rHuEPO-treated groups. Hepcidin synthesis was increased in rHuEPO200 group, and repressed in the rHuEPO400 and rHuEPO600 groups. Our study showed that when a high erythropoietic stimulus occurs, hepcidin synthesis is mainly regulated by TSAT; however, when the erythropoiesis rate reaches a specific threshold, extramedullary hematopoiesis is triggered, and the control of hepcidin synthesis is switched to matriptase-2, thus inhibiting hepcidin synthesis.

Isocitrate treatment of acute anemia of inflammation in a mouse model.

Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit.

Quantification of Hepcidin-related Iron Accumulation in the Rat Liver.

Hepcidin was originally detected as a liver peptide with antimicrobial activity and it functions as a central regulator in the systemic iron metabolism. Consequently suppression of hepcidin leads to iron accumulation in the liver. AbbVie developed a monoclonal antibody ([mAb]; repulsive guidance molecule [RGMa/c] mAb) that downregulates hepcidin expression by influencing the RGMc/bone morphogenetic protein (BMP)/neogenin receptor complex and causes iron deposition in the liver. In a dose range finding study with RGMa/c mAb, rats were treated with different dose levels for a total of 4 weekly doses. The results of this morphometric analysis in the liver showed that iron accumulation is not homogenous between liver lobes and the left lateral lobe was the most responsive lobe in the rat. Quantitative hepcidin messenger RNA analysis showed that the left lateral lobe was the most responsive lobe showing hepcidin downregulation with increasing antibody dose. In addition, the morphometric analysis had higher sensitivity than the chemical iron extraction and quantification using a colorimetric assay. In conclusion, the Prussian blue stain in combination with semi-quantitative and quantitative morphometric analysis is the most reliable method to demonstrate iron accumulation in the liver compared to direct measurement of iron in unfixed tissue using a colorimetric assay.

Iron and noncontrast magnetic resonance T2* as a marker of intraplaque iron in human atherosclerosis.

OBJECTIVE: Iron has been implicated in atherogenesis and plaque destabilization, whereas less is known about iron-related proteins in this disease. We compared ex vivo quantities with in vivo vessel wall T2*, which is a noncontrast magnetic resonance relaxation time that quantitatively shortens with increased tissue iron content. We also tested the hypothesis that patients with carotid atherosclerosis have abnormal T2* times vs controls that would help support a role for iron in human atherosclerosis. METHODS: Forty-six patients undergoing carotid endarterectomy and 14 subjects without carotid disease were prospectively enrolled to undergo carotid magnetic resonance imaging. Ex vivo measurements were performed on explanted plaque and 17 mammary artery samples. RESULTS: Plaques vs normal arteries had higher levels of ferritin (median, 7.3 [interquartile range (IQR), 4-13.8] vs 1.0 [IQR, 0.6-1.3] ng/mg; P < .001) and oxidized low-density lipoprotein (median, 0.17 [IQR, 0.12-0.30] vs 0.01 [IQR, 0.003-0.03] ng/mg; P < .001) as well as hepcidin (median, 8.7 [IQR, 4.6-12.4] vs 2.6 [IQR, 1.3-7.0] ng/mL; P = .03); serum hepcidin levels did not distinguish atherosclerosis patients from controls (median, 40.6 [IQR, 18.8-88.6] vs 33.9 [IQR, 17.6-55.2]; P = .42). Shorter in vivo T2* paralleled larger plaque volume (ρ = -.44; P = .01), and diseased arteries had shorter T2* values compared with controls (median, 17.7 ± 4.3 vs 23.0 ± 2.4 ms; P < .001). CONCLUSIONS: Diseased arteries have greater levels of iron-related proteins ex vivo and shorter T2* times in vivo. Further studies should help define the role of T2* as a biomarker of iron and atherosclerosis.

Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids.

Endogenous peptides that represent biological and pathological information of disease have attracted interest for diagnosis. However, the extraction of those low abundance peptides is still a challenge because of the complexity of human bodily fluids (HBF). Hepcidin, a peptide hormone, has been recognized as a biomarker for iron-related diseases. There is no rapid and reliable way to enrich them from HBF. Here we describe a peptide extraction approach based on nanoporous silica thin films to successfully detect hepcidin from HBF. Cooperative functions of nanopore to biomolecule, including capillary adsorption, size-exclusion and electrostatic interaction, were systematically investigated to immobilize the target peptide. To promote this new approach to clinical practices, we further applied it to successfully assay the hepcidin levels in HBF provided by healthy volunteers and patients suffering from inflammation. Our finding provides a high-throughput, rapid, label-free and cost-effective detection method for capturing and quantifying low abundance peptides from HBF. FROM THE CLINICAL EDITOR: Diagnosing diseases with low concentration peptide biomarkers remains challenging. This team of authors describes a peptide extraction approach based on nanoporous silica thin films to successfully detect low concentrations of hepcidin from human body fluids collected from 119 healthy volunteers and 19 inflammation patients.

Hepcidin analysis in pneumonia: Comparison of immunoassay and LC-MS/MS.

BACKGROUND: The iron-regulatory hormone hepcidin is a promising biomarker to differentiate anaemia of inflammation from iron deficiency. Plasma hepcidin concentrations increase substantially during inflammation, and the amount of smaller, non-biologically active isoforms of hepcidin increase in inflammatory conditions. These smaller isoforms are measured in some, but not all analytical methods. Thus, we evaluated the comparability of two analytical methods with different isoform selectivity during and after acute-phase pneumonia as a highly inflammatory model disease. METHODS: Blood samples from a cohort of 267 hospitalized community-acquired pneumonia patients collected at admission and a 6-week follow-up were analysed. Hepcidin was measured in plasma by an immunoassay, which recognizes all hepcidin isoforms, and a liquid chromatography tandem mass spectrometry (LC-MS/MS), which selectively measures the bioactive hepcidin-25. Additionally, a subset of serum samples was analysed by LC-MS/MS. RESULTS: Hepcidin measurements by immunoassay were higher compared with LC-MS/MS. The relative mean difference of hepcidin plasma concentrations between the two analytical methods was larger in admission samples than in follow-up samples (admission samples <200 ng/mL: 37%, admission samples >200 ng/mL: 78%, follow-up samples >10 ng/mL: 22%). During acute-phase pneumonia, serum concentrations were on average 22% lower than plasma concentrations when measured by LC-MS/MS. CONCLUSIONS: Immunoassay measured higher hepcidin concentrations compared with LC-MS/MS, with more pronounced differences in high-concentration samples during acute-phase pneumonia. These findings should be considered in local method validations and in future harmonization and standardization optimization of hepcidin measurements.

Clinical review: Biomarkers of acute kidney injury: where are we now?

The recognition that acute kidney injury (AKI) is a significant independent risk factor for morbidity and mortality has resulted in a substantial number of publications over the past 5 years or more. In no small part these have, to a degree, highlighted the inadequacy of conventional markers of renal insufficiency in the acute setting. Much effort has been invested in the identification of early, specific AKI markers in order to aid early diagnosis of AKI and hopefully improve outcome. The search for a 'biomarker' of AKI has seen early promise replaced by a degree of pessimism due to the lack of a clear candidate molecule and variability of results. We outline the major studies described to date as well as discuss potential reasons for the discrepancies observed and suggest that evolution of the field may result in success with ultimately an improvement in patient outcomes.

Optimization of micro-HPLC peak focusing for the detection and quantification of low hepcidin concentrations.

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 µL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.

Clinical Immunoassay for Human Hepcidin Predicts Iron Deficiency in First-Time Blood Donors.

BACKGROUND: Serum markers currently used as indicators of iron status have clinical limitations. Hepcidin, a key regulator of iron homeostasis, is reduced in iron deficiency (ID) and increased in iron overload. We describe the first CLIA-validated immunoassay with excellent accuracy and precision to quantify human serum hepcidin. Its diagnostic utility for detecting ID in first-time blood donors was demonstrated. METHODS: A monoclonal competitive ELISA (C-ELISA) was developed for the quantitation of human hepcidin and validated according to CLIA guidelines. Sera from nonanemic first-time blood donors (n = 292) were analyzed for hepcidin, ferritin, transferrin, and serum iron. Logistic regression served to determine the utility of hepcidin as a predictor of ID. RESULTS: The C-ELISA was specific for human hepcidin and had a low limit of quantitation (4.0 ng/mL). The hepcidin concentration measured with the monoclonal C-ELISA was strongly correlated with a previously established, extensively tested polyclonal C-ELISA (Blood 2008;112:4292-7) (r = 0.95, P < 0.001). The area under the receiver operating characteristic curve for hepcidin as a predictor of ID, defined by 3 ferritin concentration thresholds, was >0.9. For predicting ID defined by ferritin <15 ng/mL, hepcidin <10 ng/mL yielded sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin <30 ng/mL yielded sensitivity of 67.6% and specificity of 91.7%. CONCLUSION: The clinical measurement of serum hepcidin concentrations was shown to be a potentially useful tool for diagnosing ID.