ALBERT

Description: Administration in the drinking water of the orally-active iron chelator 1,2-diethyl-3-hydroxypyridin-4-one (CP94) to C57BL/10ScSn mice caused the development of hepatic protoporphyria. This was detected after 1 week and continued as long as the chelator was given (15 weeks). The more hydrophilic 1,2-dimethyl- and 1-hydroxyethyl,2-ethyl-analogues (CP20 and CP102) were also tested, but they were both inactive in inducing accumulation of protoporphyrin in the liver. Restriction of in vivo iron supply for ferrochelatase seemed a likely mode of action, but an approximately 30% decrease in activity of this enzyme was also observed when measured in vitro. Extracts of livers from mice given CP20, CP94, and CP102 showed no potential to inhibit mouse ferrochelatase, in contrast to the findings with an extract from mice treated with the known porphyrogenic chemical 4-ethyl - 3 , 5 - diethoxycarbonyl - 2 , 6 - dimethyl - 1 , 4 - dihydropyridine, -indicating that ferrochelatase inhibition did not occur by the formation of an N-ethyl-protoporphyrin derived from metabolism by cytochrome P450. CP20, CP94, CP102, and CP117 (the pivoyl ester of CP102) all caused significant depression of the levels of ferritin-iron and total nonheme iron, but only CP94 caused the significant accumulation of protoporphyrin. Protoporphyria did not occur with iron overloaded C57BL/10ScSn mice or in SWR mice that had elevated basal iron status. Although the protoporphyrin had only a small effect on the total levels of the hemoprotein cytochrome P450 in C57BL/10ScSn mice, the activity of the CYP2B isoforms of cytochrome P450 was actually induced in both strains. The results show that CP94 could cause protoporphyria in individuals of low iron status, perhaps through specifically targeting particular iron pools available to ferrochelatase and by concomitantly stimulating heme synthesis.

Description: The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Quantification of hepcidin concentrations in urine and plasma holds promise as a biomarker for diagnosis and monitoring of disorders of iron metabolism. To date, various mass spectrometry (MS, time of flight and liquid chromatography-MS/MS) as well as immunochemical (IC, competitive radio-immunoassay and ELISA) and functional methods exploiting an internal or external standard, have been developed to measure hepcidin levels in both plasma and urine. Only a few of them have been published. The hepcidin levels quantified by these methods are likely to differ substantially in analytical characteristics. Therefore, comparative studies are imperative for progress in the field by allowing meaningful exchange of hepcidin results derived from various laboratories. In this study we compared the analytical characteristics of several available urinary and plasma hepcidin assays as a first step towards standardization of hepcidin quantification methods. Six and 8 laboratories participated in an international inter-laboratory evaluation of hepcidin levels from a common human urinary (n=8) and plasma (n=7) sample set, respectively, exploiting MS (n=5) and IC (n=3) based methods. Participating laboratories were requested to perform 3 hepcidin measurements for each sample on 4 different days, making a total of 96 urine and 84 plasma measurements per laboratory. The methods differed widely in mean urine and plasma hepcidin level, i.e. range 2.9–427.1 nmol/mmol creatinine and 11.4–124.6 nM, respectively. Coefficient of variation (CV)-ranges for the between-sample and withinsample variation were 126.1–191.5 % and 10.8–33.8%, respectively, for urine and 40.4–96.6 % and 5.5–31.0%, respectively, for plasma. The ratio between-sample CV/within-sample CV varies between 15.3 and 4.1 for urine and 15.1 and 2.4 for plasma. Hepcidin results of most methods mutually correlated (plasma: R range 0.82–1.00; urine: R range 0.73–0.99). We conclude that for the various methods absolute hepcidin values differ widely, but generally correlate and the ratio between-sample and within sample CV vary greatly, suggesting relevant differences in ability to detect small differences in hepcidin concentrations. To allow future comparison, exchangeability and transferability of hepcidin data, we recommend publication of all hepcidin results according to STARD (www.stard-statement.org) to ensure complete reporting of analytical characteristics and to develop initiatives for standardization of assays.

Description: Background. Factors that determine net synthesis of hepcidin and hence iron absorption and distribution depend on a balance of competing factors which may be disease specific. Such factors include anemia, ineffective erythropoiesis (IE), transferrin saturation (Tf sat), iron overload and inflammation. Recently GDF-15, a marker of erythroid maturation and hence IE, has been linked with depression of hepcidin synthesis in vitro and showed elevated levels in beta thalassemia (Tanno et al, Nat Med, 2007). The relationship of hepcidin synthesis to iron overload in sickle cell disease (SCD) is not clear and may differ from thalassemia syndromes because IE is less marked. We wished to establish whether the dominant factors determining net hepcidin synthesis differed between patients with SCD and those with thalassemia intermedia (TI) and thalassemia major (TM). Patients and methods. Serum hepcidin was measured in hypertransfused (Hb〉9.5g/dl) patients with TM (n=18), untransfused or sporadically transfused patients with thalassemia intermedia TI (n=18), and multi-transfused patients with SCD (n=24), and related to markers of anemia, iron overload and erythroid expansion. A newly developed mass spectrometry assay (Bansal et al, Anal Biochem, 2008, In Press) was used to determine serum hepcidin. GDF-15 was measured by an ELISA assay. Multivariate analysis was performed using SIMCA-P software and partial least squares for discriminant analysis (PLS-DA), using samples from each of the clinical groups to investigate relationships between hepcidin, serum iron, non-transferrin bound iron (NTBI), transferrin saturation (Tf sat), serum ferritin, liver iron, transfusion history, erythropoietin, hemoglobin and GDF-15. Results. Serum hepcidin levels were higher in TM (13.9 ± 10.0 nmol/L) than SCD (8.51±8.16 nmol/L, p=0.043) whereas values in TI (3.82 ±3.56 nmol/L) were close to healthy controls (4.04 ± 2.06nmol/l). However, when SCD patients were matched for levels of anemia and iron load with TM, plasma hepcidin levels were similar or higher in SCD. GDF-15 values were highest for TI (11,444± 2177 ng/l), than TM (4117 ± 577 ng/l, P

Description: Background: Imbalances in iron homeostasis result in a variety of disorders. Excess iron accumulates in the circulation and tissues of patients with hereditary hemochromatosis (HH), iron-loading anemia (β-thalassemia major and intermedia (Thal), myelodysplastic syndromes (MDS) and sickle cell disease after transfusion (SCD). To prevent iron-induced tissue damage, detection of impending iron toxicity is needed before complications develop and become irreversible. Plasma non-transferrin bound iron (NTBI) and its labile (redox active) component (LPI) are thought to be potentially toxic forms of iron identified in the serum of patients with iron overload. Objective: To increase our insights into NTBI and LPI concentrations measured by the current worldwide leading analytical assays in four different categories of iron overloaded patients (HH, Thal, MDS, transfused SCD) undergoing various treatments (phlebotomies, iron chelation, red blood cell transfusions). Methods: We compared 10 different assays (5 NTBI, 1 NTBI isoform specific and 4 LPI) as part of an international inter-laboratory study. Serum samples were from 60 patients with 4 iron overload disorders. Serum samples were split into two aliquots, coded (blinded), stored at -80°C and shipped for analysis to 5 different laboratories worldwide. Laboratories performed duplicate measurements on each aliquot of a serum sample on 2 different days, resulting in a total of 4 measurements for each sample. Some laboratories provided multiple assays. Results: NTBI and LPI measurements in the serum of iron-overloaded patients showed good reproducibility with a high between-sample (range 67.1-97.2%) and a low within-sample variance (0-2.2%) relative to the total variance of each assay. Absolute NTBI and LPI levels differed considerably between assays. Four assays (2 LPI and 2 NTBI) also reported negative values. LPI levels were ± 10% of the NTBI levels. Highest levels were observed in patients with naive HH and naive Thal intermedia, transfusion-dependent MDS and transfusion-dependent Thal major. These 4 patients groups also had the highest transferrin saturation (TSAT) levels, but only 3 of them were among the groups with the highest ferritin levels. Eight (4 LPI and 4 NTBI) of the 10 assays could discriminate well between iron overload diseases. In general correlations were highest within the same group of NTBI or LPI assays. Interestingly, one of the LPI assays showed better correlations with NTBI assays (range rs=0.85-0.90) than with the other LPI assays (range rs=0.61-0.77). In contrast, one of the NTBI assays showed better correlations with LPI assays (range rs=0.67-0.75) than with the other NTBI assays (range rs=0.50-0.59). The assays show a hyperbolic relation with TSAT; NTBI and LPI concentrations only substantially increase above a certain TSAT level of ~70% and ~ 90%, respectively. This is illustrated for both a representative NTBI (Figure 1A) and LPI assay (Figure 1B). This relation does not exist between any of the assays and ferritin (Figure 1C,D). Conclusions: While NTBI and LPI values of various assays are well correlated and discriminate between iron overload disorders, absolute values differed considerably between assays. Both standardization of assays and clinical outcome studies to determine clinically relevant toxic thresholds are needed. At present TSAT may provide a useful alternative in the clinical management of patients with iron overload. Figure 1: Relation between representative assays and TSAT, Ferritin. Assay results are given for day 2 as duplicate measurements (circle and square). Figure 1:. Relation between representative assays and TSAT, Ferritin. Assay results are given for day 2 as duplicate measurements (circle and square). Disclosures De Swart: Novartis Europe: Research Funding. Swinkels:Novartis Europe: Research Funding.