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ORIGINAL RESEARCH ARTICLE

Effects of Iron and Phytic Acid on Production of Extracellular Radicals by Enterococcus faecalis

Danny R. Moore^*, Yashige Kotake and Mark M. Huycke^*,1

^* The Muchmore Laboratories for Infectious Diseases Research, Research Service, Department of Veterans Affairs Medical Center, and Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

To whom requests for reprints should be addressed at ¹ Medical Service (111), 921 N.E. 13th Street, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104. E-mail: mark-huycke{at}ouhsc.edu

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide, hydrogen peroxide, and hydroxyl radical while colonizing the intestinal tract. To determine whether dietary factors implicated in colorectal cancer affect oxidant production by E. faecalis, radicals were measured in rats colonized with this microorganism while on diets supplemented with iron or phytic acid. Hydroxyl radical activity was measured by assaying for aromatic hydroxylation products of D-phenylalanine using reverse-phase high-performance liquid chromatography and electrochemical detection. In vitro, as expected, iron enhanced, and phytic acid decreased, hydroxyl radical formation by E. faecalis. For rats colonized with E. faecalis given supplemental dietary iron (740 mg elemental iron as ferric phosphate per kg diet) or phytic acid (1.2% w/w), no differences were found in concentrations of urinary ortho- or meta- isomers of D-phenylalanine compared to rats on a basal diet. Aqueous radicals in colonic contents were further assessed ex vivo by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. Mixtures of thiyl (sulfur-centered) and oxygen-centered radicals were detected across all diets. In vitro, similar spectra were observed when E. faecalis was incubated with hydrogen sulfide, air-oxidized cysteine, or an alkylsulfide, as typical sulfur-containing compounds that might occur in colonic contents. In conclusion, intestinal colonization with E. faecalis in a rat model generates both thiyl and oxygen-centered radicals in colonic contents. Radical formation, however, was not significantly altered by short-term dietary supplementation with iron or phytic acid.

Key Words: Enterococcus faecalis • free radicals • dietary iron • dietary phytic acid • aromatic hydroxylation • electron spin resonance spectroscopy