Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples

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.

Improved Glycemic Control and Vascular Function in Overweight and Obese Subjects by Glyoxalase 1 Inducer Formulation

“…Safety assessment of tRES-HESP coformulation was assessed by electrocardiogram and analysis of blood markers. Plasma methylglyoxal and glycation and oxidation adducts in plasma protein and urine (second void after overnight fast) were assayed by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) (14,15).…”

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cellsin vitro

“…The mESC media and hypoxic atmosphere were changed daily and mESCs were passaged every 2 -3 days when 80-90% confluent. To impose dicarbonyl stress on mESCs, we added exogenous high purity MG. High purity MG was prepared and purified as described [23]. To avoid phenotypic drift or differentiation of ESCs in culture, batches of new mESCs were revived from cryostorage every 3 months and used for experiments at ≤ passage 15. mESC viability was assessed as the percentage of cells that exclude Trypan blue and was determined after 3, 6 and 12 days of culture under 3% and 20 % oxygen atmosphere conditions.…”

“…Activities of Glo1 and Glo2 were assayed spectrophotometrically, Glo1 protein and Glo1 mRNA were assayed by Western blotting and RT-PCR, and MG, glycation adduct MG-H1 and creatinine were assayed by stable isotopic dilution analysis liquid chromatographytandem mass spectrometry (LC-MS/MS), as described [23,[27][28][29][30]. MG reductase activity was determined spectrophotometrically as described [31] with minor modification: using 1 mM MG as substrate and 50 mM sodium phosphate buffer, pH 7.4 at 37°C, as the assay buffer.…”

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

“…AGE Deposition on Extracellular Collagen Substrate-The unhydrated forms of MG are hydrophobic and thus can pass through membranes (50,51). In addition, extracellular deposition of MG and GO adducts contributes to tissue damage with the modified proteins having relatively long lifetimes (52).…”