ABSTRACT
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.
Subject(s)
Neutrophils/physiology , Reactive Oxygen Species/analysis , Adult , Humans , In Vitro Techniques , Kinetics , Luminescent Measurements , Luminol , Photometry/methods , Probability , Time FactorsABSTRACT
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.
Subject(s)
Luminescent Measurements , Neutrophils/metabolism , Adult , Erythrocyte Count , Humans , In Vitro Techniques , Kinetics , Leukocyte Count , Luminol , Methods , Models, Biological , Oxidation-ReductionABSTRACT
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.
