ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures

Abstract

Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

Dicarbonyl Stress at the Crossroads of Healthy and Unhealthy Aging

“…A similar increase was confirmed in a later study in fasting serum samples from healthy and T2D patients, by the gas chromatography–mass spectrometry (GC/MS) methodology [88]. Although LC/MS is considered the current golden standard method for MGO detection, an ELISA-based assay has just been tested and validated for quantifying MGO levels in both plasma and cell culture [89], offering a new operationally simple screening tool. A more recent investigation performed in “newly diagnosed” T2D patients demonstrated a significant 1.62 fold increase of MGO plasma levels already at early stage of diabetes in the absence of diabetic complications [90].…”

A Ratiometric Fluorescence Probe for Selective Detection of ex vivo Methylglyoxal in Diabetic Mice

“…The measurement results were not significantly different from the proposed method, indicating the authenticity of the MGO content, and it was consistent with the reported results. [40] Therefore, the DAN‐based ratiometric probe can be considered as one of the potential methods for determining MGO in biological samples.…”

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS

“…It is well known that o-phenylenediamine (OPD) is the most commonly used derivatization reagent for quantitative α-dicarbonyl compounds [113][114][115][116]. OPD reacts with α-dicarbonyl compounds, such as methylglyoxal, to produce stable 2-methylquinoxaline with high MS detection sensitivity [117]. However, when it is used to determine the content of α-dicarbonyl compounds in biological samples with high levels of sugar, such as honey, OPD may undergo Maillard reaction with reducing sugars to form α-dicarbonyl compounds, thereby, affecting quantification.…”

“…As compared with the widely used DNPH, MPIA derivatives show higher sensitivity, which is probably attributed to the strong proton affinity of the lone pairs on imidazole nitrogen atom and the high hydrophobicity of MPIA derivatives.Different from monoamines, diamines react with α-dicarbonyl compounds to form cyclic compounds, so that a single derivatized product can be obtained without a reducing reagent.It is well known that o-phenylenediamine (OPD) is the most commonly used derivatization reagent for quantitative α-dicarbonyl compounds[113][114][115][116]. OPD reacts with α-dicarbonyl compounds, such as methylglyoxal, to produce stable 2-methylquinoxaline with high MS detection sensitivity[117]. However, when it is used to determine the content of α-dicarbonyl compounds in biological samples with high levels of sugar, such as honey, OPD may undergo Maillard reaction with reducing sugars to form α-dicarbonyl compounds, thereby, affecting quantification.…”