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 alloxan added. The higher the concentration of alloxan in relation to the initial concentration of GSH, the faster GSH oxidation proceeds, as well as oxygen consumption, and therefore, formation of reactive oxygen species. The highest rates of GSH oxidation, i. e. GSSG formation, were found at concentration ratios of between 2.0 mmol/L GSH and 0.2 and 0.04 mmol/L alloxan, respectively. 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 oxidized to one molecule of GSSG, and during each cycle one molecule of oxygen is reduced simultaneously to one molecule of hydrogen peroxide. In total, therefore, one molecule of alloxan oxidizes at least 50 molecules of GSH and forms about 25 molecules of hydrogen peroxide.

Key words:

Alloxan - GSH - GSSG - HPLC - ROS

References

• 1 Grankvist K, Marklund S L, Sehlin J, Taljedahl I B. Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islets in vitro. .  Biochem J. 1979;  182 17-25
• 2 Munday R. Dialuric acid autoxidation. Effect of transition metals on the reaction rate and on the generation of “active oxygen” species.  Biochem Pharmacol. 1988;  37 409-413
• 3 Winterbourn C C, Munday R. Glutathione-mediated redox cycling of alloxan. Mechanism of SOD inhibition and metal-catalyzed OH^· formation.  Biochem Pharmacol. 1989;  38 271-277
• 4 Lenzen S, Munday R. Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin.  Biochem Pharmacol. 1991;  42 1385-1391
• 5 Brömme H J, Ebelt H, Peschke D, Peschke E. Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin.  Cell Mol Life Sci. 1999;  55 487-493
• 6 Kosower N S, Kosower E M. The glutathione status of cells.  Int Rev Cytol. 1978;  54 106-160
• 7 Halliwell B, Gutteridge J MC. Biologically relevant metal ion-dependent hydroxyl radical generation. An update.  FEBS Lett. 1992;  307 108-112
• 8 Brömme H J, Mörke W, Peschke E, Ebelt H, Peschke D. Scavenging effect of melatonin on hydroxyl radicals generated by alloxan.  J Pineal Res . 2000;  29 201-208
• 9 Patterson J W, Lazarow A, Levey S. Reactions of alloxan and dialuric acid with the sulfhydryl group.  J Biol Chem. 1949;  177 197-204
• 10 Halliwell B. Drug antioxidant effects. A basis for drug selection?.  Drugs. 1991;  42 569-605
• 11 De Vos A, Heimberg H, Quartier E, Huypens P, Bouwens L, Pipeleers D, Schuit F. Human and rat beta cell differ in glucose transporter but not in glucokinase gene expression.  J Clin Invest. 1995;  96 2489-2495
• 12 Johnson J H, Newgard C B, Milburn J L, Lodish H F, Thorens B. The high K_m glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence.  J Biol Chem. 1990;  265 6548-6551
• 13 Malaisse W J, Malaisse-Lagae F, Sener A, Pipeleers D G. Determinants of the selective toxicity of alloxan to pancreatic B cell.  Proc Natl Acad Sci USA. 1982;  79 927-930
• 14 Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues.  Free Radic Biol Med. 1996;  20 463-466
• 15 Frerichs H, Creutzfeldt W. Der experimentelle chemische Diabetes. In: Dörtzbach H (ed). Handbuch der experimentellen Pharmakologie, Vol. 32/1, Insulin 1.  Berlin:; Springer, 1971: 159-202
• 16 Bijsterbosch M K, Duursma A M, Bouma J MW, Gruber M. The plasma volume of the Wistar rat in relation to the body weight.  Experientia. 1981;  37 381-382
• 17 Navarro J, Obrador E, Pellicer J A, Aseni M, Vina J, Estrela J M. Blood glutathione as an index of radiation-induced oxidative stress in mice and humans.  Free Radic Biol Med. 1997;  22 1203-1209
• 18 Resnik R A, Wolff A R. The reaction of alloxan with glutathione and protein.  Arch Biochem Biophys. 1956;  64 33-50
• 19 Nishikawa M, Sato E F, Utsumi K, Inoue M. Oxygen-dependent regulation of energy metabolism in ascites tumor cells by nitric oxide.  Cancer Res. 1996;  56 4535-4540
• 20 Richter C. Reactive oxygen and nitrogen species regulate mitochondrial Ca²⁺ homeostasis and respiration.  Biosci Reports. 1997;  17 53-66
• 21 Ross D A, Jackson R C, Weber G, Morris H P. Decreased content of reduced and oxidized nicotinamide-adenine dinucleotide phosphate in rat hepatomas.  Cancer Biochem Biophys. 1982;  6 61-64