A simple, highly sensitive and improved method for the measurement of bleomycin-detectable iron: the 'catalytic iron index' and its value in the assessment of iron status in haemochromatosis.
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In the presence of ferrous ions (Fe(2+)), the anti-tumour agent bleomycin will induce DNA degradation. Degradation of DNA into substances detectable by the thiobarbituric acid test has been used previously for the detection of iron in a form that is capable of catalysing the formation of the potentially harmful hydroxyl free radical. In the present paper, we describe the application of the ethidium-binding assay of DNA damage to the measurement of bleomycin-detectable iron, comparing its performance with the conventional method in the assessment of iron standard solutions and plasma samples from haemochromatosis patients. The ethidium-binding assay proved to be more responsive than the thiobarbituric acid test in the detection of DNA damage induced by very low concentrations of iron, but became saturated at higher iron concentrations. Agreement between the two versions of the assay in the identification of plasma samples containing bleomycin-detectable iron was good, but agreement on the actual concentrations of such iron in the positive samples was poor. This discrepancy is believed to be due to interference with the thiobarbituric acid assay by plasma. Consequently, it was not possible to obtain reliable estimates of free iron concentrations in plasma when using the conventional version of the bleomycin assay. We have devised a parameter of iron status called the catalytic iron index. For healthy, non-haemochromatotic individuals, the mean value of this parameter was found to be 0.81 (range 0.78-0.84; n=20). Elevated values were observed in some plasma samples from haemochromatosis patients, but these showed no correlation with serum ferritin levels. In contrast, correlations were seen with both serum iron and transferrin saturation levels, but only when these were above the normal range. (+info)
Hemoglobinopathies in the Hamilton region. II. Thalassemia traits and iron therapy.
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Between July 1973 and July 1974 all adult patients with hypochromic anemia and a mean corpuscular volume of 75 mum3 or less were screened for hemoglobinopathies. Of the 490 patients 105 had beta-thalassemia trait, 11 had alpha1-thalassemia trait, 4 had hemoglobin Lepore trait and 1 had hemoglobin H disease. Of 48 inpatients whose charts were reviewed 19 had been on oral iron therapy and 7 of them had been given iron intramuscularly. Of 27 outpatients interviewed 10 had been on intermittent iron therapy for 18 months or more; 4 had been given at least 1 g of intramuscular iron. Iron deficiency was not documented in any of these patients. Iron deficiency should be diagnosed by means other than the presence of a hypochromic picture in the peripheral blood before iron therapy is instituted, particularly in communities with a large population of Mediterranean or South-East Asian origin. (+info)
Mouse strain differences determine severity of iron accumulation in Hfe knockout model of hereditary hemochromatosis.
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Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/-), and knockout (Hfe -/-) mice from each strain. Although the Hfe -/- mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe -/- AKR mice that were 2.5 or 3.6 times higher than those of Hfe -/- C3H or Hfe -/- C57BL/6 mice, respectively. The Hfe -/- mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH. (+info)
High-performance multiplex SNP analysis of three hemochromatosis-related mutations with capillary array electrophoresis microplates.
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An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population. (+info)
Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.
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BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized. (+info)
Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.
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We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations. (+info)
Granulopoietic progenitors in suspension culture: a comparison of stimulatory cells and conditioned media.
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Kinetic studies have been carried out to investigate the functional heterogeneity previously observed in populations of human marrow or peripheral blood cells separated by velocity sedimentation. The results obtained confirm the earlier results, in that slowly-sedimenting cells were found to stimulate both colony formation by granulopoietic progenitors and an increase in numbers of granulopoietic progenitors in suspension culture, while rapidly-sedimenting cells stimulated only colony formation and not increased progenitors in suspension cultures. Investigations of the properties of media conditioned by these two subpopulations of cells revealed no clear differences between them; both stimulated suspension cultures as well as colony formation, and both lost the former activity, but not the latter, after dialysis. The results contribute to the evidence that more than one process is regulated in cultures of granulopoietic progenitor cells. (+info)
Iron overload without the C282Y mutation in patients with epilepsy.
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To test the hypothesis that iron overload predisposes to epilepsy, transferrin saturation in 130 patients with epilepsy and sex and age matched 128 control subjects without epilepsy were studied. Mean transferrin saturation was significantly higher in the epilepsy group (39.9 (SD 19.6)%) than in the control group (29.1 (SD 14.9)%). Abnormally high transferrin saturations (men>60%, women>48%) were found in 10 patients with epilepsy but in only one subject without epilepsy. Antiepileptic drugs did not affect the transferrin saturation. Of the 11 with abnormally high transferrin saturation, two with epilepsy were heterozygotic for H63D in the haemochromatosis gene but no patient had the C282Y mutation. These results indicate that iron overload other than the C282Y mutation underlies epilepsy. (+info)