Simultaneous Quantitative Determination of Alloxan, GSH and GSSG by HPLC. Estimation of the Frequency of Redox Cycling Between Alloxan and Dialuric Acid

Abstract

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.

Design of a Sensitive and Selective Voltammetric Sensor Based on a Cationic Surfactant-Modified Carbon Paste Electrode for the Determination of Alloxan

“… 2 , 3 However, the concurrent existence of reducing agents such as glutathione and AL, which creates a redox cycle where AL is reduced to dialuric acid, which then endures autoxidation back to AL in the presence of suitable electron acceptors such as oxygen. 4 As a ramification of this redox cycling, reactive oxygen species such as superoxide radical anion (O 2 – ), hydroxyl radical (OH), hydrogen peroxide (H 2 O 2 ), and the oxidized form of glutathione will be formed. Because the catalase activity is very low in pancreas, H 2 O 2 accumulates and in the existence of a ferrous ion chelate, Fenton-reaction takes place where extremely reactive hydroxyl radicals (OH) are formed.…”

“…It is assumed that because of the uptake of AL by pancreatic β-cells, it comes in contact with the reducing agent such as reduced glutathione, which is present in eukaryotic cells. Because of the interaction between AL and reducing species such as glutathione, reactive oxygen species will be formed, which can be assigned to the cytotoxic nature of AL potentially leading to selective necrosis of β-cells. , However, the concurrent existence of reducing agents such as glutathione and AL, which creates a redox cycle where AL is reduced to dialuric acid, which then endures autoxidation back to AL in the presence of suitable electron acceptors such as oxygen . As a ramification of this redox cycling, reactive oxygen species such as superoxide radical anion (O 2 – ), hydroxyl radical (OH), hydrogen peroxide (H 2 O 2 ), and the oxidized form of glutathione will be formed.…”

Electrochemical Detection of Alloxan on Reduced Graphene Oxide Modified Glassy Carbon Electrode

“…Many researchers understand the severity of the alloxan‐diabetes and discover several methods to detect the level of alloxan. Those methods are liquid chromatography with tandem mass spectrometry (LS‐MS‐MS) , high‐performance liquid chromatography (HPLC) , flow injection analysis (FIA) , and electrochemical techniques . Among these analytical methods, electrochemical techniques are simple, more efficient, low cost, high sensitivity, and easy to handling.…”