IN-VITRO FREE-RADICAL METABOLISM OF PHENOLPHTHALEIN BY PEROXIDASES

Phenolphthalein, a widely used laxative, is the active ingredient in m ore than a dozen commercial nonprescription formulations. Fast-flow EP R studies of the reaction of phenolphthalein with horseradish peroxida se (HRP) and hydrogen peroxide permit the direct detection of two free radicals. One has EPR parameters characteristic of phenoxyl radicals. The other has a broad unresolved spectrum, possibly arising from free radical polymeric products of the initial phenoxyl radical. EPR spin- trapping studies of incubations of phenolphthalein with lactoperoxidas e, reduced glutathione (GSH), and hydrogen peroxide with 5,5-dimethyl- 1-pyrroline N-oxide (DMPO) demonstrate stimulated production of DMPO/( .)SG compared with an identical incubation lacking phenolphthalein. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed by a sequence of reactions init iated by the glutathione thiyl radical. Enhanced production of DMPO su peroxide radical adduct is also found in a system of phenolphthalein, NADH, and lactoperoxidase. In this system the phenolphthalein phenoxyl radical abstracts hydrogen from NADH to generate NAD(.), which is nor spin trapped by DMPO, but reacts with molecular oxygen to produce the superoxide radical detected by EPR. In the absence of DMPO, the oxyge n consumption is measured using the Clark-type electrode. Production o f ascorbate radical anion is also enhanced in a system of phenolphthal ein, ascorbic acid, hydrogen peroxide, and lactoperoxidase. Ascorbate inhibits oxygen consumption when phenolphthalein is metabolized in the presence of either glutathione or NADH by reducing radical intermedia tes to their parent molecules and forming the relatively stable ascorb ate anion radical. The detection of enhanced free radical production i n these three systems, a consequence of futile metabolism (or redox cy cling), suggests that phenolphthalein may be a significant source of o xidative stress in physiological systems, Parallel EPR and oxygen cons umption studies with phenolphthalein glucuronide give analogous result s, but with lesser enhancement of free radical production.