Microtiter plate assay for phagocyte-derived taurine-chloramines.
Despite their potential importance, the role of phagocyte-derived chloramines ("long-lived oxidants") has not yet been investigated in inflammatory or infectious diseases. We have developed a sensitive spectrophotometric microtiter plate assay for chloramines based on their capacity to oxidize potassium iodide (KI). Consistent levels of endogenous chloramines were detected in normal human polymorphonuclear neutrophil (PMN) supernatants after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Exogenous taurine strongly enhanced chloramine secretion and was used to quantify the chlorinating potential of PMN. Taurine-chloramines were also detectable in monocyte supernatants, although in smaller amounts. The specificity of the KI assay was assessed both in terms of effect of compounds inhibiting (KBr) or interacting with (sodium azide and catalase) chloramine formation and by showing that PMN from patients with chronic granulomatous disease, due to a hereditary lack of oxidative response capacity, were unable to produce chloramines. Taurine-chloramine levels secreted by PMA (but not zymosan)-stimulated PMN were closely related to the cellular luminol-amplified chemiluminescence (CL) responses although the CL assay failed to detect chloramines in PMN supernatants. We consider that this KI assay should be of use in studying the role of long-lived phagocyte-derived oxidants in clinical medicine.
Witko
V
V
INSERM U25, Hôpital Necker, Paris, France.
Nguyen
A T
AT
Descamps-Latscha
B
B
D002700:Chloramines / Q000032:analysis* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D008165:Luminol; D010084:Oxidation-Reduction; D010586:Phagocytes / Q000378:metabolism*; D011193:Potassium Iodide / Q000378:metabolism; D013053:Spectrophotometry; D013654:Taurine / Q000378:metabolism
D002700:Chloramines; D005609:Free Radicals; D011193:Potassium Iodide; D013654:Taurine; D008165:Luminol