Determination of absolute hydrogen peroxide concentration by spectrophotometric method

There is considerable current interest in oxidative stress applicable to humans. One important reactive oxygen species generated in the body is H2O2. It was reported that substantial amount of H2O2 was excreted in urine, even in neonates. Therefore, it was hypothesized earlier that measurement of H2O2, especially in urine, could be a valuable biomarker for the generation of H2O2 and the degree of oxidative stress in vivo. The FOX-2 method has been used earlier to detect H2O2 in urine . The basic principle of this method is oxidation of ferrous ions by the sample oxidizing agents to ferric ions, which bind with xylenol orange to give a coloured complex. Gupta et al. had developed the basic method in 1975 which was later on elaborated by Wolf and coworkers. Though the method has been used to detect H2O2, hydroperoxides can also oxidize ferrous ions to ferric ions. Therefore, this method has been used successfully to measure plasma hydroperoxide concentration. H2O2 is possibly absent in normal plasma due to the presence of its degradative systems like catalase. In the FOX-2 method 90 μl of sample, 10 μl of methanol and 900 μl of xylenol orange (Sigma Chemical Co.) reagent containing ferrous ions were added successively and absorbance was noted at 560 nm (ref. 3). Therefore, the classical FOX-2 method measured any substance that was converting Fe to Fe, irrespective of their chemical nature. Ten μl of methanol was replaced by 10 μl of 10 mM triphenylphosphine earlier to reduce plasma hydroperoxides, and this modification of FOX-2 was used to measure plasma hydroperoxide concentration more accurately. In this correspondence we have modified the method by performing the assay in the presence or absence of 10 μl of catalase. This was done by replacement of 10 μl of methanol with 10 μl of catalase solution to measure absolute H2O2 concentration in biological samples. Catalase reagent was prepared by dissolving 1 mg of catalase powder (22,000 U/mg from Sigma) in 10 ml of 25 mM phosphate buffer pH 7.0, containing 2 mg/ml bovine serum albumin and stored in aliquots at –20°C. Catalase is an antioxidant enzyme catalysing the following reactions.