Changes in phagocyte activity in patients with ischaemic stroke.

The activity of peripheral phagocytes to generate reactive oxygen species (ROS) was studied in healthy individuals and patients with ischaemic stroke. The aim was to clarify the relationship between phagocyte activity, the time elapsed after the onset of disease and stroke severity. The total and extracellular production of ROS were evaluated by luminol chemiluminescence. Simultaneously the plasma oxidant activity was determined. When stimulated by opsonized zymosan, phagocytes in patients with stroke (regardless of its severity) showed fast activation. The total ROS generation increased over time in all stroke cases studied. However, the extracellular ROS generation was found to be greater in patients with severe stroke than in those with mild neurological deficiency. When stimulated by formyl-methionyl-leucyl-phenylalanine, the total oxidative phagocyte capacity (regardless of stroke severity) increased over time, but there was no change in the amount of extracellularly generated ROS. In patients with stroke the oxidant activity of plasma was enhanced. We conclude that circulating phagocytes in patients with ischaemic stroke are primed for enhanced ROS production by opsonin receptor-mediated stimulation and for increased secretion of myeloperoxidase by opsonin receptor-independent stimulation. The enhanced extracellular generation of ROS through opsonin receptor-dependent stimulation may be considered an oxidative stress biomarker in cerebral ischaemia.

Functional states of polymorphonuclear leukocytes determined by chemiluminescent kinetic analysis.

During the respiratory burst, upon stimulation with both soluble and particulate matter, polymorphonuclear leukocytes (PMN) generate reactive oxygen species (ROS) and emit chemiluminescence (CL) as a result of metabolic activation. The measurement of CL has been demonstrated to be a useful tool for in vitro assessment of the opsonophagocytic function of PMN. Using component analysis of CL kinetics, we characterized the functional state of PMN by three parameters of the respiratory burst: capacity, effectiveness and velocity (CEV space). The possibility of delimiting eight different functional states of PMN is discussed. The CL kinetics shown by blood PMN in different functional states was analysed, and revealed six out of eight functional states. We conclude that CEV-estimated functional states of PMN are relative, depending on both PMN readiness to generate ROS and conditions of the CL test.

Model components of luminol chemiluminescence generated by PMNL.

The production of activated oxygen species (AOS) by neutrophils (PMNL) is thought to play a key role in the host defence against invading microorganisms. However, the oxygen metabolites are toxic not only to the invading bacteria but also to the surrounding tissue. The oxidative metabolites production can be evaluated by means of chemiluminescent methods. In this study, the possibility of a new analytical approach for quantitative assessment of chemiluminescent kinetics (AOS generation) of isolated PMNL was estimated. Based on the assumption that the kinetics of luminol-amplified chemiluminescence (LCL) of stimulated PMNL possesses a time-probabilistic nature, this kinetics was described with three components. These components, obtained from different investigated systems, were analyzed and a conclusion was made that the first and the second component represent the processes resulting in extra-and intracellular myeloperoxidase (MPO)-dependent light emission (AOS generation), respectively. The second component was found to be completely dependent on the stimulus ingestion. The third component was not completely MPO-dependent and complicated for interpretation. This component was weakly dependent on the stimulus ingestion, and presents at least some intracellular processes different from those presented by the second component. A conclusion is made that the examined approach for analysis of LCL kinetics allows an assessment of extra-and intracellularly generated quantities of AOS by stimulated PMNL. The assessment could be done for emitting systems in which no additional modifications are used.

A new approach for analysis of chemiluminescent kinetics of activated phagocytes in blood.

Luminol chemiluminescence (LCL) is a simple, sensitive and time-saving tool to elucidate the oxidative activity of polymorphonuclear leukocytes (PMNL). In this study, a new approach for analysing the LCL kinetics, recorded from stimulated PMNL, and for a more accurate elucidation of their functional state, is proposed. This approach is based on the proposal that the LCL kinetics of stimulated PMNL is a result of time-probabilistic nature of the processes, leading to light emission. On this basis the LCL response was described by the parameters of a Poisson-type distribution. Relationships between these parameters and some factors influencing LCL kinetics (number of PMNL and erythrocytes, temperature, stirring) were investigated. The model descriptions of the LCL responses obtained had two components (modes). Their development in time was different. It was suggested, that the two phases of the model LCL response were connected with extracellular and intracellular LCL. The terms and the advantages of this approach for analysis of phagocyte oxidative capacity are discussed.

Dependence of whole blood luminol chemiluminescence on PMNL and RBC count.

Luminol chemiluminescence (LCL) of whole blood as response to zymosan stimulation was used to assess the activity of polymorpho-nuclear leukocytes (PMNLs). Since PMNL and red blood cells (RBCs) are the primary components determining LCL response, the influence of PMNL and RBC count on the response was investigated over wide ranges of cell count and time interval. A linear relationship was found between maximum LCL intensity (Imax) (reflecting PMNL activity) and PMNL count. This allows a proper correction of Imax to be made as related to the sample PMNL count. This correction was valid for PMNL concentrations from 3.10(3) PMNL/ml to 6.10(5) PMNL/ml. When concentrations range from 6.10(3) PMNL/ml to 3.10(4) PMNL/ml the relationship was valid for a time interval round the peak. During the same interval the inhibitory effect of RBC count was studied. A correction of LCL was found possible for different RBC concentration ranging from 2.5.10(6) RBC/ml to 10(8) RBC/ml. These independent corrections of LCL response to PMNL and RBC counts were applied to diluted whole blood samples. The common correction of Imax was applicable in dilutions not less than 1:50. To correct light intensity for the time interval around Imax, the dilutions used had to be not less than 1:200. The results obtained permit an objective assessment of PMNL activity to be made by testing whole blood samples from different individuals and/or samples diluted to different extents.

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes.

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.

[Formation of the superoxide anion radical during the oxidation of biogenic amines catalyzed by mitochondrial monoamine oxidase]. / Ob obrazovanii anion-radikala superokisi pri okislenii biogennykh aminov, kataliziruemom monoaminoksidazoi mitokhondrii.

The studies on the activity of monoamine oxidase from human placenta, using 2-phenylethylamine as a substrate, corroborate the hypothesis on the possible superoxide radical generation upon FAD oxidation at the second (aerobic) stage of monoamine oxidase reaction. It has been shown that hydrogen peroxide, but not other activated O2 forms, was the end product of this reaction. No superoxide radical generation took place in such systems. And therefore, the induction of lipid peroxidation in the presence of catalase was impossible in mitochondrial membranes containing monoamine oxidase and amines oxidized by it.

[Study of monoamine oxidase from human placenta mitochondria by the chemoluminescence method]. / Issledovanie monoaminoksidazy mitokhondrii platsenty cheloveka metodom khemiliuminestsentsii.

The activity of monoamine oxidase from human placenta mitochondria was determined with 2-phenylethylamine and benzylamine as substrates by the generation of hydrogen peroxide in a conjugated luminol-peroxidase system, using the chemiluminescence method. The monoamine oxidase was found to oxidize at a high rate MAO substrates and revealed high sensitivity to clorgyline, a specific inhibitor of monoamine oxidase type A. It was shown that the use of the chemiluminescence technique for determining the monoamine oxidase activity gives the results that are fully consistent with those obtained by other methods.

The interaction of copper chloride with erythrocyte membrane as a source of activated oxygen species. A chemiluminescent study.

The possibility for the generation of activated oxygen species during the interaction between copper chloride and erythrocyte membranes was investigated. A chemiluminescent method for detecting superoxide radicals and hydrogen peroxide was used. It was found that the interaction of CuCl2 with erythrocyte membrane is accompanied with O-2 and H2O2 generation. On the base of this result it is proposed that the activated oxygen species generated by CuCl2-membrane interaction may be able to initiate peroxidative breakdown processes in erythrocytes eventually leading to haemolysis.

A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates.

A chemiluminescent method for measuring the concentration of activated oxygen species (O-2 and H2O2) is described. Its main features are: high sensitivity (10(-9) M H2O2), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.