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Differentiating between intra‐ and extracellular chemiluminescence in diluted whole‐blood samples
Author(s) -
RÁJECKÝ M.,
LOJEK A.,
ČÍŽ M.
Publication year - 2012
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/j.1751-553x.2011.01370.x
Subject(s) - extracellular , luminol , chemistry , chemiluminescence , reactive oxygen species , intracellular , horseradish peroxidase , superoxide dismutase , catalase , biochemistry , biophysics , chromatography , biology , enzyme
Summary Introduction: The differentiation between extra‐ and intracellular production of reactive oxygen species (ROS) in whole blood was measured by luminol‐ and isoluminol‐enhanced chemiluminescence (CL). Methods: Azide (total CL inhibition), azide + horseradish peroxidase (HRP, restoring extracellular CL), superoxide dismutase + catalase (depleting extracellular ROS) and HRP (enhancing extracellular CL) were used to modulate luminol‐ and isoluminol‐enhanced CL (10 −6 –10 −3 m luminophores) of 125× diluted whole blood which was activated by both calcium ionophore A23187 (Ca‐I) and opsonized zymosan particles (OZP) separately. Results: Both activators stimulated intra‐ and extracellular production of ROS. Luminol‐enhanced CL of Ca‐I‐activated samples detected the intracellular ROS, and with the addition of HRP detected the extracellular CL as well. CL enhanced with isoluminol in concentrations of 10 −4 m or less was mostly extracellular. There was a mixture of intra‐ and extracellular CL in OZP‐activated samples, probably because of the ingestion of luminophore molecules. Conclusion: Measurement of Ca‐I‐activated CL enhanced with 10 −4 m luminol is recommended for the detection of intracellular ROS. The addition of HRP leads to the detection of overall ROS production while the OZP‐activated system with its addition of HRP can only be used to detect overall ROS production. Ca‐I‐activated CL enhanced with 10 −4 m isoluminol and with addition of HRP is recommended for the detection of extracellular CL.