Hydrogen peroxide has been found in the aqueous humor of several animal species including man, at the low uM level (5 to 90 uM). In every case reported H202 determination was made with the dichlorophenolindophenol (DCPIP) method, which involves the horseradish peroxidase-catalyzed oxidation of DCPIP by H202. Results obtained with this method are, however, highly questionable on the following bases: 1) ascorbate already present in the aqueous humor or exogenously added as part of the procedure to fully reduce DCPIP, can be spontaneously degraded to products that apparently include H202; 2) the enzyme itself, once incorporated to the reaction mixture, can catalyze further degradation of ascorbate, now with H202 consumption. The chances are therefore that these complex interactions introduce serious errors in the estimation of H202 with the DCPIP method. The role of H202 as a hypothetical oxidative agent contributing to lens aging and senile cataractogenesis critically depends on the reliability of the reported values. Strategies are proposed to unambiguously establish the levels of H202 in the aqueous humor. Two existing, potentially valuable reactions for H202 assay have been preliminarily converted into HPLC (high performance liquid chromatography) procedures to increase their sensitivity and specificity to the required levels. These are the H202/ peroxidase-dependent oxidation of Scopoletin, and the direct reaction between Hydroxynapthaldehyde Thiosemicarbazone (HNTS) and H202. Both reactions require additional modifications to be useful. The Scopoletin method will require the use of a different peroxidase which does not accept ascorbate as substrate, such as yeast cytochrome c peroxidase, or the removal of ascorbate by pretreatment of the aqueous humor with ascorbate oxidase or an anion-exchanger. The HNTS procedure may require the previous removal of aqueous humor components also reacting with HNTS in order to avoid chromatographic interferences. In both methods fluorescence detection of column eluates should be used. Finally, quantification of H202, if necessary, will be made by measuring the levels of peroxidase-H202 or catalase-H202 intermediates with dual wavelength spectrophotometry. Fractionation of aqueous humor by HPLC to separate H202 prior to analysis will also be considered.
| Garcia-Castineiras, S (1998) [Hydrogen peroxide in the aqueous humor: 1992-1997] P R Health Sci J 17:335-43 |
| Garcia-Castineiras, S; Velazquez, S; Martinez, P et al. (1992) Aqueous humor hydrogen peroxide analysis with dichlorophenol-indophenol. Exp Eye Res 55:9-19 |
