Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells

Abstract

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Figure 1

Reaction with and recovery of methylglyoxal from reduced glutathione. (A) Methylglyoxal (20 μM) was derivatized with o-PD (175 μM) in PCA (0.5 M). At each time, 2-MQ content was determined by HPLC analysis. (B) The methylglyoxal in an equilibrium mixture of reduced glutathione (2 mM) and methylglyoxal (20 μM) was derivatized with o-PD (175 μM) in PCA (0.5 M). At each time, 2-MQ content was determined by HPLC analysis. (C) An equilibrium mixture of reduced glutathione (2 mM) and methylglyoxal (20 μM) was incubated in PCA (0.5 M). At each time, the methylglyoxal-reduced glutathione adduct was removed by solid-phase extraction and the remaining free methylglyoxal was derivatization by o-PD (175 μM) for 2 h followed by 2-MQ determination by HPLC analysis.

Figure 2

Kinetics of methylglyoxal recovery from cell extracts in the presence of o-PD. (A) Samples were incubated with o-PD (200 μM) for the indicated times before HPLC analysis. Adjustment for nucleic acid degradation was done. After 24 h, this adjustment was equivalent to 12.5 μM methylglyoxal. (B) Control samples were incubated for the indicated times before the PCA precipitate was removed by centrifugation (12,000 × g, 10 min) and o-PD (200 μM) was added for 2 h to derivatize the free methylglyoxal that was present followed by HPLC analysis. Samples were adjusted for nucleic acid degradation. After 24 h, this adjustment was equivalent to 3.95 μM methylglyoxal. All samples were whole CHO cell extracts from a common source and were incubated at 20°C in PCA (0.45 M). Each data point represents mean ± σ (n = 2 or 3). (Inset) Initial recovery of methylglyoxal. The samples analyzed at t = 0 are equivalent to samples assayed with the standard assay.

Figure 3

Recovery of methylglyoxal from cell extracts at various concentrations of o-PD. (A) 2-MQ recovery. (B) 2,3-Dimethylquinoxaline recovery. 2,3-Dimethylquinoxaline is the o-PD derivative of 2,3-butanediol. All samples were whole CHO cell extracts from a common source. Samples were derivatized at 20°C in PCA (0.45 M) for 24 h with o-PD at the indicated concentrations. After 24 h, the samples were analyzed for 2-MQ and 2,3-dimethylquinoxaline content by HPLC. Each data point represents the mean ± 2σ (n = 6).