Pro-oxidant : antioxidant balance in health and disease and the role of uric acid in antioxidant defence : analytical and clinical aspects

Abstract

The human body contains pro-oxidants and antioxidants work against these pro-oxidants. A balance between pro-oxidants and antioxidants is essential to determine the body status in terms of health and disease. Uric acid (or urate) has antioxidant properties in in vitro test systems, but may act in vivo as a pro-oxidant, depleting other antioxidants and increasing risk of disease. Allantoin is the product of non-enzymatic oxidation of urate, and measuring allantoin, or allantoin:urate ratio, may help assess in vivo pro-oxidant : antioxidant balance. The aims of this study were to develop, modify or evaluate tests of oxidant : antioxidant balance and to use these tests to assess and compare this balance in health and disease, make recommendation regarding the effect of anticoagulant used, storage condition on plasma for selected tests. In addition, the role of urate in antioxidant defence was explored. Laboratory techniques used in the study involved the FRAP/FRASC (ferric reducing antioxidant power /ascorbate) assay for total antioxidant power of plasma/ascorbate, the PeroXOquant and TBARSsf (thiobarbituric acid reacting substances measured by synchronous fluorimetry) tests for lipid peroxidation in plasma, HPLC-UV (high-performance liquid chromatography with ultraviolet detection) for plasma allantoin and urate, enzymatic method for urate in plasma and in urine, HPLC-UV for 帢-tocopherol, flow cytometry for membrane peroxidation in red blood cells (RBCs), comet assay for DMA damage of lymphocytes and other biochemical tests such as the assay for creatinine. Results indicate that ascorbate shows constant antioxidant efficiency in plasma, is less stable in plasma than in pure water, and higher temperature will increase the rate of degradation. Ascorbate is also less stable in EDTA than in heparinised plasma. Plasma total antioxidant power, measured by the FRAP assay, is greater in men than in women. Of the several tests modified and evaluated in this study, the PeroXOquant assay was found unreliable. Both ascorbate and 帢-tocopherol showed protection against membrane peroxidation of RBCs induced by cumene hydroperoxide (CumOOH). Apart from the antioxidative function, ascorbate and 帢-tocopherol also exhibited pro-oxidative property in the study of DNA damage initiated by hydrogen peroxide (H2O2). With specific reference to urate, this was found to protect against DNA damage initiated by H2O2 and CumOOH-mediated lipid peroxidation on RBC membranes. Excretion of urate in healthy subjects was not to change significantly over the day. Plasma allantoin was higher in subjects with diabetes mellitus or rheumatoid arthritis, supporting its use as an index of oxidative stress. In conclusion, measurement of ascorbate or total antioxidant power of body fluids should be performed as soon as possible after sampling (stored in ice-water or at 4 C) and heparin should be the anticoagulant of choice. All the in vitro findings of urate support its role as an important antioxidant in antioxidant defence, but further work is recommended with regard to the oxidation products of urate.