Haemin-induced cell death in human monocytic cells is consistent with ferroptosis

Background

Iron overload is a major issue for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, and the haemin in turn induces cell death and the generation of reactive oxygen species (ROS) in both murine macrophages and human monocytic THP-1 cells. This haemin-induced cell death process has been shown to be iron-dependent. Thus, we hypothesized that haemin-induced THP-1 cell death is a result of ferroptosis, an iron-dependent mechanism of cell death regulation.

铁过载诱导活性氧物质生成对骨髓造血功能影响的体外实验研究

目的 建立体外铁过载骨髓造血细胞模型,检验铁过载对细胞活性氧物质(ROS)水平的影响以及ROS升高对骨髓造血功能的影响。方法 在骨髓单个核细胞培养的过程中添加枸橼酸铁铵(FAC),使细胞铁过载,检验这一过程中细胞ROS水平、细胞凋亡水平、造血细胞集落形成和CD 34+细胞计数的变化。再用去铁胺(DFO)祛铁或抗氧化剂N-乙酰半胱氨酸(NAC)清除过多的ROS后,检测上述指标的变化。结果 ①在培养液中加入不同浓度FAC培养不同时间,发现骨髓造血细胞内可变铁池(LIP)水平升高,且具有时间和浓度依赖性,在含400μmoL/L FAC的培养液中培养24h时LIP水平达到最高。②在400μmol/L FAC浓度下,培养骨髓造血细胞24h后骨髓造血细胞内总的ROS、粒细胞和红细胞内ROS显著升高,分别为对照组的1.77、1.75和2.12倍。与对照组比较,DFO和NAC处理后均能明显降低细胞内ROS水平(P＜0.05)。③对骨髓细胞造血功能的检测发现FAC组细胞凋亡比例[(24.80±2.99)％]较对照组[(8.90±0.96)％]显著升高;造血细胞集落形成单位(CFUE、CFU-GM、BFU-E和CFU-mix)计数明显低于对照组(P值均＜0.05);CD34+细胞比例[(0.39±0.07)％]较对照组[(0.91±0.12)％]也显著降低。且这些损伤都可以通过DFO和NAC处理而部分恢复。结论 铁过载通过诱导ROS生成影响骨髓造血功能,这种损伤可以通过祛铁和抗氧化处理减轻。可能为治疗铁过载患者骨髓造血功能低下寻找新的靶点。     

铁过载骨髓造血细胞体外模型的建立及其对造血的影响

本研究通过铁与骨髓单个核细胞共培养,建立铁过载骨髓造血细胞模型并观察其对造血功能的影响.在体外培养骨髓单个核细胞的过程中,在培养液中添加枸橼酸铁铵(FAC),使细胞铁过载,建立铁过载骨髓造血细胞模型,然后检验这一过程中造血细胞的凋亡水平、造血集落形成(CFU-E、CFU-GM、BFU-E和CFU-mix)和CD34+细胞的计数变化.再用去铁胺(DFO)祛铁,观察上述指标的变化.结果发现:在不同时间加入培养液中不同浓度FAC,骨髓造血细胞内可变铁池(LIP)水平升高,且具有时间和浓度依赖性,在含400 μmol/L FAC的培养液中培养24小时时LIP水平达到最高.骨髓细胞造血功能检测表明,FAC组细胞凋亡比例(24.8 ±2.99%)较对照组(8.9±0.96%)显著升高(P＜0.01);造血集落形成单位计数明显低于对照组(P＜0.05);CD34+细胞比例(0.39±0.07%)与对照组(0.91±0.12%)相比也显著降低(P＜0.01).这些损伤影响可以通过DFO的处理部分消除.结论:在体外培养过程中添加铁能诱导骨髓单个核细胞铁过载,建立铁过载骨髓造血细胞模型,并能引起骨髓造血功能损伤,而这种损伤作用可以通过祛铁法减轻.本模型为深入研究铁过载对骨髓细胞造血功能的作用机制提供实验方法,并为骨髓造血功能低下的铁过载病人治疗提供新的靶点.     

Sensitive method for nontransferrin-bound iron quantification by graphite furnace atomic absorption spectrometry

OBJECTIVE: To establish a sensitive method for measuring nontransferrin-bound iron (NTBI) in serum samples using graphite furnace atomic absorption spectrometry (GFAAS). DESIGN AND METHODS: Nontransferrin-bound iron (NTBI) was chelated using nitrilotriacetic acid (NTA) and then ultrafiltered according to the method employed by Singh et al. [1]. Serum ultrafiltrates were diluted eightfold with distilled water. NTBI from the Fe-NTA complex present in the serum ultrafiltrate was measured using GFAAS. RESULTS: Nontransferrin-bound iron (NTBI) and other parameters were measured in seven patients diagnosed with hereditary hemochromatosis by liver biopsy. Total serum iron, NTBI and transferrin saturation values (ranging from 87% to 90%) were elevated for three of the seven hemochromatosis patients tested before therapeutic phlebotomy. Six of the seven hemochromatosis patients had undergone phlebotomy and revealed normal total serum iron, NTBI and transferrin saturation values. Nine test subjects (not diagnosed with hemochromatosis) with abnormally high total serum iron and/or ferritin concentrations exhibited normal NTBI values (< or =0.14 micromol/L to 0.29 micromol/L). The detection limit was 0.1 micromol/L for a 25 microL injection volume. CONCLUSIONS: The GFAAS method presented here provides a sensitive assay to quantitate NTBI in serum samples. The method developed is 4 to 5 times more sensitive than the only other GFAAS method [2] and more than an order of magnitude more sensitive than other colorimetric methods [1,3]. Improvement in sensitivity over the other GFAAS method [2] may be accounted for by differences in sample preparation between this method and that of Nielsen et al. [2]. Serum ultrafiltrates in this study were diluted eightfold with distilled water and mixed with a magnesium nitrate matrix modifier before GFAAS analysis. NTBI results obtained from this study indicate that the plasma iron pool in hemochromatosis patients awaiting phlebotomy increases to a level at which transferrin's ability to bind iron becomes exhausted and elevated NTBI levels appear in the serum. NTBI can mediate the production of reactive oxygen species and may cause organ damage associated with iron overload.     

Non-transferrin-bound iron is present in serum of hereditary haemochromatosis heterozygotes

BACKGROUND: Hereditary haemochromatosis (HH) is a common autosomal recessive disease. Recently, HH heterozygosity has been identified as an independent risk factor for myocardial infarction and cardiovascular mortality. Iron may play an important role in atherogenesis by catalyzing peroxidation of low-density-lipoprotein (LDL), an essential step in atherogenesis. In iron overload conditions, non-transferrin-bound iron (NTBI) is found in serum, which can catalyze lipid peroxidation. We investigated whether sera of HH heterozygotes contain more NTBI than sera of normal controls. METHODS: In 27 treated HH homozygotes, 22 HH heterozygotes and 17 healthy control subjects, conventional parameters of iron status (serum iron, transferrin saturation, serum ferritin) were measured. NTBI was detected using HPLC after addition of nitrilotriacetic acid and pretreatment with cobalt. RESULTS: The conventional parameters of iron status were similar in the HH heterozygous group and the control group. NTBI was significantly higher in homozygotes compared to heterozygotes (1.79 micromol L-1 vs. 0.51 micromol L-1, 95% CI of the difference = 0.6-1.95, P < 0.001), and controls (1.79 micromol L-1 vs. - 0.3 micromol L-1, 95% CI of the difference = 1.36-2.81, P < 0.001). The difference in NTBI between the heterozygous subjects and control subjects was also significant (0.51 micromol L-1 vs. - 0. 3 micromol L-1, 95% CI of the difference = 0.05-1.57, P < 0.05). CONCLUSION: Phlebotomy treated HH homozygotes maintain a high and potentially harmful serum NTBI. HH heterozygotes have a higher serum NTBI than normal controls. The reported increased risk of cardiovascular events in heterozygous haemochromatosis may be explained by NTBI-catalyzed LDL peroxidation.     

Interference of deferasirox with assays for serum iron and serum unsaturated iron binding capacity during iron chelating therapy

BackgroundDeferasirox (DFX) is an oral iron chelator that is used worldwide for the treatment of iron overload. Although serum ferritin level is usually measured as a marker of the efficacy of DFX, we sometimes experienced unexplainable changes in other serum markers for iron. We hypothesized that photometric assays for serum iron (sFe) and unsaturated iron binding capacity (UIBC) might be affected by DFX.MethodsMeasurement of sFe and UIBC was performed using 4 different assay systems. The samples were prepared by adding 0–300 μM DFX to pooled human serum or 15 randomized human serum samples. In some experiments, DFX-iron complex (DFX-Fe) was prepared by mixing iron ammonium citrate solution and DFX solution.ResultsMeasurement of sFe was influenced by DFX-Fe, while iron-free DFX showed no effect on the value of sFe; DFX-Fe was measured as sFe, undistinguishable from transferrin-bound iron. On the other hand, measurement of serum UIBC was influenced by DFX itself; DFX might have been bound to iron in the reagent used for the assay, leading to an increase in UIBC values.ConclusionsDFX affected the sFe and UIBC assay systems. We must be careful in observing these markers during iron chelation therapy with DFX.     

Oxidative status and malondialdehyde in beta-thalassaemia patients

Background In β‐thalassaemia syndromes, decreased or impaired biosynthesis of β‐globin leads to accumulation of unpaired α‐globin chains. Moreover, the iron overload in β‐thalassaemia patients generates oxygen‐free radicals and peroxidative tissue injury. The aim of this study was to detect and correlate iron overload parameters with the oxidative stress and the antioxidant capability in β‐thalassaemia patients. Design Serum iron, transferrin saturation, serum ferritin, nontransferrin‐bound iron (NTBI), levels of serum free and total (free + bound) malondialdehyde (MDA) and total peroxyl radical‐trapping antioxidant parameter (TRAP) were evaluated in 21 regularly transfused β‐thalassaemia major (TM) patients, 13 untransfused β‐thalassaemia intermedia (TI) patients and 17 healthy controls. Blood from the TM patients was drawn 48 h after the last desferoxamine (20–40 mg kg^?1) infusion and just before transfusion. Results Free and total MDA and NTBI levels were higher in the TM patients than in the TI. In the TM patients the free MDA levels correlated positively with serum iron (r = +0·3, P = 0·0006), whereas the total MDA correlated positively with NTBI (r = +0·45, P = 0·037). However, a negative correlation was observed between TRAP and NTBI (r = ?0·4, P = 0·0006). In the TI patients there was no significant correlation between free or total MDA and TRAP or NTBI. Conclusions Our results confirm the peroxidative status generated by iron overload in thalassaemia patients and highlight the rapid formation of marked amounts of free MDA despite the chelation therapy in TM patients.     

Regulation of transferrin receptor expression and ferritin content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and downregulation by interferon gamma.

We have investigated the regulation of key human iron binding proteins in mononuclear phagocytes by IFN gamma and iron transferrin. In a previous study, we demonstrated that IFN gamma downregulates the expression on human monocytes of transferrin receptors, the major source of iron for the cell. In the present study, we show that IFN gamma also downregulates the intracellular concentration of ferritin, the major iron storage protein in the cell. By radioimmunoassay, the mean ferritin content of nonactivated monocytes was 361 +/- 107 fg/monocyte (mean +/- SEM) whereas the mean ferritin content of IFN gamma-activated monocytes was 64 +/- 13 fg/monocyte, an 82% reduction with activation (P < 0.01, t test). Consistent with its downregulating effect on these iron proteins, IFN gamma treatment also results in decreased iron incorporation. IFN gamma-activated monocytes incorporated 33% less iron from 59Fe-transferrin than nonactivated monocytes (P < 0.05, t test). Gel filtration chromatography revealed that incorporated iron is located primarily in ferritin in both nonactivated and IFN gamma-activated monocytes. Ferritin in IFN gamma-activated monocytes is saturated with approximately three times as much 59Fe as ferritin in nonactivated monocytes. We have also explored the effect of iron transferrin on transferrin receptor expression and intracellular ferritin content in human monocytes. We have found that iron transferrin markedly upregulates both transferrin receptor expression and intracellular ferritin content in both nonactivated (2.3- and 1.3-fold, respectively) and IFN gamma-activated (3.4- and 2.9-fold, respectively) monocytes. This study demonstrates that transferrin receptor expression and intracellular ferritin content in human monocytes is unidirectionally and coordinately upregulated by iron transferrin and unidirectionally and coordinately downregulated by IFN gamma.     

Bleomycin-detectable iron assay for non-transferrin-bound iron in hematologic malignancies.

BACKGROUND: A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method. METHODS: The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis. RESULTS: The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 micromol/L) and a high (1.5 micromol/L) contrtrol, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 micromol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay. CONCLUSIONS: The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.     

Myocardial iron loading in patients with thalassemia major on deferoxamine chelation.

BACKGROUND: Heart failure secondary to myocardial iron loading remains the leading cause of death in thalassemia major (TM). We used cardiovascular magnetic resonance (CMR) to assess the prevalence of myocardial iron overload and ventricular dysfunction in a large cohort of TM patients maintained on conventional chelation treatment with deferoxamine. METHODS: A mobile CMR scanner was transported from London, UK, to Sardinia, Italy where 167 TM patients were assessed for myocardial iron loading, B-natriuretic peptide (BNP), and ferritin. In patients with myocardial iron loading CMR assessments of ventricular function were also made. RESULTS: Myocardial iron loading (T2* < 20 ms) was present in 108 (65%) patients, which was severe (T2* < 8 ms) in 22 (13%). Impaired (< 56%) left ventricular (LV) ejection fraction (EF) was present in 5%, 20% and 62% of patients with mild, moderate or severe iron loading. Increasing myocardial iron was related to impaired LVEF (Rs = 0.57, p < 0.001), weakly related to serum ferritin (Rs = -0.34, p < 0.001), and not related to liver iron (Rs = 0.11, p = 0.26). BNP was weakly related to myocardial iron (Rs = -0.35, p < 0.001) and was abnormal in only 5 patients. CONCLUSIONS: Myocardial siderosis was found in two-thirds of thalassemia major patients on maintenance deferoxamine treatment. This was combined with a high prevalence of impaired LV function, the severity of which tracked the severity of iron deposition. BNP was not useful to assess myocardial siderosis.     

Common presence of non-transferrin-bound iron among patients with type 2 diabetes

OBJECTIVE: Recently, we reported increased cardiovascular disease mortality among supplemental vitamin C users with type 2 diabetes in a prospective cohort study. Because vitamin C may cause oxidative stress in the presence of redox active iron, we hypothesized that non-transferrin-bound iron (NTBI), a form of iron susceptible to redox activity, may be present in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: We measured serum NTBI levels using high-performance liquid chromatography in 48 patients with known diabetes (at least 5 years duration since diagnosis), 49 patients with newly diagnosed diabetes, and 47 healthy control subjects (frequency matched on age and sex). RESULTS: NTBI was commonly present in diabetes: 59% in newly diagnosed diabetes and 92% in advanced diabetes. Mean NTBI values varied significantly between the three groups, with the highest values being observed in patients with known diabetes and the lowest in the control subjects (0.62 +/- 0.43 vs. 0.24 +/- 0.29 vs. 0.04 +/- 0.13 micromol/l Fe). Serum total iron or percent transferrin saturation were very similar among the three groups, yet NTBI was strongly associated with serum total iron (r = 0.74, P < 0.01) and percent transferrin saturation (r = 0.70, P < 0.01) among the patients with known diabetes. CONCLUSIONS: Consistent with our hypothesis, these data demonstrate the common existence of NTBI in type 2 diabetic patients with a strong gradient with severity. Prospective cohort studies are required to clarify the clinical relevance of increased NTBI levels.     

Improvement in oxidative stress and antioxidant parameters in β-thalassemia/Hb E patients treated with curcuminoids

ObjectivesTo evaluate the hematological profile, oxidative stress, and antioxidant parameters in β-thalassemia/Hb E patients treated with curcuminoids for 12 months.Design and methodsTwenty-one β-thalassemia/Hb E patients were given 2 capsules of 250 mg each of curcuminoids (a total of 500 mg) daily for 12 months. Blood was collected every 2 months during treatment and 3 months after withdrawal and was determined for complete blood count, malonyldialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) in red blood cells (RBC), and non-transferrin bound iron (NTBI) in serum.ResultsThe increased oxidative stress in β-thalassemia/Hb E patients was shown by higher levels of MDA, SOD, GSH-Px in RBC, serum NTBI, and lower level of RBC GSH. Curcuminoids administration resulted in improvement of all the measured parameters as long as they were administered. After 3 months withdrawal of treatment, all parameters returned close to baseline levels.ConclusionCurcuminoids may be used to ameliorate oxidative damage in patients with β-thalassemia/Hb E disease.     

载超顺磁性氧化铁和DiI荧光高分子微球巨噬细胞MR成像

目的 制备载超顺磁氧化铁(SPIO)纳米粒和DiI荧光高分子微球(DiI-SPIO-PLGA),探讨其作为MRI对比剂体外巨噬细胞成像的效果和作为荧光示踪剂体外示踪巨噬细胞的可行性。 方法 采用双乳化法制备DiI-SPIO-PLGA微球,并检测其理化性质。培养小鼠RAW264.7巨噬细胞,与DiI-SPIO-PLGA微球共孵育12 h后行普鲁士蓝染色和荧光显微镜观察,将吞噬了微球的细胞和空白细胞分别重悬于0.5 ml 1%琼脂糖Eppendof管中,行MR扫描。 结果 所得样品为外壳装载SPIO颗粒和DiI荧光的微球,粒径(868.00±68.73)nm。普鲁士蓝染色结果显示,几乎所有细胞内都有蓝染颗粒分布,部分区域聚集成堆;倒置荧光显微镜下可见细胞内有大量红色微球。MRI显示吞噬了DiI-SPIO-PLGA微球的实验组管内信号显著降低,管内信号值/背景信号值明显低于对照组(P<0.05)。 结论 DiI-SPIO-PLGA微球能有效加强MR成像效果,荧光信号强烈,可同时作为MRI阴性对比剂和荧光示踪剂。     

Serum ferritin in the assessment of liver iron overload and iron removal therapy in porphyria cutanea tarda

Serum ferritin, an index of iron stores, was studied in 60 patients with porphyria cutanea tarda (PCT), in 21 patients who had other liver diseases without siderosis (cirrhosis [LC] and chronic active hepatitis [CAH]), and in 32 patients with associated liver siderosis (alcoholic LC, LC and CAH in minor thalassemia). Ferritin levels were higher in patients with porphyria than in healthy controls and patients without liver siderosis (P less than 0.001), whereas no statistical difference was observed between patients with porphyria and those with liver siderosis. Because iron removal is considered the treatment of choice for PCT, some patients with PCT underwent phlebotomy and others received chelating therapy with subcutaneous infusion of deferoxamine. Follow-up of the patients showed a correlation between serum ferritin level and urinary porphyrin excretion; when the clinical and biochemical syndrome became normal, serum iron and ferritin had fallen to normal values (t test pair data analysis before and after: P less than 0.001 in each group). No appreciable difference was found between controls and patients with PCT whose conditions had been normalized, irrespective of the chronic liver damage always present in PCT. Our results suggest that serum ferritin increase in PCT is related more to liver iron overload than to liver damage, and ferritin follow-up is recommended to indicate the exhaustion of hepatic iron stores during iron depletion therapy, as well as to detect an early replenishment after remission.     

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

While hemochromatosis is characterized by sequestration of iron-protein complexes in hepatocyte lysosomes, little is known about the effects of excess iron on these organelles. Therefore, we studied the effects of experimental iron overload on hepatocyte lysosomal structure, physicochemical properties, and function in rats fed carbonyl iron. A sixfold increase (P less than 0.0001) in hepatic iron and a fivefold increase in lysosomal iron (P less than 0.01) was observed after iron loading; as a result, hepatocyte lysosomes became enlarged and misshapen. These lysosomes displayed increased (P less than 0.0001) fragility; moreover, the fluidity of lysosomal membranes isolated from livers of iron-loaded rats was decreased (P less than 0.0003) as measured by fluorescence polarization. Malondialdehyde, an end product of lipid peroxidation, was increased by 73% (P less than 0.008) in lysosomal membranes isolated from livers of iron-overloaded rats. While amounts of several individual fatty acids in isolated lysosomal membranes were altered after iron overload, cholesterol/phospholipid ratios, lipid/protein ratios, double-bond index, and total saturated and unsaturated fatty acids remained unchanged. The pH of lysosomes in hepatocytes isolated from livers of iron-loaded rats and measured by digitized video microscopy was increased (control, 4.70 +/- 0.05; iron overload, 5.21 +/- 0.10; P less than 0.01). Our results demonstrate that experimental iron overload causes marked alterations in hepatocyte lysosomal morphology, an increase in lysosomal membrane fragility, a decrease in lysosomal membrane fluidity, and an increase in intralysosomal pH. Iron-catalyzed lipid peroxidation is likely the mechanism of these structural, physicochemical, and functional disturbances.     

Influences of sleep and the circadian rhythm on iron-status indices

ObjectivesThe aim was to study the influence of sampling time, food intake and sleep on tests used to screen for and monitor conditions of iron deficiency and overload.Design and methodsThe 24 h variations of iron, transferrin, transferrin saturation (TS) and ferritin were studied in seven healthy individuals during standardised food intake, and during night or day sleep.ResultsIron and TS showed clear diurnal variations, with peaks at 12.6 h and 12.8 h respectively, during night sleep, and at 19.7 h and 19.3 h, respectively, during day sleep. Ferritin did not demonstrate any circadian variation. Transferrin and ferritin were unaffected by sleep-condition. Meals did not have any effect except a slight decline of transferrin.ConclusionsTime of day and sleeping patterns had great influence on iron and TS, whereas no or only minor effects are seen on the concentration of ferritin and transferrin. Meals have limited effects.     

Imaging monocytes with iron oxide nanoparticles targeted towards the monocyte integrin MAC‐1 (CD11b/CD18) does not result in improved atherosclerotic plaque detection by in vivo MRI

Imaging of macrophages with superparamagnetic iron oxide particles (SPIO) has been performed to improve detection of atherosclerotic plaque inflammation in human and mouse studies by molecular magnetic resonance imaging (MRI). Since affinity of the monocyte/macrophage integrin MAC‐1 (CD11b/CD18) is upregulated in inflammation, we generated a contrast agent targeting CD11b (CD11b‐SPIOs) for improved macrophage detection in plaques. CD11b‐SPIOs and non‐targeted SPIOs (control‐SPIOs) were incubated in vitro with human monocytes/macrophages. As quantified by SPIO‐induced MRI signal extinction, intracellular iron‐content was significantly higher in monoytes/macrophages incubated with CD11b‐SPIO than with control‐SPIO in vitro (p < 0.05), suggesting an improved uptake of CD11b‐SPIOs into monocytes. Therefore, the aortic arch (AA) and vessel branches of ApoE^?/?‐knockout mice on a Western‐type diet were imaged before and 48 h after contrast agent injection of either CD11b‐SPIOs or control‐SPIOs, using a 9.4 T animal MRI system. The SPIO‐induced change in the MRI signal was quantified, as well as the macrophage‐content by anti‐CD68 immunhistochemistry and the iron‐content by Prussian‐blue staining. However, SPIO‐induced signal extinction in in vivo‐MRI was similar in CD11b‐SPIO and control‐SPIO‐injected animals, with a non‐significant trend towards an improved uptake of CD11b‐SPIOs in the subclavian artery and subsections of the AA. These data correlated well with the results obtained by histology. Although in vitro MRI‐data indicated an increased uptake of targeted CD11b‐SPIOs in monocytes/macrophages, in vivo mouse data do not allow improved atherosclerotic plaque detection compared WITH non‐targeted SPIOs. Therefore, CD11b‐targeted MRI contrast labelling of monocytes/macrophages does not seem to be a successful strategy in stable atherosclerotic plaques such as found in the ApoE^?/?‐knockout‐model. However, the impressive correlation between MRI and histology data encourages further development of inflammation‐ and plaque‐specific contrast agents for vulnerable plaque imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Prevalence, causes and effects of increased iron storage in patients with kidney transplantation

Patients on chronic hemodialysis often need blood transfusions due to erythropoietin deficiency. Even after successful kidney transplantation iron overload may persist. Former histological studies have revealed siderosis of the liver in 69% of all patients whose serum ferritin was above 1100 ng/ml. The aim of the present study was to evaluate the influence of iron overload on liver function. In 146 symptom free patients with renal allografts serum ferritin was determined to detect possible iron overload. Serum ferritin between 4 and 5480 ng/ml were found (women: 358.7 +/- 105.3; men 282.4 +/- 63.3 ng/ml; x +/- SEM). Twelve patients (8.1%) had ferritin levels higher than 1100 ng/ml. These twelve patients as well as another group of eight patients with renal allografts whose serum ferritin was known to be higher than 1100 ng/ml were included for further evaluation. Their data were matched and compared with those of a control group also patients with renal allograft (same age and sex) whose serum ferritin was lower than 1100 ng/ml. Transaminases (SGPT 22.6 +/- 3.6 vs. 15.4 +/- 6.0 U/l; SGOT 14.7 +/- 2.0 vs. 13.0 +/- 4.8 U/l) and plasma glucose (90.5 +/- 7.1 vs. 76.8 +/- 3.7 mg/dl) were found to be significantly higher (p less than 0.05) in patients with serum ferritin levels above 1100 ng/ml. Elevated transaminases were significantly more frequent in patients with high serum ferritin (9 vs. 2; p less than 0.02) as compared with the control. Ferritin levels significantly correlated with the number of preceding blood transfusions (p less than 0.002). Hbs-persistence was detected in six out of 20 patients with high ferritin levels but only in one out of 20 in the control group (p less than 0.05) whereas anti-Hbs prevalence was not different in the two groups. These data indicate that chronic iron overload should be considered as a possible cause of chronic liver disease in patients with renal allografts.     

Serum hepcidin concentration in chronic haemodialysis patients: associations and effects of dialysis, iron and erythropoietin therapy

Background Hepcidin, a liver‐derived peptide induced by iron overload and inflammation, is a major regulator of iron homeostasis. As hepcidin decreases gastrointestinal iron absorption and recirculation from monocytes, over‐expression is associated with the development of anaemia. Methods We studied the associations between circulating hepcidin levels and various laboratory parameters related to anaemia and/or inflammation in 20 patients on chronic haemodialysis. Furthermore, we determined the impact of dialysis and iron and/or erythropoietin (rhEpo) supplementation therapy on hepcidin serum concentrations. The patients were withheld from iron and rhEpo for 2 weeks before study entry. Hepcidin was measured by liquid chromatography‐mass spectrometry (LC‐MS/MS); serum iron and haematological parameters, cytokines and pro‐hepcidin by commercially available enzyme‐linked immunosorbent assays (ELISA) or standard automated methods. Results While hepcidin levels at baseline were not correlated to pro‐hepcidin, interleukin‐6 or transforming growth factor‐beta concentrations, we found significant associations with reticulocyte count (r = ?0·55; P = 0·015), serum iron (r = 0·7; P = 0·004) and ferritin levels (r = 0·63; P = 0·004) and transferrin saturation (r = 0·69, P = 0·001). Dialysis using either a high or a low flux biocompatible dialyser resulted in a significant decrease of hepcidin concentrations, which returned to pre‐dialysis values before the next dialysis session. When studying the effects of anaemia treatment, we observed a significant reduction of hepcidin levels following administration of rhEpo but not iron. Conclusions Hepcidin levels in stable haemodialysis patients appear to reflect systemic iron load, but not inflammation. Due to the negative association between reticulocyte counts and hepcidin, the reduction of circulating hepcidin concentrations by dialysis and/or rhEpo treatment may positively affect erythropoiesis.