In spite of longstanding interest in the role of biopolymer glycation in human disease, no generally applicable method for the quantitative determination of CEdG has been described. A 32
P post-labeling assay has been used to estimate endogenous levels of CEdG in human buccal epithelial cells of 2–3/107
). However, although the post-labeling method offers potentially great advantages in sensitivity, a major drawback is that direct analyte verification is usually not possible. Moreover, post-labeling is prone to artifacts and false positives, and may lead to inaccurate estimation of adduct levels due to several factors including RNA contamination (29
). An immunoaffinity-based method for the detection of CEdG using a polyclonal antibody coupled to a diode array HPLC platform has more recently been described by the Pischetsreider group (30
). This approach was used to provide the first demonstration of CEdG in human urine and cultured smooth muscle cells. In some cases, peak identity was confirmed by LC-MS/MS, but quantitation was not practical owing to the imprecise nature of immunoaffinity chromatography. A monoclonal-based immunohistochemical detection method has also been reported and was used to demonstrate elevated levels of CEdG in aorta and kidney of diabetic patients relative to normal controls (31
). However, antibody-based assays are primarily of value in qualitative and comparative determinations of adduct abundance. For quantitative determinations LC-ESI-MS/MS in conjunction with the isotope dilution method is rapidly becoming the standard approach for the quantitative analysis of DNA adducts (32
). Although structural analyses of DNA-AGEs by LC-ESI-MS/MS have been previously described and recently reviewed (34
), to the best of our knowledge no reliable quantitative methods for CEdG measurement have appeared, presumably due to a lack of suitable isotopically enriched standards. To address this issue we synthesized 15
N-enriched isotopomers of CEdG differing from the unlabelled adducts by 5 amu, which provided sufficient mass resolution for accurate and reproducible quantitation using the stable isotope dilution method.
The ability to simultaneously resolve and quantitate both diastereomers of CEdG provided two independent parameters for assessing DNA glycation levels within a single sample. The biological significance of the CEdG diastereomer ratio in vivo
is presently unclear, but could reflect stereochemical biases in adduct repair or polymerase bypass. Examination of the CEdG stereoisomer distribution in vivo
by LC-ESI-MS/MS would only be meaningful if the rate of stereochemical interconversion was negligible. Superseding the issue of stereochemical integrity is that of overall adduct stability. Loss of the CEG base from either stereoisomer during workup would result in the generation of abasic sites leading to an underestimation of true nucleoside adduct levels. This was of particular concern since CEdG has been purported to undergo depurination more readily than dG at elevated temperatures (35
). We decided to quantify the extent of depurination and racemization by monitoring free base formation and isomer interconversion under acidic conditions at 37°C rather than at non-physiological temperatures. The data in suggest that the CEdG diastereomers possess similar stability, and are slightly more resistant to depurination under acidic conditions than dG. This fact, together with the prohibitive barrier to stereochemical interconversion, indicates that determination of CEdG diastereomer ratios may be plausibly used in quantitative biomarker studies.
One important confounding factor in the quantitation of adducts resulting from oxidative or oxoaldehyde DNA modification is artifactual product formation during sample isolation and workup. For example, the problems surrounding the measurement of 8-oxo-dG using GC-MS and/or mildly oxidizing workup conditions have been detailed previously (36
). In the case of CEdG adducts, the presence of MG during the workup could complicate the accurate determination of endogenous levels. The high background levels of CEdG detected in reagent grade calf thymus DNA prompted us to examine the effects of carbonyl scavenger addition prior to the enzymatic digestions. Scavengers such as AG and D-P react rapidly with MG and other oxoaldehydes to yield aminotriazines and thiazolidines respectively () which are relatively unreactive electrophiles (24
). D-penicillamine has been reported to react with MG 60χ faster than AG (24
), and thus may be more advantageous for CEdG determinations requiring DNA isolation from complex tissue matrices.
Reactions of carbonyl scavengers AG and D-P with MG yield isomeric aminotriazines and 2-acylthiazolidine, respectively.
We suggest that MG bound reversibly to proteins (23
) was predominantly responsible for the formation of DNA glycation artifacts observed during the isolation and workup of dsDNA. Extraction and workup procedures which expose DNA for extended periods to cell lysates and partially purified enzyme reagents increase the probability for the ex vivo
formation of CEdG, necessitating the need for carbonyl scavengers. This hypothesis is consistent with a previously published report showing that MG-BSA conjugates prepared by incubating MG with BSA can be used as reagents to induce DNA damage in cultured mammalian cells (39
).. The data in suggest that the addition of AG or D-P can largely eliminate artifactual CEdG formation. Minimizing exposure to proteins by shortening the enzymatic lysis and hydrolysis/dephosphorylation steps may also reduce the requirement for carbonyl scavengers.
We are currently examining a diverse array of tumor and corresponding control tissues in order to determine whether the trends noted in the breast cancer specimen are a general feature of tumors which display elevated levels of glycolysis. The finding of significantly lower CEdG in breast tumors relative to adjacent normal tissue can potentially be explained by the observation that glycolytic cancers possess lower levels of MG as a result of overexpression of the glyoxalase system. This highly evolutionarily conserved system consists of two non-homologous zinc metalloenzymes Glo1 and 2, which act sequentially to convert MG into lactate using reduced glutathione (GSH) as a catalytic cofactor (40
Glo1/2 are overexpressed ~ 3–5× in many breast cancers relative to normal mammary tissue, and enhanced expression of either one or both enzymes has also been observed in prostate, kidney, lung, colon, stomach, brain and ovarian cancers (42
). This is believed to be a metabolic adaptation to counter the pro-apoptotic effect of MG accumulation in glycolytic tumors. This phenomenon has formed the basis for the proposal to develop Glo1 inhibitors as cancer therapeutics (43
). One potential application of the quantitative LC-ESI-MS/MS method would be in monitoring the efficacy of glyoxalase inhibitors, which would be expected to induce a dose dependent increase in CEdG levels.
Methylglyoxal is a potent mutagen in mammalian cells, inducing G>T and G>C transversions, as well as a large number (50%) of multibase deletions (5
). Since 89% of the base substitution mutations are observed at guanosine, and CEdG is the predominant adduct formed from reaction of MG with DNA, it appears likely that this pattern of transversions arises from CEdG. Evidence for this has recently been obtained via primer extension assays using oligonucleotide templates containing CEdG. (45
). The presence of CEdG in DNA has also been shown to induce single-strand breaks, suggesting an alternative mechanism by which this adduct may contribute to genetic instability (46
Embryonic hyperglycemia has been shown to result in a high frequency of congenital malformations leading to perinatal death; and transgenic (lacI) mouse models have implicated a prominent role for DNA damage and mutagenesis in glucose-associated embryopathy (47
). Conditions which result in the impairment of glucose regulation such as diabetes and metabolic syndrome have been shown to significantly increase the risk for cancers of the breast, liver, pancreas, colon, cervix and endometrium (49
). Although the mechanistic reasons for this are as yet unclear, it is conceivable that genetic instability induced by CEdG may be involved.
The availability of a quantitative LC-MS/MS method for the measurement of CEdG complements methods currently available for protein AGEs (7
), and should allow for a more comprehensive evaluation of the role of nucleotide glycation in human metabolic disease.