The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

Abstract

To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.

Optical probes for detection and quantification of neutrophils’ oxidative burst. A review

“…This difference could be related to the fact that isoluminol only detects extracellular production of reactive species [72]. The extracellular origin of isoluminol chemiluminescence was also confirmed by its almost complete blockade in the presence of SOD and catalase [80]. Since the response to PMA as well as to fMLP was almost totally inhibited by SOD, Lundqvist and Dahlgren [76] concluded that isoluminol mainly detects cellular release of O 2…”

“…Isoluminol differs from luminol not with respect to the basic mechanism for generation of light but only in respect to the position of the amino group in the phthalate ring of the molecule, which makes isoluminol less lipophilic and thus less permeable to biological membranes than luminol (Fig. 3) [76,80]. In general, the intensity of isoluminol-enhanced chemiluminescence in leukocytes stimulated by different activators is markedly lower when compared with luminol-enhanced chemiluminescence [72].…”

“…The absence or very low levels of chemiluminescence obtained when stimulating cells isolated from patients with CGD reveals the importance of NADPH oxidase in the luminol chemiluminescence [23,64]. Due to its chemical structure, namely the small size and lipophilicity, luminol can diffuse across biological membranes and therefore allow the detection of both extra and intracellular production of reactive species [76,77,80]. To measure specifically the intracellular reactive species, SOD and catalase, membraneimpermeable enzymes, should be added to reaction mixture to remove secreted O 2…”

Role of nanostructured gold surfaces on monocyte activation and Staphylococcus epidermidis biofilm formation

“…Next, we analyzed the activity of the cells by measuring the luminol-mediated chemiluminescence (detecting both intra-and extracellular ROS) 42 cell types that adhere to the surface of implants after insertion in vivo. The oxidative response and secretion of molecules are influenced by both the properties of the material and the component of their microenvironment.…”

A new chemiluminescence paradox: selective inhibition of isoluminol‐amplified activity in phagocytes by peptides from annexin AI

“…This difference between the two molecules does not change the light-generating mechanism, even though the physicochemical properties differ and the quantum yield from luminol is somewhat higher than that of isoluminol (3). Isoluminol is more hydrophilic since the polarity increases as the amino group moves away from the first carbon atom (2,4). As a consequence, isoluminol is less able to transverse biological membranes and is therefore used to detect the release of NADPH oxidase-derived superoxide ions.…”