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Updated: Apr 30, 2026
Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples
Published on: July 28, 2016
Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during
Keiichi Moriguchi1, Norikazu Ohno
1Department of Oral Anatomy and Research Institute of Advanced Oral Science, School of Dentistry, Aichi-Gakuin University.
Abstract:
Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.
Insights
Polymorphonuclear leukocytes (PMNs) generate hydrogen peroxide (H2O2) during phagocytosis. Optimal fixation conditions are crucial for accurately measuring H2O2 production and visualizing its release into phagosomes.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) are key immune cells that eliminate pathogens via phagocytosis.
- Phagocytosis involves the production of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), by the NADPH oxidase complex.
- Understanding H2O2 generation dynamics is crucial for comprehending innate immune responses.
Purpose of the Study:
- To quantify H2O2-dependent fluorescence in PMNs during opsonized zymosan A (OPZ) phagocytosis under various glutaraldehyde (GA) fixation conditions.
- To visualize the cellular mechanisms of H2O2 generation and release into phagosomes using electron microscopy.
Main Methods:
- PMN phagocytosis of OPZ was induced, followed by fixation with varying concentrations (0.5%, 1%, 10%) and durations (1-30 min) of glutaraldehyde.
- H2O2 production was measured using 2'-7'-dichlorofluorescin diacetate fluorescence with a microplate reader.
- Electron microscopic cytochemistry was employed to visualize H2O2 generation and vesicle fusion events.
Main Results:
- A 0.5% GA concentration yielded the highest H2O2-dependent fluorescence across all fixation times, suggesting higher concentrations inhibit phagocytosis and ROS production.
- Electron microscopy revealed PMN attachment to particles and initial mild secretory vesicle deposits within 1 minute.
- Within 10 minutes, engulfed particles showed enhanced reaction deposits in vesicles that fused with the phagosomal membrane, releasing free radicals into the phagosome.
Conclusions:
- Glutaraldehyde fixation concentration significantly impacts H2O2 measurement in phagocytic PMNs.
- Secretory vesicles play a critical role in H2O2 production and delivery to phagosomes during phagocytosis.
- The study provides insights into the spatiotemporal dynamics of ROS generation during phagocytosis.
