Naomi Meyerstein

New Mechanistic Aspects of the Fenton Reaction

The kinetics of the Fenton reaction was studied in detail. A second reaction step in the presence of excess H2O2 is attributed to formation of the complex Fe(III)(-O2H)(aq). Therefore, the reaction of Fe(H2O)(6)(2+) with Fe(III)(-O2H)(aq) in the presence of Fe(II) to form Fe(III)(aq) (k=(7.7+/-1.5) x 10(5) M(-1) s(-1)) may contribute to the overall Fenton reaction, and could account for some of the debate in the literature concerning its detailed mechanism. If this is correct for LFe(III)(-O2H)(aq) also, then it might be of significant biological importance. The activation parameters DeltaH(not equal), DeltaS(not equal), and DeltaV(not equal) for the Fenton reaction were measured under various experimental conditions, and are used in the mechanistic interpretation.

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.

Acoustic cavitation in phacoemulsification: chemical effects, modes of action and cavitation index

High-intensity ultrasound (US) energy (HIUE) has been extensively used in the last 3 decades in a wide range of surgical procedures, including phacoemulsification. The generation of radicals and sonoluminescence (SL) by application of continuous-wave (CW) HIUE to an aqueous medium under conditions simulating cataract phacoemulsification surgery is demonstrated by electron paramagnetic resonance (EPR) spectroscopy and a sensitive photon-detecting system. The findings provide direct evidence for the generation of acoustic cavitation in the simulated intraocular environment, pointing out that generation of acoustic cavitation in clinical phacoemulsification and other surgical applications of US is possible. The findings imply that the effects of acoustic cavitation in aqueous medium may contribute to the endothelial damage observed clinically following phacoemulsification cataract surgery. Saturation of the irrigating solution with various gases modifies the acoustic cavitation. Saturation of the irrigating solution with CO2 practically eliminates acoustic cavitation, with the concomitant elimination of radicals and SonL. CO2 may be utilized clinically to suppress acoustic cavitation in phacoemulsification and other medical applications. A cavitation index (CI) is introduced for the purpose of standardizing phacoemulsification instrumentation and other medical US devices that employ HIUE.

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.

Protective effect of free-radical scavengers on corneal endothelial damage in phacoemulsification

PURPOSE: To examine the role of the water‐soluble antioxidants glutathione and ascorbic acid in the irrigating solution on corneal endothelial cells following exposure to high‐intensity ultrasound energy. SETTING: Goldschleger Eye Research Institute, Sheba Medical Center, Tel‐Aviv University, Tel Aviv, Israel. METHODS: Thirty‐two rabbit eyes were subjected to prolonged exposure to the phacoemulsification device in the anterior chamber. The eyes were divided into 4 groups that differed only in the composition of the irrigating solution applied to the eyes: balanced salt solution (BSS) BSS Plus BSS containing additional soluble components including glutathione, BSS with 10−3 M of oxidized glutathione (GSSG), and BSS with 10−2 M of ascorbic acid. Specular microscopy was performed preoperatively and 1 week after surgery. RESULTS: The BSS group exhibited the highest endothelial cell loss (19.3%), followed by the BSS Plus group (10.6%), the GSSG group (5.2%), and the ascorbic acid group (0.9%). An overall difference was found between the groups (F = 11.046, P<.0001), and all groups demonstrated a statistically significant difference from the control BSS group (P<.02, P = .001, and P<.0001, respectively). CONCLUSIONS: Damage to the cornea is largely due to the free radicals generated by high‐intensity ultrasound energy during phacoemulsification. Adding the antioxidants ascorbic acid and GSSG to the irrigation solution significantly reduced the endothelial corneal cell damage. Ascorbic acid in the concentration of 10−2 M had the highest protective effect; thus, it should be evaluated for clinical use.

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties.

The kinetics of reduction of the radical R ”, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl, by blood and its components were studied using the EPR technique. The results demonstrate that R ” is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R ” . The observed first order rate of disappearance of the nitroxide radical, k , is: k blood > k eryth > k plasma and k blood ; k eryth + k plasma . The results demonstrate that: The erythrocytes catalyze the reduction of R ” by ascorbate. The rate of reduction of the radical is high though it does not penetrate the cells. In human erythrocytes there is an efficient electron transfer route through the cell membrane. The study points out that R ” is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.

Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes

The lateral diffusion coefficients (D) and the mobile fractions of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) and of membrane proteins labelled with fluorescein isothiocyanate, were measured by fluorescence photobleaching recovery on erythrocytes from healthy persons and from a hereditary spherocytosis patient. Measurements of lipid probe mobility were performed on ghosts labelled by NBD-PE exclusively at the external monolayer, or at both sides of the membrane. Our results indicate the following: (1) The mean values and the temperature dependence of D are different at the external and internal membrane leaflets. (2) In both normal and HS ghosts the mobile fraction of NBD-PE in the external monolayer does not depend significantly on temperature. On the other hand, the mobile fraction in the internal monolayer is reduced as the temperature is decreased. (3) At low temperatures, the mobile fraction of NBD-PE in the internal monolayer of spherocytic ghosts is significantly lower than the mobile fraction in the internal monolayer of normal ghosts. (4) No differences were observed between the mobilities of membrane proteins in normal and in spherocytic ghosts. However, differences were observed between the two cell populations in the temperature-dependence of the intrinsic fluorescence of unlabelled membrane proteins. The implications of these results for membrane phospholipid asymmetry and for cytoskeletal interactions with the internal lipid monolayer are discussed.

Differential effects of lipids on the osmotic fragility of hamster erythrocytes

1. 1. There was no difference in osmotic potential between erythrocytes of control and heat-exposed hamster, thus the increased red cell osmotic fragility of the heat-exposed animal is related to membrane properties of the erythrocytes. 2. 2. Linolenoyl sorbitol at 4 μg/ml (9 · 10−6 M) increased the osmotic stability of the erythrocytes of both animal groups in a parallel fashion. This lipid did not modify the frequency distribution of hemolysis of the erythrocytes. 3. 3. At 65% hemolysis, added linolenoyl and stearoyl sorbitol similarly protected both groups of erythrocytes. At 95% hemolysis, specific interactions were apparent: linolenoyl sorbitol (at 6 μg/ml) induced almost full protection against osmotic hemolysis of both groups of erythrocytes. Stearoyl sorbitol (at 6 μg/ml) was only partially effective, and the protection afforded to the erythrocytes from heat-exposed animals was particularly limited. 4. 4. Linolenoyl sorbitol expanded the erythrocyte membrane at hypotonic media more effectively than stearoyl sorbitol. 5. 5. The data support the hypothesis that the unsaturated acyl groups of the membrane lipids contribute to higher osmotic stability of the erythrocyte.