Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds to and kills some Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. GNB contain endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic lipid A component. The possibility that MPO and EPO bind and inhibit the endotoxin of GNB was tested by mixing MPO or EPO with LPS or lipid A and measuring for inhibition of endotoxin activity using the chromogenic Limulus amebocyte lysate (LAL) assay. The endotoxin-inhibiting activities of MPO and EPO were also tested in vivo using an LPS 90% lethal dose (LD90) mouse model studied over a five-day period. Mixing MPO or EPO with a fixed quantity of LPS from Escherichia coli O55:B5 or with diphosphoryl lipid A from E. coli F583 inhibited LAL endotoxin activity in proportion to the natural log of the MPO or EPO concentration. MPO and EPO enzymatic activities were not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. Both MPO and EPO increased mouse survival in the LPS LD90 model. In conclusion, MPO and EPO nonenzymatically inhibited in vitro endotoxin activity using the LAL assay, and MPO and high-dose EPO significantly increased mouse survival in a LPS LD90 model, and such survival was increased in a dose-dependent manner.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic leukocyte 21 haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds and 22 kills specific Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. 23 Endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic Lipid A component, is a 24 characteristic of all GNB. The possibility that haloperoxidases bind to and inhibit the endotoxin 25 of GBN was considered and tested by contacting MPO and EPO with LPS and Lipid A and 26 measuring for inhibition of endotoxin activity using either the in vitro gel or chromogenic 27 Limulus amebocyte lysate (LAL) assays. Contacting MPO and EPO with LPS purified from 28 Escherichia coli O55:B5 and with diphosphoryl Lipid A purified from E. coli F583 inhibited 29 their endotoxin activities in proportion to the natural log of the MPO or EPO concentration. 30 Although MPO is less cationic than EPO, MPO consistently demonstrated inhibition of 31 endotoxin activity that is about threefold superior to EPO. Haloperoxidase enzymatic activity 32 was not required for inhibition, and MPO haloperoxidase action did not increase endotoxin 33 inhibition. MPO and EPO inhibition of LPS endotoxin activity was also measured using a 90% 34 lethal dose (LD90) mouse model studied over a five-day period. Based on Kaplan Meier survival 35 analysis, MPO significantly increased mouse survival in a dose-dependent manner. EPO was less 36 effective. In conclusion, contacting MPO and EPO with LPS and Lipid A inhibits in vitro 37 endotoxin activities, but inhibition is independent of haloperoxidase enzymatic function. MPO 38 significantly increases mouse survival against LPS in an in vivo LD90 endotoxin model. 39 40 41 42 Introduction:

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