Spectrophotometric Assay for the Quantification of Plasma Ethanol Levels in Mice through Chromium-Ethanol Oxidation-Reduction Reaction

Abstract

The quantification of blood/plasma ethanol concentration (BEC/PEC) is of great importance in experiments involving basic research, clinical studies, and bioethanol production. Traditional methods commonly used to measure BEC can be expensive and require high-cost equipment and qualified labor. The aim of this study was to develop a low-cost method that can be performed with simple infrastructure commonly available in research laboratories. For this, we developed a protocol to quantify PEC in mice, using the method of reduction of potassium dichromate by ethanol. However, this oxidation-reduction (redox) reaction is not specific to ethanol. Thus, the PEC was measured following a sequence of chemical reactions to eliminate the reductive interfering substances presented in the samples. Firstly, we evaluated the sensitivity of the dichromate reactive to ethanol and to different reducing substances found in the plasma, in order to determine which the main interfering substances are. Next, once the main interfering substances were determined in the dichromate reduction, plasma was assayed for PEC. First, mice received intraperitoneally (i.p.) saline (basal reading, 0% ethanol) or ethanol injections (0.5, 1, 2, 3, and 4 g/kg) and had their plasma collected. After plasma deproteinization and plasma glucose oxidation, it was mixed with the dichromate/acetic acid reactive, and then the products of the redox reaction were determined by the spectrophotometric method. Then, we determined the PEC with the same plasma samples using a commercial ethanol assay kit as a positive control. We found an excellent correlation between the administered ethanol doses and PECs in both the methods analyzed. The values of PEC found in the dichromate reaction method were similar to those obtained in the literature with the same ethanol doses, and to the commercial enzyme activity assay. Therefore, despite the need for a background reading, this method can be successfully applied to determine PEC using low-cost chemical reagents.