Fluorescence‐based assay for reactive oxygen species: a protective role for creatinine

Abstract

Attack by reactive oxygen species leads to a decay in phycoerythrin fluorescence emission. This phenomenon provides a versatile new assay for small molecules and macromolecules that can function as protective compounds. With 1-2 × 10⁻⁸ m phycoerythrin, under conditions where peroxyl radical generation is rate-limiting, the fluorescence decay follows apparent zero-order kinetics. On reaction with HO·, generated with the ascorbate-Cu²⁺ system, the fluorescence decays with apparent first-order kinetics. Examination of the major components of human urine in this assay confirms that at physiological concentrations, urate protects against both types of oxygen radicals. A novel finding is that creatinine protects efficiently by a chelation mechanism against radical damage in the ascorbate-Cu²⁺ system at creatinine, ascorbate, and Cu²⁺ concentrations comparable to those in normal urine. Urate and creatinine provide complementary modes of protection against reactive oxygen species in the urinary tract.— Glazer, A. N. Fluorescence-based assay for reactive oxygen species: a protective role for creatinine. FASEB J. 2: 2487-2491; 1988.