Evaluation of leucomethylene blue as a probe for assessing antioxidant activity reveals a potential application in the assessment of male fertility

Abstract

The oxidation of leucomethylene blue (LMB) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) are colorigenic reactions, generating methylene blue (M⁺) and the ABTS^•+ cation radical, respectively. In this study, we have analyzed the conditions under which these probes become oxidized and explored their application in the assessment of antioxidant activity. Using hematin as a catalyst, LMB responded to hydrogen peroxide and cumene hydroperoxide, whereas ABTS could only respond to the latter. However, in the presence of horse radish peroxidase, both probes responded specifically and dose-dependently to hydrogen peroxide. ABTS and LMB could also be oxidized in the anodic chamber of an electrochemical cell, permitting both pre-activation assays of free radical formation and post-activation assessments of free radical scavenging activity. In the pre-activation mode, both ABTS and LMB successfully revealed DMSO's capacity to inhibit free radical formation, in contrast to the ‘green’ solvent Cyrene™, which was virtually devoid of such activity. In the post-activation mode, the LMB oxidation product, methylene blue, was shown to be particularly sensitive to the 2-electron reducing properties of vitamin C. In contrast, ABTS responded more sensitively to compounds, like resveratrol, that used hydrogen atom transfer and one electron reduction to achieve their antioxidant action. Only LMB-based antioxidant assessments correlated with sperm motility (P < 0.001), suggesting this probe's sensitivity to 2-electron reduction may find particular application in diagnostic andrology. Evidently, not all redox sensors are created equal, so in future assessments of antioxidant activity, it will be important to match the chemistry of the probe with its analytical application.