Glyoxalase- and methylglyoxal-related research has required the development of quantitative and reliable techniques for the measurement of methylglyoxal-derived glycation adducts of protein and DNA. There are also other glycation adducts, oxidation adducts and nitration adducts of proteins and oxidation adducts of DNA. Proteolysis of protein releases glycation, oxidation and nitration free adducts (glycated, oxidized and nitrated amino acids) in plasma and nuclease digestion of DNA releases glycated and oxidized nucleosides into plasma and other body fluids for excretion in urine. The gold standard method for quantifying these adducts is stable isotopic dilution analysis LC-MS/MS. Protein and DNA adduct residues are determined by assay of enzymatic hydrolysates of protein and DNA extracts prepared using cocktails of proteases and nucleases respectively. Free adducts are determined by analysis of ultrafiltrates of plasma, urine and other physiological fluids. Protein damage markers (13 glycation adducts, five oxidation adducts and 3-nitrotyrosine) and DNA damage markers (three glycation adducts and one oxidation adduct) are quantified using 25 μg of protein, 10 μg of DNA or 5 μl of physiological fluid. Protein and nucleotide AGE (advanced glycation end-product) assay protocols resistant to interferences is described.

The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure.

Novel sensing technologies for liquid biopsies offer promising prospects for the early detection of metabolic conditions through omics techniques. Indeed, high-field nuclear magnetic resonance (NMR) facilities are routinely used for metabolomics investigations on a range of biofluids in order to rapidly recognise unusual metabolic patterns in patients suffering from a range of diseases. However, these techniques are restricted by the prohibitively large size and cost of such facilities, suggesting a possible role for smaller, low-field NMR instruments in biofluid analysis. Herein we describe selected biomolecule validation on a low-field benchtop NMR spectrometer (60 MHz), and present an associated protocol for the analysis of biofluids on compact NMR instruments. We successfully detect common markers of diabetic control at low-to-medium concentrations through optimised experiments, including α-glucose (≤2.8 mmol/L) and acetone (25 µmol/L), and additionally in readily accessible biofluids, particularly human urine. We present a combined protocol for the analysis of these biofluids with low-field NMR spectrometers for metabolomics applications, and offer a perspective on the future of this technique appealing to ‘point-of-care’ applications.