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.
Highlights
Methylglyoxal (MG) is a highly reactive α-oxoaldehyde metabolite ubiquitous in all living organisms [1]
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
To prevent MG from forming advanced glycation end-products (AGEs), most organisms have a glyoxalase system, which in humans consist of two cytosolic enzymes; glyoxalase 1 (GLO1) and glyoxalase 2 (GLO2) [12]
Summary
Methylglyoxal (MG) is a highly reactive α-oxoaldehyde metabolite ubiquitous in all living organisms [1]. Elevated levels of MG has been linked to a vast amount of different ageing-related pathologies, including Alzheimer's and Parkinson's disease [6], diabetic complications [7], oxidative stress [8], and reduced longevity in simple organisms [9]. The toxicity of MG is primarily due to its ability to form advanced glycation end-products (AGEs); non-enzymatic post-translational protein modifications that may disrupt normal protein function [10,11]. The glyoxalase system is the primary pathway responsible for the detoxification of MG by converting it into non-toxic D-lactate using reduced gluthatione (GSH) as a cofactor [13,14]. While the glyoxalase system is the major detoxification pathway, AKR activity may be physiologically relevant as increased AKR activity e.g. have been linked to reduced diabetic complications [16]. As more than 99% of the MG in cells is estimated to be reversibly bound to thiols, primarily GSH, and a range
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