Methylglyoxal (MG) is a sugar degradation product, which is endogenously formed by fragmentation of triose phosphates during glycolysis, ketone body metabolism of acetone, and catabolism of threonine. Food, beverages, and medical products are important exogenous sources with concentrations of up to 100 microM MG. MG is a reactive dicarbonyl compound, which easily modifies amino groups of proteins (glycation reaction) and thereby induces proinflammatory responses. Moreover, increased mutation frequencies in mammalian cells after treatment with MG have been reported, which are caused by stable modifications of DNA bases. Thus far, two types of adducts have been identified, which are formed during the reaction of free guanine or 2'-deoxyguanosine with high MG concentrations. In this study, we investigated the prolonged exposure of DNA to physiological MG concentrations. DNA was incubated with MG, enzymatically hydrolyzed to release the free nucleosides, and then analyzed by LC-MS/MS. We detected four products, which were derived from the reaction of 2'-deoxyguanosine and 2'-deoxyadenosine with 1 and 2 equiv of MG each. The adducts with 1 equiv of MG were identified as N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and N6-(1-carboxyethyl)-2'-deoxyadenosine. LC-MS/MS was optimized for these compounds, and incubation of DNA was repeated using physiological concentrations of 10 microM MG. Thereby, CEdG proved to be the most sensitive and suitable marker for the reaction of DNA with MG (negative MRM mode, three mass transitions [M - 1](-) 338-->178, 338-->106, and 338-->149).
Advanced glycation end‐products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2‐carboxyethyl‐2′‐deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)‐MS/MS. In resting cells, a 1.7‐fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose‐dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG‐modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.
Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.
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