During phagocytosis by polymorphonuclear leukocytes (PMN), oxygen is reduced to hydrogen peroxide (H202) and other free radicals. Polymorphonuclear leukocytes utilize the oxygen by-products for the peroxidative dependent killing of ingested bacteria. Previous studies have indicated that the elaboration of H202 will attenuate phagocytosis and chemotaxis of normal PMN, (1) compared to those activities of PMN maintained in an anaerobic environment. In addition, the H202 released into the extracellular media from phagocytizing PMNs will rapidly oxidize glutathione in glucose 6-phosphate dehydrogenase deficient red cells leading to a shortening of their in vivo survival (2). At least two biochemical defense mechanisms are available in the cytosol of cells to defend against the toxic effects of H202; these include catalase and the maintenance of adequate GSH levels by enzymes associated with the glutathione pathway. To explore the relative contribution of each of these enzyme systems, we quantitated the activities of enzymes responsible for disposal of H202 in the PMN obtained from four different mammalian species.Materials and methods. Human PMN were isolated from peripheral blood as previously described (3). Guinea pig, mouse and rat PMN were collected from the peritoneal cavities of these animals 18 hours after injection of 12% caseinate (4). All PMN samples were then pelleted by a 200g 10-min centrifugation. Red cells were lysed from these pellets by a 20 sec exposure to distilled water and isotonicity was then restored. The PMN samples were suspended in Krebs-Ringer phos-' This work has been supported by grants from the National Institutes of Health Nos. PHS R01 A1 10892-05, PHS ROI A1 13586-02 and H L 19779-03, and a grant from the Riley Memorial Association. Dr. Boxer is an Established Investigator of the American Heart Association. phate buffer (KRP) pH 7.4 at a cell concentration of I x lo8 cells/ml. Cell samples were sonicated at an output setting of 3 for 60 sec with a Sonifier Cell Disrupter model W140, Heat Systems-Ultrasonics Inc.Catalase, glutathione peroxidase (GPX), glutathione reductase (GR) and myeloperoxidase assays were determined on cellular sonicates. The sonicates were clarified by centrifugation at 27,000g for 10-min and the supernatant was employed for these enzyme determinations except for myeloperoxidase. The 27,OOOg pellet was resuspended in KRP buffer pH 7.4 and then sonicated an additional 60 sec at an output setting of 5. The sonicated granular fraction was used for the determination of myeloperoxidase activity.Catalase activity was quantitated by recording the rate of decrease in absorbance of H202 at 240 nm (E240m = 43.6 M-' cm-') (6). The reactions were performed at 25" in 3.0 ml cuvettes containing 20 mM H202 in 50 mM phosphate buffer pH 7.0 and were started with 25-100 pg of sample protein.Glutathione peroxidase activity was measured by observing the rate of oxidation of NADPH at 340 nm (EMo-= 6.22 x lo3 M-'cm-') at 25" by the method of Paglia (7). 100-500 pg sample protein was adde...
Early and effective cytoreduction for high peripheral white blood cell counts in pediatric patients with acute leukemia may be helpful in preventing complications secondary to hyperviscosity. It also may be a useful adjunct to systemic chemotherapy. As an alternative to automated apheresis for this purpose, manual exchange transfusion is efficacious and does not require hemapheresis instrumentation and disposables and the related special staff. Two patients, a neonate with acute myeloblastic leukemia and a white blood cell count of 422.2 k/microliter as well as a 2 1/2-year-old with an admission diagnosis of acute promyelocytic leukemia and a white blood cell count of 617.4 k/microliter, underwent manual exchange hemotherapy for acute cytoreduction. The procedures were tolerated well, and significant leukocyte removal was achieved, with the respective leukocyte reductions being 81.1 and 68.7%. The techniques available for pediatric cytoreduction are compared, with emphasis on their efficiency and safety and appropriateness for very small children.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.