Simultaneous quantitative determination of alloxan, GSH and GSSG by HPLC. Estimation of the frequency of redox cycling between alloxan and dialuric acid

This in vitro study compares the frequency of redox cycling between alloxan and dialuric acid at different initial ratios of glutathione and alloxan. Alloxan oxidizes GSH to GSSG. The rate of GSH oxidation at a given initial GSH concentration of 2.0 mmol/L depends on the initial concentration of all oxan added. The higher the concentration of alloxan in relation to the init ial concentration of GSH, the faster GSH oxidation proceeds, as well as oxy gen consumption, and therefore, formation of reactive oxygen species. The h ighest rates of GSH oxidation, i.e. GSSG formation, were found at concentra tion ratios of between 2.0 mmol/L GSH and 0.2 and 0.04 mmol/L alloxan, resp ectively. Because 0.04 mmol/L alloxan oxidizes 2.0 mmol/L GSH completely, a frequency of at least 25 cycles between alloxan and dialuric acid within 3 hours can be assumed. During each redox cycle, two molecules of GSH are ox idized to one molecule of GSSG, and during each cycle one molecule of oxyge n is reduced simultaneously to one molecule of hydrogen peroxide. In total, therefore. one molecule of alloxan oxidizes at least 50 molecules of GSH a nd forms about 25 molecules of hydrogen peroxide.