Reticulocyte hemoglobin content in the evaluation of iron status of hemodialysis patients.

The assessment of iron status for hemodialysis patients has been hindered by the inaccuracy of commonly used diagnostic tests. A novel assay, the reticulocyte hemoglobin content (CHr), has recently been found to sensitively detect functional iron deficiency among nonuremic patients treated with recombinant erythropoietin (rHuEPO). The purpose of this study was to evaluate the CHr for the assessment of iron status in hemodialysis patients. One hundred sixty-four stable hemodialysis patients had a mean CHr of 27.5 +/- 2.8 pg with a normal distribution of values. The mean CH (mature red cell hemoglobin content) was 26.4 +/- 2.4 pg. There was a close correlation between CHr and CH (r = 0.86, P < 0.0001). A significant subgroup of patients (12.2%) had CHr values < CH. These patients had recent increases in rHuEPO dose, and a lower mean transferrin saturation and hematocrit, suggesting the recent onset of functional iron deficiency due to the increase in rHuEPO dose. In the second phase of the study, 32 patients were randomly selected to receive treatment with a single dose infusion of 1,000 mg of intravenous iron dextran (IVFe). Patients were classified as iron deficient (N = 7) if they responded with a significant reticulocytosis (sustained 1 basis point increase in corrected reticulocyte index within 2 weeks). All other patients were classified as iron replete (N = 25). A CHr < 26 pg at baseline predicted iron deficiency with a sensitivity of 100%, specificity of 80%. The serum ferritin, transferrin saturation and percentage of hypochromic red blood cells all were less accurate. The time to correction of iron deficiency at the level of the reticulocyte was found to be within 48 hours as measured by correction of the mean CHr to > 26 pg, and by the shift of the vast majority of the reticulocyte population to CHr > 26 pg within this time span. We conclude that CHr < 26 pg is an accurate measure of iron status in hemodialysis patients, that a CHr value < CH indicates the acute onset of iron deficiency, and that a single dose infusion of intravenous iron results in correction of iron deficiency at the level of the reticulocyte within 48 hours.

Effects of subcutaneous recombinant human erythropoietin in normal subjects: development of decreased reticulocyte hemoglobin content and iron-deficient erythropoiesis.

The present study evaluates the properties of the reticulocytes produced in healthy volunteers after treatment with different regimens of recombinant human erythropoietin (r-HuEPO). Twenty-four subjects were randomly assigned to one of three different subcutaneous (SC) r-HuEPO (Protcrit; Ortho Biotech) administration protocols (I: 300 U/kg on days 1, 4, 7, 10; II: 400 U/kg on days 1, 5, 9; III: 600 U/kg on days 1, 10) with oral iron supplementation (Niferex; 150 mg, twice a day). The characteristics of the reticulocytes produced were examined with a flow cytometry method that allows measurements of individual reticulocyte cell volume, hemoglobin concentration, and hemoglobin content. Administration of SC r-HuEPO was associated with a significant increase in the production of reticulocytes. The hemoglobin content of reticulocytes (CHr, in picograms of hemoglobin per cell) in the three groups was 28.5 +/- 1.0, 28.2 +/- 0.5, and 28.5 +/- 1.3, respectively, at baseline, decreased to 24.6 +/- 1.6 (p < 0.001), 24.5 +/- 2.3 (p < 0.001), and 27.5 +/- 1.8 (not significant) at day 10, and returned to baseline after r-HuEPO was discontinued (28.8 +/- 0.9, 28 +/- 0.8, and 28.8 +/- 1.4, respectively, at day 22). The percentage of reticulocytes with cell hemoglobin content less than 23 pg was taken as an indicator of iron-deficient erythropoiesis. At baseline, 5.6% +/- 2.7%, 6.9% +/- 3.4%, and 8.3% +/- 3.8% of reticulocytes had less than 23 pg hemoglobin in groups I, II, and III, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional electrophoretic analysis of spore proteins of the Microsporida.

Microsporida are potentially useful as biological control agents for insects of economic and medical importance. Prior to their responsible use, however, an accurate and reliable means of identification to the species and subspecies level is required. Current methods used for identification are not adequate, due to variability of identifiable characters and to the occurrence of dimorphism. Recently, progress has been made in the use of biochemical characteristics to support the more traditional methods of distinguishing between morphologically similar species. We report on an improved method of characterization of microsporidan spore proteins, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This method increased the number of spore polypeptides resolved from Nosema locustae spore protein extracts 2-3-fold over 1-dimensional PAGE. Also, each of the 2D-PAGE spore protein fingerprints of the species examined, namely Nosema locustae, Nosema bombycis, and Vairimorpha necatrix, were unique and differences in their spore protein composition were easily determined. The major structural proteins of Nosema locustae spores co-electrophoresed with alpha and beta tubulin from calf brain and had similar pI and molecular weight values as reported for tubulin in other species. Each species' 2D-PAGE fingerprint contained a few polypeptides that were present in relatively high concentration and these polypeptides may represent the major proteins of the structural components of the spore.

Cell type-specific association between two types of spectrin and two types of intermediate filaments.

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside-out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such associations may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.

Association of spectrin with desmin intermediate filaments.

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.

Functional characterization of human erythrocyte spectrin alpha and beta chains: association with actin and erythrocyte protein 4.1.

Human erythrocyte spectrin alpha and beta chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of alpha-beta heterodimers with sedimentation characteristics identical with native alpha-beta dimers. The binding of 125I-labeled band 4.1 to alpha and beta chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. It was found that both alpha and beta chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified beta chains but not alpha chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified alpha chains, beta chains, and recombinant alpha-beta heterodimers to F-actin was measured in the presence of band 4.1. We found that alpha or beta chains separately exhibited no band 4.1 dependent association with F-actin but that alpha-beta heterodimers formed by recombination of the chains did. We conclude that spectrin binding to F-actin in the presence of band 4.1 requires the participation of both of spectrin's polypeptide chains.