Red Blood Cell Defects Research Articles

Vesiculation of healthy and defective red blood cells

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

A report of two cases of splenectomy in children younger than two years old with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common pediatric hemolytic anemia and one of the most common types of familial hemolytic anemia caused by congenital red blood cell defects. HS often presents clinically as childhood anemia, jaundice, and progressive splenomegaly. It is generally accepted that splenectomy is the most effective treatment for HS, but development of overwhelming infections with symptoms such as chills, high fever, and septicemia may occur because of immune dysfunction after splenectomy. As a result, the generally proposed age for children to undergo splenectomy is after 5 years of age. Splenectomy in younger HS patients is rarely reported. Here, however, we report 2 cases of HS in children younger than 2 years of age (15 months and 18 months) who underwent splenectomy; we also report on their follow-up. • We report two cases of HS in children <2 yrs who underwent splenectomy. • Splenectomy in younger HS patients is rarely reported. • Splenectomy can be a solution for children <2 yrs with severe HS or hemolytic crisis.

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels

Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology.

Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. Materials and Methods. Three major TPMT single-nucleotide polymorphisms (TPMT∗2, ∗3B, and ∗3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT∗3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT∗2, ∗3A, or ∗3B alleles were observed in this Korean population. Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

A new paradigm in the diagnosis of hereditary hemolytic anemia

A new paradigm in the diagnosis of hereditary hemolytic anemia

Possible Links between Sickle Cell Crisis and Pentavalent Antimony

For over 60 years, pentavalent antimony (Sb(v)) has been the first-line treatment of leishmaniasis. Sickle cell anemia is a disease caused by a defect in red blood cells, which among other things can cause vasooclusive crisis. We report the case of a 6-year-old child with leishmaniasis who during treatment with meglumine antimoniate developed a sickle cell crisis (SCC). No previous reports describing the relationship between antimonial drugs and sickle cell disease were found. Reviews of both the pathophysiology of SCC and the mechanism of action of Sb(v) revealed that a common pathway (glutathione) may have resulted in the SCC. ChemoText, a novel database created to predict chemical-protein-disease interactions, was used to perform a more expansive and systematic review that was able to support the association between glutathione, Sb(v), and SCC. Although suggestive evidence to support the hypothesis, additional research at the bench would be needed to prove Sb(v) caused the SCC.

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29(-/-) embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.

G6PD A−variant influences the antibody responses to Plasmodium falciparum MSP2

High antibody levels directed to Plasmodium falciparum merozoite surface proteins (MSP), including MSP2, as well as genetically related red blood cell defects, have previously been found to be associated with protection against malaria. Here, our main objective was to study the changes in MSP2-specific total IgG, IgG1 and IgG3 responses during a malaria transmission season in order to assess the impact of sickle-cell, α(+)-thalassemia and G6PD variants on antibody kinetics. Repeated parasitological assessments of a cohort of children were conducted during an 8-month period. Antibody responses to recombinant MSP2/3D7 and MSP2/FC27 proteins were measured at the beginning and at the end of transmission season. We found that (i) the period of last Plasmodium falciparum infection during the transmission season was associated with IgG3 anti-MSP2 change. Compared to the IgG3 levels of children infected in January 2003 (end of transmission season), the IgG3 level of children decreased with the length of the period without infection, (ii) G6PD A− carriers had a lower increase of IgG3 levels to MSP2/FC27 and MSP2/3D7 during the transmission season than the noncarriers. This latter finding is suggestive of qualitative and/or quantitative reduction of exposure to malarial antigens related to this genetic variant, leading to weaker stimulation of specific antibody responses. We speculate that cell-mediated immune activity may explain the clinical protection afforded by this genetic trait.

Pleiotropic effects of intravascular haemolysis on vascular homeostasis

The breakdown of senescent or defective red blood cells releases red cell contents, especially haemoglobin, which scavenges nitric oxide (NO) and decomposes to haem and free iron. These are potent oxidants, all of which have promoted the evolution of inducible and vasculoprotective compensatory pathways to rapidly clear and detoxify haemoglobin, haem and iron. Chronic haemolytic red cell disorders as diverse as sickle cell disease, thalassaemia, unstable haemoglobinopathy, cytoskeletal defects and enzymopathies have been linked to a clinical constellation of pulmonary hypertension, priapism, leg ulceration and possibly cerebrovascular disease and thrombosis. Besides free haemoglobin, haemolysis has been associated with extracellular arginase that limits substrate availability to NO synthase, endogenous inhibitors of NO synthase activity, and inappropriate activation of haemostatic pathways. This article reviews the haemolytic disorders that have been reported to manifest vascular complications, and explores the speculative possibility that haemolysis mediates some of the vascular complications of inflammation and diabetes.

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice

beta-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate beta-globin chain production. The excessive unbound alpha-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis. In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival. The alpha/beta-globin chain ratio in beta(IVSII-654)-thalassemic mice, 1.26+/-0.03, was significantly higher than that of wild type mice, 0.96+/-0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human beta(E)-globin transgene, with a more balanced globin chain synthesis, 0.92+/-0.05. Moreover, transgenic mice harboring 8 extra copies of the human beta(E)-globin transgene showed inversely imbalanced alpha/beta-globin synthesis ratio, 0.83+/-0.01, that resulted in a mild beta-thalassemia phenotype due to the excessive beta-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of beta(IVSII-654)-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice. Imbalanced alpha/beta-globin chain as a consequence of either reduction or enhancement of beta-globin chain synthesis can cause abnormal red blood cell properties in mouse models.

Genetic loss of KCa3.1 causes subtle erythrocyte macrocytosis and progressive splenomegaly

KCa3.1, also known as Gardos channel in erythrocytes, is considered to be a major regulator of red blood cell (RBC) volume by mediating K+ efflux. However, the functional importance of KCa3.1 in RBC in vivo is still incompletely understood. Here we used KCa3.1‐/‐ mice to investigate the consequences of KCa3.1 deficiency for RBC metrics, functions and sequestration. RBCs of KCa3.1‐/‐ mice of all ages were mildly macrocytic but their biconcave appearance preserved. RBCs number, total hemoglobin and hematocrit were unchanged in the adult KCa3.1‐/‐ mice and increased in the premature KCa3.1‐/‐ mice. Filterability, Ca2+ dependent volume decrease and osmotic tolerance of RBCs lacking KCa3.1 were noticeably reduced when compared to RBC of wt‐mice. Deformability to increasing shear stress was unchanged. Strikingly, KCa3.1‐/‐ mice developed progressive splenomegaly which was considerable in the &gt;6‐month‐old mice and was paralleled by increased iron deposition in the aged‐mice presumably as a consequence of enhanced RBC sequestration. Injections of the KCa3.1‐blocker TRAM‐34 also produced mild splenomegaly in wt‐mice. We conclude that genetic deficit of erythroid KCa3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly.

Disruption of the Gardos channel (KCa3.1) in mice causes subtle erythrocyte macrocytosis and progressive splenomegaly

Gardos channel, the erythrocyte Ca(2+)-activated K(+) channel (K(Ca)3.1), is considered a major regulator of red blood cell (RBC) volume by mediating efflux of potassium and thus cell dehydration and shrinkage. However, the functional importance of K(Ca)3.1 in RBC in vivo is incompletely understood. Here, we used K(Ca)3.1(-/-)-mice to investigate the consequences of K(Ca)3.1 deficiency for RBC indices, functions, and sequestration. RBCs of K(Ca)3.1(-/-)-mice of all ages were mildly macrocytic but their biconcave appearance being preserved. RBC number, total hemoglobin, and hematocrit were unchanged in the adult K(Ca)3.1(-/-)-mice and increased in the premature K(Ca)3.1(-/-)-mice. Filterability, Ca(2+)-dependent volume decrease and osmotic tolerance of RBCs lacking K(Ca)3.1 were noticeably reduced when compared to RBC of wild-type littermates. Deformability to increasing shear stress was unchanged. Strikingly, K(Ca)3.1(-/-)-mice developed progressive splenomegaly which was considerable ( approximately 200% of controls) in the >6-month-old mice and was paralleled by increased iron deposition in the aged mice presumably as a consequence of enhanced RBC sequestration. Daily injections of the K(Ca)3.1-blocker TRAM-34 (120 mg/kg) also produced mild splenomegaly in wild-type mice. We conclude that genetic deficit of erythroid K(Ca)3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly.

Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice

The mouse response to acute Salmonella typhimurium infection is complex, and it is under the influence of several genes, as well as environmental factors. In a previous study, we identified two novel Salmonella susceptibility loci, Ity4 and Ity5, in a (AcB61 × 129S6)F2 cross. The peak logarithm of odds score associated with Ity4 maps to the region of the liver and red blood cell (RBC)–specific pyruvate kinase (Pklr) gene, which was previously shown to be mutated in AcB61. During Plasmodium chabaudi infection, the Pklr mutation protects the mice against this parasite, as indicated by improved survival and lower peak parasitemia. Given that RBC defects have previously been associated with resistance to malaria and susceptibility to Salmonella, we hypothesized that Pklr is the gene underlying Ity4 and that it confers susceptibility to acute S. typhimurium infection in mice. Using a fine mapping approach combined with complementation studies, comparative studies, and functional analysis, we show that Pklr is the gene underlying Ity4 and that it confers susceptibility to acute S. typhimurium infection in mice through its effect on the RBC turnover and iron metabolism.

Pathophysiology of Thrombocytopenia and Anemia in Mice Lacking Transcription Factor NF-E2

AbstractExpression of the p45 subunit of transcription factor NF-E2 is restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia, resulting from a late arrest in megakaryocyte differentiation, and a number of red blood cell defects, including anisocytosis and hypochromia. Here we report results of studies aimed to explore the pathophysiology of these abnormalities. Mice lacking NF-E2 produce very few platelet-like particles that display highly disorganized ultrastructure and respond poorly to platelet agonists, features consistent with the usually lethal hemorrhage in these animals. Thrombocytopenia was evident during fetal life and was not corrected by splenectomy in adults. Surprisingly, fetal NF-E2–deficient megakaryocyte progenitors showed reduced proliferation potential in vitro. Thus, NF-E2 is required for regulated megakaryocyte growth as well as for differentiation into platelets. All the erythroid abnormalities were reproduced in lethally irradiated wild-type recipients of hematopoietic cells derived from NF-E2-null fetuses. Whole blood from mice lacking p45 NF-E2 showed numerous small red blood cell fragments; however, survival of intact erythrocytes in vivo was indistinguishable from control mice. Considered together, these observations indicate a requirement for NF-E2 in generating normal erythrocytes. Despite impressive splenomegaly at baseline, mice lacking p45 NF-E2 survived splenectomy, which resulted in increased reticulocyte numbers. This reveals considerable erythroid reserve within extra-splenic sites of hematopoiesis and suggests a role for the spleen in clearing abnormal erythrocytes. Our findings address distinct aspects of the requirements for NF-E2 in blood cell homeostasis and establish its roles in proper differentiation of megakaryocytes and erythrocytes.

Pathophysiology of Thrombocytopenia and Anemia in Mice Lacking Transcription Factor NF-E2

Expression of the p45 subunit of transcription factor NF-E2 is restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia, resulting from a late arrest in megakaryocyte differentiation, and a number of red blood cell defects, including anisocytosis and hypochromia. Here we report results of studies aimed to explore the pathophysiology of these abnormalities. Mice lacking NF-E2 produce very few platelet-like particles that display highly disorganized ultrastructure and respond poorly to platelet agonists, features consistent with the usually lethal hemorrhage in these animals. Thrombocytopenia was evident during fetal life and was not corrected by splenectomy in adults. Surprisingly, fetal NF-E2–deficient megakaryocyte progenitors showed reduced proliferation potential in vitro. Thus, NF-E2 is required for regulated megakaryocyte growth as well as for differentiation into platelets. All the erythroid abnormalities were reproduced in lethally irradiated wild-type recipients of hematopoietic cells derived from NF-E2-null fetuses. Whole blood from mice lacking p45 NF-E2 showed numerous small red blood cell fragments; however, survival of intact erythrocytes in vivo was indistinguishable from control mice. Considered together, these observations indicate a requirement for NF-E2 in generating normal erythrocytes. Despite impressive splenomegaly at baseline, mice lacking p45 NF-E2 survived splenectomy, which resulted in increased reticulocyte numbers. This reveals considerable erythroid reserve within extra-splenic sites of hematopoiesis and suggests a role for the spleen in clearing abnormal erythrocytes. Our findings address distinct aspects of the requirements for NF-E2 in blood cell homeostasis and establish its roles in proper differentiation of megakaryocytes and erythrocytes.

Compartmentalization of the erythrocyte membrane by the membrane skeleton: intercompartmental hop diffusion of band 3.

Recent developments in microscopic instrumentation and probes have allowed the observation and manipulation of the movement of membrane proteins and lipids in the plasma membrane at the level of single molecules. These experiments are performed by tracking small colloidal gold particles attached to specific membrane proteins and lipids (single particle tracking) (Jacobson et al., 1995 ; Sheets et al., 1995 ; Kusumi and Sako, 1996 ; Saxton and Jacobson, 1997 ) or by dragging particle–protein complexes along the surface of the plasma membrane using laser tweezers (also termed an “optical trap” or OT) (Edidin et al., 1991 ; Kusumi and Sako, 1996 ; Kusumi et al., 1998 ). In optimal cases, a single protein molecule is attached to a 40-nm-diameter colloidal gold probe (Tomishige et al., 1998 ). These single molecule experiments have demonstrated that most membrane-spanning proteins do not undergo simple diffusion, but that a significant number are locally slowed or stopped, some are temporarily confined, and others are transported unidirectionally (Kusumi et al., 1993 ; Sheets et al., 1997 ; Sako et al., 1998 ; Simson et al., 1998 ). Previously, these processes had not been observed, because the methods used, e.g., fluorescence recovery after photobleaching, recorded the ensemble-averaged behavior of a large number of molecules. The underlying cellular mechanisms that induce nonrandom diffusion have remained largely unknown; however, we and others found that properties of the membrane skeleton network fundamentally affect the movement and distribution of certain membrane proteins, such as transferrin receptor, α2-macroglobulin receptor, MHC class I molecules, E-cadherin, and band 3, through corralling and binding effects imposed by the membrane skeleton network (Edidin et al., 1991 ; Luna and Hitt, 1992 ; Kusumi et al., 1993 ; Sako and Kusumi, 1994 , 1995 ; Sako et al., 1998 ; Tomishige et al., 1998 ). The results are particularly clear with band 3 in human erythrocyte ghost membranes, where the specimen consists of a lipid bilayer and its underlying membrane skeleton, which has been biochemically and biophysically well characterized (Golan, 1989 ; Bennett, 1990 ; Bennett and Gilligan, 1993 ; Mohandas and Evans, 1994 ). The results obtained by these studies support a “membrane skeleton fence model” in which the cytoplasmic portion of a membrane protein collides with the membrane skeletal “meshwork” because of steric hindrance, thus resulting in the temporal confinement of the protein within the mesh (compartment, Figure ​Figure1A).1A). Membrane proteins escape from these compartments and hop to adjacent ones because of the dynamic properties of the membrane skeleton. We predict that time-dependent fluctuations in the distance between the membrane and the skeleton are large or the membrane–skeleton network connections form and break continuously, as expected in a chemical equilibrium, providing membrane proteins with the opportunity to pass through the mesh barrier. In the case of band 3, macroscopic diffusion within the erythrocyte membrane occurs as the result of a series of such intercompartmental hops. Figure 1 (A) The model to explain the confined diffusion of band 3 in the membrane (the membrane skeleton fence model). (B) Movement of gold particles attached to band 3 in the erythrocyte membrane, observed with a temporal resolution of 8 ms (4 times greater ... The scientific content of this work has been published previously (Tomishige et al., 1998 ), and the purpose of the present essay is to give the reader a glimpse of the raw data from this study, as recorded by both normal and high-speed video microscopy. We hope the video sequences attached to this article will help the viewing audience develop a feel for how band 3 interacts with the membrane skeleton and undergoes intercompartmental hop diffusion within the erythrocyte ghost membrane.

Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.

A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.

Amelioration of effects of severe dietary copper deficiency by food restriction in rats

To test the effect of food restriction on responses to dietary copper deficiency, male weanling rats were fed two amounts of dietary copper via five dietary-intake regimens ranging from ad libitum to 70% of ad libitum intake. Copper-deficient rats exhibited characteristic signs, including depressed organ copper content, reduced activity of copper-dependent enzymes, enlarged hearts, and anemia. Food restriction attenuated the cardiac enlargement, red blood cell defects, and reduction of superoxide dismutase activity in copper-deficient rats. Mineral and enzyme assays suggested that possible mechanisms for this amelioration are the correction of copper status and/or the improvement of antioxidant status. Also, food restriction depressed serum cholesterol and enhanced cytochrome c oxidase activity in both copper-adequate and copper-deficient rats, which compensated for effects of copper deficiency. A second experiment illustrated that the mortality associated with severe copper deficiency was also inhibited by food restriction.

Spectrophotometric determination of oxalate in whole blood

Whole blood oxalate concentrations have been reported to be higher than serum or plasma concentrations [l-3], however, this has not been confirmed by more recent, reliable methods. Measurement of oxalate in whole blood involves all of the same problems associated with oxalate determination in serum or plasma and the color of blood can provide an additional drawback. Recently, Costello and Landwehr [4] reviewed many methods currently available to measure oxalate concentrations in serum and they note that the discrepancy between values obtained by direct methods and isotope-dilution procedures has narrowed considerably. Mean serum or plasma oxalate concentrations have now been demonstrated to range between 1.25 and 4.01 pmol/l using a variety of enzymic methods [4], while in vivo isotopic studies consistently demonstrate lower mean oxalate values, < 1.83 pmol/l [4]. It can be concluded that reliable determination of serum oxalate is now both possible and necessary in a variety of clinical conditions [4]. The determination of oxalate in whole blood is of interest for many reasons. First, it is not known whether the oxalate concentration in whole blood is higher than serum in healthy normals or in patients with hyperoxalemia. Second, such methodology would appear to be extremely useful in the investigation of the reported oxalate transport defect in red blood cells in idiopathic calcium oxalate nephrolithiasis described by Baggio et al. [5]. Finally, if serum and whole blood oxalate levels are found to be similar then less blood volume would necessarily be required for routine measurements employing current modified serum methods.