title: Differentiating between intra- and extracellular chemiluminescence in diluted whole-blood samples.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Rájecký
authors.forename: A
authors.initials: A
authors.lastname: Lojek
authors.forename: M
authors.initials: M
authors.lastname: Cíž
mesh_terms: D001769:Blood; D001773:Blood Cells; D006801:Humans; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000031:analogs & derivatives; D017382:Reactive Oxygen Species / Q000032:analysis*
chemical_list: D017382:Reactive Oxygen Species; C032487:isoluminol; D008165:Luminol