Dalia Mazor

EFFECT OF RADIATION ON RED CELL MEMBRANE AND INTRACELLULAR OXIDATIVE DEFENSE SYSTEMS

Ionizing radiation is currently used for prevention of transfusion associated graft versus host disease (TAGVHD). As radiation damage is associated with the production of activated oxygen species, the aim of this study was to observe the immediate effect of ionizing radiation on red cell membrane and intracellular oxidative defense systems. Neonatal and iron deficiency (IDA) cells, known for their increased sensitivity to oxidative stress, were chosen and compared with normal cells. Irradiation was performed in doses of 1500 cGy, 3000 cGy and 5000 cGy. GSH and methemoglobin levels and the activity of different antioxidant enzymes, measured under optimal in vitro conditions, were preserved in all cells after irradiation. Only radiation at the highest does of 5000 cGy, caused significant potassium leakage in neonatal cells and insignificant increase in IDA cells. Thus, cells with increased sensitivity to oxidative stress are more susceptible to damage by ionizing radiation than normal cells.

Iron Deficiency Anemia: Recovery from in vitro Oxidative Stress

Red blood cells in iron deficiency anemia (IDA) have a decreased activity of essential antioxidant enzymes. The present study examined the effect of in vitro exposure to oxidative agents in IDA cells and their recovery capacity. Red cells of 26 IDA patients and 10 healthy subjects were examined. Cells of IDA patients had higher levels of reduced glutathione (GSH), and normal methemoglobin and malonyldialdehyde (MDA) levels. Exposure to butyl hydroperoxide revealed a dose-dependent sensitivity in IDA cells, with extensive GSH depletion and increased MDA levels. These changes were partially reversible by incubation with dithiothreitol. Exposure to phenazine methosulfate, to produce intracellular superoxide ions, resulted in moderate GSH depletion and methemoglobin production. IDA cells were more sensitive than control cells to high concentrations of this substance. This effect was further augmented by preincubation with a superoxide dismutase inhibitor. Our data demonstrate that IDA cells are more susceptible to oxidation but have good capacity for recovery.

Oxidative Stress in Newborn Erythrocytes

ABSTRACT: Phenylhydrazine (PHZ) exposure is used to study in vitro red cell aging mechanisms dependent on Hb oxidation. The effect of PHZ on normal neonatal red blood cells was studied in unseparated blood and after separation into light and heavy cells. PHZ caused more extensive morphologic changes in neonatal than in adult red blood cells. PHZ exposure of neonatal cells caused less reduced glutathione depletion than in adult cells. Although we found the same total amount of oxidized Hb in both cells, a well defined oxidation product of Hb was demonstrated by Mössbauer spectra only in neonatal cells. This oxidation product was not methemoglobin but a trivalent, high-spia iron compound. All neonatal cell populations were more sensitive to PHZ than were adult ones, as demonstrate by the presence of Heinz bodies at low PHZ concentration, which did not affect adult cells. These studies demonstrate greater sensitivity of neonatal cells to PHZ in all densityseparated populations.

Antioxidant status in pediatric acute lymphocytic leukemia (ALL) and solid tumors: the impact of oxidative stress.

Pediatric ALL patients are subjected to an aggressive and continuous chemotherapy protocol, while solid tumor patients have a less intensive treatment. We studied the antioxidant status of children from the two groups and hypothesized that the antioxidant status will differ in concert with their treatment.

Quantitative determinations of microcytic-hypochromic red blood cell population and glycerol permeability in iron-deficiency anemia and β thalassemia minor

Abstract The Hl/H2 Technicon automated cell analyzer measures, in addition to the usual red blood cell (RBC) parameters, subpopulations of microcytic (M) and hypochromic (H) red blood cells. The M/H ratio may be useful in the differential diagnosis of iron-deficiency anemia (IDA) and β thalassemia minor (Thal). Thirty-three iron-deficient patients and 26 thalassemia patients were studied. The M/H ratio was found to be higher in thalassemia patients than in IDA patients. Using a cut-off point of 1.9 M/H ratio, the calculated discriminant efficiency was 88%. When glycerol lysis values were determined at 70 s as a cut-off point, the discriminant efficiency was slightly higher, at 91%. Thus, the combination of the M/H ratio and the glycerol lysis time (GLT) improves the discriminant efficiency and provides a good diagnostic tool to differentiate between the two microcytic-hypochromic anemias. The study suggests that the M/H ratio together with the GLT could serve as a useful screening tool, prior to the application of other more sophisticated methods.

EFFECT OF VITAMIN K1 ON GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENT NEONATAL ERYTHROCYTES IN VITRO

AIM To determine whether vitamin K1, which is routinely administered to neonates, could act as an exogenous oxidising agent and be partly responsible for haemolysis in glucose-6-phosphat-dehydrogenase (G-6-PD). METHODS G-6-PD deficient (n=7) and control (n=10) umbilical cord blood red blood cells were incubated in vitro with a vitamin K1 preparation (Konakion). Two concentrations of Vitamin K1 were used, both higher than that of expected serum concentrations, following routine injection of 1 mg vitamin K1. Concentrations of reduced glutathione (GSH) and methaemoglobin, indicators of oxidative red blood cell damage, were determined before and after incubation, and the mean percentage change from baseline calculated. RESULTS Values (mean (SD)) for GSH, at baseline, and after incubation with vitamin K1 at concentrations of 44 and 444 μM, respectively, and percentage change from baseline (mean (SD)) were 1.97 + 0.31 μmol/g haemoglobin, 1.89 ± 0.44 μmol/g (-4.3 ± 13.1%), and 1.69 ± 0.41 μmol/g (-14.5 ±9.3%) for the G-6-PD deficient red blood cells, and 2.27 ± 0.31 μmol/g haemoglobin, 2.09 ± 0.56 μmol/g (−7.2 ± 23.2%), and 2.12 ± 0.38 μmol/g (−6.0 + 14.1%) for the control cells. For methaemoglobin (percentage of total haemoglobin), the corresponding values were 2.01± 0.53%, 1.93 ± 0.37% (−0.6 ± 17.4%) and 2.06 ± 0.43% (5.7 ± 14.2%) for the G-6-PD deficient red blood cells, and 1.56 ± 0.74%, 1.70 ± 0.78% (12.7 ± 21.9%), and 1.78 ± 0.71% (20.6 ± 26.8%) for the control red blood cells. None of the corresponding percentage changes from baseline was significantly different when G-6-PD deficient and control red blood cells were compared. CONCLUSIONS These findings suggest that G-6-PD deficient red blood cells are not at increased risk of oxidative damage from vitamin K1.

Erythrocyte agglomeration and survival studies in citrate-phosphate-dextrose (CPD) units.

ZusammenfassungIn einer früheren Arbeit wurde über eine vermehrte Agglomerationsneigung von in CPD aufbewahrten Erythrozyten berichtet. In der jetzt vorliegenden Arbeit wurden als Ausdruck des Alterungsprozesses von Blutkonserven folgende Werte geprüft: Agglomeration der Erythrozyten; freies Hämoglobin; ATP; osmotische Resistenz und bestimmte Enzymaktivitäten. Anschließend wurden die gelagerten Erythrozyten mit51Cr markiert, und ihre 24-Stunden-Überlebensrate wurde durch Autotransfusion bestimmt. Der Agglomerationstest war bereits positiv, wenn die Überlebensrate noch 70 % betrug. Frische Blutkonserven waren durch hohe ATP-Spiegel und negative Agglomeration, alte Konserven durch gegenteilige Befunde gekennzeichnet. Bei mittlerer Aufbewahrungsdauer waren ATP-Spiegel und Agglomeration nicht zuverlässig korreliert. Der Agglomerationstest scheint den Alterungsgrad von Erythrozytenkonserven zuverlässig anzuzeigen. Jedoch scheinen noch weitere Untersuchungen nötig, bevor entschieden werden kann, ob dieser Test auch zuverlässig mit der Lebensfähigkeit der Erythrozyten nach Transfusion korreliert ist.SummaryWe have previously reported increased tendency to agglomeration in CPD-stored erythrocytes. In the present study we investigated the deterioration pattern of fresh units, as detected by agglomeration, free hemoglobin levels, ATP levels, osmotic fragility, and certain enzymatic activities. When the negative test was converted to a positive one, the 24-h survival values of these units were determined by autotransfusion. A positive agglomeration occurred while viability was still above 70%. Fresh units had high ATP levels and negative agglomeration while old units had low ATP levels and positive agglomeration. However, in intermediate-storage units, no definite correlation between ATP and agglomeration was found.Agglomeration seems to reflect the degree of changes in stored erythrocytes. However, further studies are needed before this test may serve for prediction of post-transfusion viability.

Effect of Vitamin K on Neonatal Erythrocytes

This study investigated the possible oxidative effect of vitamin K3 (menadione) and Vitamin K1 (Konakion) on neonatal erythrocytes by controlled in vitro exposure. Menadione caused only mild morphological changes and did not decrease ATP levels. However, it oxidized intracellular hemoglobin to methemoglobin in neonatal cells more than in adult cells. Reduced glutathione contents were higher in neonatal cells, but less available for antioxidant protection. Konakion did not increase methemoglobin levels in newborn infants after a prophylactic injection. In vitro exposure to Konakion did not affect reduced glutathione and ATP levels, nor did it oxidize hemoglobin. However, extensive morphological changes were observed, attributed to the effect of its solvent. Therefore, it seems that menadione, which is no longer administered to newborns, causes oxidative stress in neonatal cells whereas Konakion, the current vitamin K1, does not, either in in vitro studies or by therapeutic administration.

Liquid Stored Erythrocytes: Agglomeration, Morphology, Sialic Acid, and Toluidine Blue Uptake Studies* **

SummaryIncreased tendency to reversible agglomeration in erythrocytes in outdated units has been previously demonstrated. This phenomenon occurred while post transfusion viability was still acceptable. In the present study erythrocytes of units with early agglomeration were investigated and compared with those with late agglomeration. There was no morphological characterization of these erythrocytes and their sialic acid content and the toluidine blue uptake were not lower than in late agglomeration. Thus, it seems that agglomeration reflects membranal changes, but is not directly related to the sialic acid content of the membrane or to its toluidine blue uptake.