Abstract
This protocol describes a method for the detection and quantification of methylglyoxal (MG), the major physiological substrate of the cytosolic glyoxalase system. Accumulation of MG, also called dicarbonyl stress, is implicated in tissue damage in aging and disease. Measurement of MG is important in physiological studies, in the development of glyoxalase 1 (Glo1) inducer and inhibitor therapeutics, and in the characterization of medical products, especially dialysis fluids, and of thermally processed foods and beverages. MG can be derivatized with 1,2-diaminobenzene (DB), resulting in an adduct that can be detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification is achieved by stable isotopic dilution analysis with [(13)C3]MG. Pre-analytic processing at ambient temperature, under acidic conditions with peroxidase inhibition, avoids artifactual overestimation of MG. Estimates obtained from physiological samples can be validated by kinetic modeling of in situ rates of protein glycation by MG for confirmation of the results. This procedure was developed for the analysis of cultured cells, plasma and animal tissue samples, and it can also be used to analyze plant material. Experimental measurement requires 4.5 h for sample batch pre-analytic processing and 30 min per sample for LC-MS/MS analysis.
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