Differentiating between intra- and extracellular chemiluminescence in diluted whole-blood samples

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 125x 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.