Studies on the chlorinating activity of myeloperoxidase.

Two methods were utilized to demonstrate the peroxidation of chloride ion to a free species (HOCl or Cl2) by myeloperoxidase. The peroxidase caused the volatilization of radioactivity from soultions containing hydrogen peroxide and [36Cl]NaCl, and catalyzed the formation of HOCl when solutions contianing these components were passed through a Millipore filter to which the peroxidase was adsorbed. In this flow system, 90 mug of canine myeloperoxidase generated 80 muM HOCl in the presence of 200 muM H2O2 at a rate corresponding to a turnover of 100 min-1. Under these conditions, o-tolidine, whose oxidation can be coupled to Cl- peroxidation in free solution, did not accelerate turnover. In contrast to chloroperoxidase and horseradish peroxidase, myeloperoxidase does not utilize chlorite for chlorination reactions. This oxidant inactivates the enzyme. At low pH, chloride ion suppresses the oxidation of myeloperoxidase (to the stable compound II) by both hydrogen peroxide and hypochlorite. Acceptor chlorination is therefore not a rate-controlling reaction in the myeloperoxidase mechanism, and the potential of the functional peroxidase couple is higher than the HOCl/Cl- couple under chlorinating conditions. The product-forming step may be a reverse of compound I formation at the expense of HOCl, rather than the chlorination of Cl- by a chloroperoxidase-like chlorinating intermediate. [1]

Myeloperoxidase and Cardiovascular Disease

Myeloperoxidase (MPO) is a leukocyte-derived enzyme that catalyzes the formation of a number of reactive oxidant species. In addition to being an integral component of the innate immune response, evidence has emerged that MPO-derived oxidants contribute to tissue damage during inflammation. MPO-catalyzed reactions have been attributed to potentially proatherogenic biological activities throughout the evolution of cardiovascular disease, including during initiation, propagation, and acute complication phases of the atherosclerotic process. As a result, MPO and its downstream inflammatory pathways represent attractive targets for both prognostication and therapeutic intervention in the prophylaxis of atherosclerotic cardiovascular disease.

Myeloperoxidase-catalyzed reactions have been attributed to potentially proatherogenic biological activities throughout the evolution of cardiovascular disease, including during initiation, propagation, and acute complication phases of the atherosclerotic process. As a result, myeloperoxidase and its downstream inflammatory pathways represent attractive targets for both prognostication and therapeutic intervention in the prophylaxis of atherosclerotic cardiovascular disease. [2]

Assay method for myeloperoxidase in human polymorphonuclear leukocytes

A simple assay method for measuring myeloperoxidase (MPO) has been developed. MPO is found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence of H2O2 and halide ions. This improved assay method is based on work of Andrews and Krinsky using tetramethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPO under optimal conditions of TMB at 1.6 mm, H2O2 concentration of 0.3 mm, pH 5.4, and incubation temperature of 37°C, sensitivity of MPO measurements increased eightfold in comparison with the original TMB method. A method has been established to determine absorbance at 655 nm of the reaction mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer. An attempt was also made to raise the sensitivity by using 3,3′-dimethyoxybenzidine (DMB), a carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB method. [3]

Respiratory Burst Enzymes and Oxidant-antioxidant Status in Nigerian Children with Sickle Cell Disease

Aim: To measure respiratory burst enzymes, pro-oxidants, antioxidants and red cell indices in Nigerian children with sickle cell disease (HbSS) below five years of age and compared with apparently healthy children with normal haemoglobin (HbAA).

Method: A total of 45 subjects were recruited which included 23 children (age range 10 – 48 months) with HbSS and 22 children (age- and sex- matched) with HbAA. Blood samples were collected and red cell indices were determined using automated haematology analyser while serum superoxide dismutase (SOD), glutathione peroxidise (GSH-Px) and myeloperoxidase (MPO) activities were measured using ELISA kits. Serum malondialdehyde (MDA), hydrogen peroxide (H2O2), glutathione S transferase (GST), catalase (Cat), xanthine oxidase (XO) and glutathione (GSH) were measured with colorimetric techniques. MPO, SOD and Cat represented respiratory burst enzymes; MDA, H2O2 and XO were measured as pro-oxidants while GSH, GST and GSH-Px were the measured antioxidants.

Results: Mean concentration of malondialdehyde was significantly reduced (5.56±1.12nmol/L vs. 6.46±1.11nmol/L, P=.04) in HbSS children compared with HbAA children. Similarly, mean serum activity of myeloperoxidase in HbSS children was significantly reduced compared with HbAA children (66.12±13.34U/mL vs 77.02±13.54U/mL, P=.03). However, there were no significant differences in mean concentration of serum glutathione, hydrogen peroxide; serum activities of glutathione peroxidase, catalase, superoxide dismutase, xanthine oxidase and glutathione S transferase in HbSS children compared with HbAA children

Conclusion: HbSS children in this population did not demonstrate raised oxidative stress. [4]

Alteration of Testicular Macrophage Morphology and Associated Innate Immune Functions in Cadmium Intoxicated Swiss Albino Mice

Aims: The present study investigates in a mouse model the extent of immunomodulatory effects after exposure to cadmium chloride (in vivo) in the testes.

Study Design: Experimental study.

Place and Duration of Study: Department of Biotechnology, Assam University, Silchar, Assam, India; between may 2010 and march 2012.

Methodology: LD50 was determined and the percent mortality of mice was plotted against their respective decreasing levels of cadmium chloride. To elucidate the immunomodulatory effects of cadmium chloride, Swiss albino mice were divided into two groups: the 1st group was intraperitonially injected with cadmium chloride (0.35 mg/kg b.w.) and the 2nd group with isotonic saline solution for 15 days. The isolated testicular macrophages were used to determine the morphological alteration as well as cell function studies such as phagocytosis, intracellular killing capacity, myeloperoxidase, nitric oxide release and TNF-α release assay from cadmium chloride -treated and control group of adult male Swiss albino mice.

Results: The present work shows that cadmium chloride is responsible for a significant alteration in morphology from 22.2 ± 0.05% to 60.1 ± 1.19% (P**), degenerative changes in scanning electron microscopy and reduced cell function such as phagocytosis (from 21000 ± 577.35 to 7100 ± 115.47; P**), myeloperoxidase release (from 46.8 ± 0.872 µM to 30.23 ± 1.041 µM; P*), nitric oxide release (from 11 ± 1.53 to 5 ± 1.2; P*) and the intracellular killing capacity was also reduced significantly (P**) in testicular macrophages probably by increasing oxidative damage. It also shows that TNF-α increases with cadmium chloride treatment (from 164 ± 4.62 to 235 ± 5.2; P*).

Conclusion: Thus it can be concluded that the toxic potential of cadmium chloride causes morphological changes as well as alterations in cell function in macrophages, rendering the animals more prone to infection, all of which may bear particular significance in heavy metal induced infertility. [5]

  Reference

[1] Harrison, J.E. and Schultz, J., 1976. Studies on the chlorinating activity of myeloperoxidase. Journal of Biological Chemistry, 251(5), pp.1371-1374.

[2] Nicholls, S.J. and Hazen, S.L., 2005. Myeloperoxidase and cardiovascular disease. Arteriosclerosis, thrombosis, and vascular biology, 25(6), pp.1102-1111.

[3] Suzuki, K., Ota, H., Sasagawa, S., Sakatani, T. and Fujikura, T., 1983. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Analytical biochemistry, 132(2), pp.345-352.

[4] Adelakun, A., Ajani, O., Ogunleye, T., Disu, E., Kosoko, A. and Arinola, G. (2014) “Respiratory Burst Enzymes and Oxidant-antioxidant Status in Nigerian Children with Sickle Cell Disease”, Biotechnology Journal International, 4(3), pp. 270-278. doi: 10.9734/BBJ/2014/7411.

[5] Chakraborty, S. and Sengupta, M. (2013) “Alteration of Testicular Macrophage Morphology and Associated Innate Immune Functions in Cadmium Intoxicated Swiss Albino Mice”, Journal of Advances in Medicine and Medical Research, 4(1), pp. 451-467. doi: 10.9734/BJMMR/2014/5498.

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