A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.
Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.
Haqqani
A S
AS
Department of Biochemistry, Microbiology, and Immunology, University of Ottawa and the Ottawa Regional Cancer Center, Ottawa, Ontario, K1H 8L6, Canada.
Sandhu
J K
JK
Birnboim
H C
HC
D001965:Bromides / Q000378:metabolism*; D047209:Eosinophil Peroxidase; D018922:HL-60 Cells; D006801:Humans; D008163:Luminescent Measurements; D008165:Luminol / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000032:analysis*; D010544:Peroxidases / Q000032:analysis
D001965:Bromides; D008165:Luminol; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase