Determination of urinary glyoxal and methylglyoxal by high-performance liquid chromatography

Abstract

Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase high-performance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100-120 mOsm/kg H2O) under acidic conditions (pH 4.5) at 60 degrees C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within- and between-day relative standard deviations of less than 5%. Urinary levels (mean +/- standard deviation, n = 16) of glyoxal and methylglyoxal in healthy subjects were 4.7 +/- 1.35 microg/mg creatinine, 2.2 +/- 0.65 microg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.

Capillary gas chromatographic determination of methylglyoxal from serum of diabetic patients by precolumn derivatization with 1,2-diamonopropane

“…Various reactive carbonyl compounds with low molecular weight have been proposed to play an important role in pathogenesis of many chronic and age related diseases as carbonyl stress [1,2]. Chronic hyperglycemia leading to tissue damage is the primary etiological factor in the pathogenesis of diabetic macrovascular disease and may contribute to macrovascular complications [3][4][5].…”

Methylglyoxal in food and living organisms

“…Most IS used in the published methods are structurally related to MG such as 2,3-butanedione [26,29,33], 2,3-pentanedione [34], 3,4-hexanedione [35], or 2,3-hexanedione [36,37]. These IS can be added to the sample as a nonderivatized compound or as a compound previously derivatized with corresponding DA.…”

“…Therefore, the incubations had to be performed in the dark and even in the atmosphere of nitrogen. It has been shown that some com-pounds that stabilize diamino derivatives (such as b-mercaptoethanol [40]) significantly decrease MG level [26], and that even the osmolality of incubation mixtures has the same effect on the derivatization process [34].…”

“…When the amount of MG is very low, preliminary concentration of the sample is necessary. This can be performed by liquid-liquid [34,35,38,39] or SPE [29,32,33,36] with additional evaporation of organic solvent or by freeze drying of samples deproteinized by TFA [26]. HPLC analysis was usually performed on the RP HPLC columns which were eluted with water solutions of acids or buffers in combinations with methanol or ACN under the gradient or isocratic conditions, while GC analysis was performed on fused-silica capillary columns with He as carrier gas.…”

“…The methods of MG quantification in human and/or mouse whole blood [29,33,36,40], human plasma [26,35,38], human urine [34,43], yeast cell culture [33], Chinese hamster ovary (CHO) cells [32], rat heart, liver, kidney, and aorta [36,39] are presented in Table 1. As body fluids and tissues (except for urine) have a high content of protein, deproteinization is an unavoidable step in sample processing and all the above-mentioned facts connected with deproteinization should be considered.…”