Quantitation by Liquid Chromatography-Nanoelectrospray Ionization-High Resolution Tandem Mass Spectrometry of DNA Adducts Derived from Methyl Glyoxal and Carboxyethylating Agents in Leukocytes of Smokers and Non-smokers
A liquid chromatograpy-nanoelectrospray Ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method was developed for quantitation of the DNA adducts 7-(2\xe2\x80\x99-carboxyethyl)guanine (7\xe2\x80\x932\xe2\x80\x99-CEG) and N2-(1\xe2\x80\x99-carboxyethyl)guanine (N2-1\xe2\x80\x99-CEG), as their methyl esters, in human leukocyte DNA from smokers and non-smokers. 7\xe2\x80\x932\xe2\x80\x99-CEG has been previously identified in all human liver samples analyzed and is formed from an unknown carboxyethylating agent while N2-1\xe2\x80\x99-CEG is formed from the advanced glycation endproduct methyl glyoxal. The method was applied for the analysis of these two DNA adducts in leukocyte DNA from 20 smokers and 20 non-smokers, in part to test the hypothesis that 7\xe2\x80\x932\xe2\x80\x99-CEG could be formed by endogenous nitrosation, as previously observed in rats treated with nitrosodihydrouracil and nitrite. Levels of 7\xe2\x80\x932\xe2\x80\x99-CEG (mean \xc2\xb1 S.D.) were 0.6 \xc2\xb1 0.2 pmol/\xce\xbcmol dG in smokers and 0.5 \xc2\xb1 0.2 pmol/\xce\xbcmol dG in nonsmokers, while those of N2-1\xe2\x80\x99-CEG were 5.4 \xc2\xb1 1.9 pmol/\xce\xbcmol dG in smokers and 5.6 \xc2\xb1 2 pmol/\xce\xbcmol dG in non-smokers. These results did not support our hypothesis that endogenous nitrosation of dihydrouracil in smokers leads to higher levels of 7\xe2\x80\x932\xe2\x80\x99-CEG in leukocyte DNA than in non-smokers. However the study provides the first data on levels of these DNA adducts in human leukocyte DNA, and the LC-NSI-HRMS/MS method developed for their quantitation could be important for future studies of DNA damage by methyl glyoxal.
Introduction<!>Blood samples.<!>Chemicals, Enzymes, and Chromatography Supplies.<!>Sample Preparation for Analysis of 7\xe2\x80\x932\xe2\x80\x99-CEG and N2-1\xe2\x80\x99-CEG<!>Analysis of DNA for 7\xe2\x80\x932\xe2\x80\x99-CEG and N2-1\xe2\x80\x99-CEG.<!>Method characterization<!>Statistical analysis<!>Results<!>Discussion
<p>In 2019, more than 42,000 new cases of liver cancer and greater than 31,000 deaths from this disease were expected in the U.S. Known risk factors for liver cancer in the U.S. include excess body weight, type 2 diabetes, chronic infection with hepatitis B or C virus, heavy alcohol consumption, and tobacco smoking (https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2019/cancer-facts-and-figures-2019.pdf). Multiple studies have reported an association between cigarette smoking and liver cancer [1]. The association is plausible because cigarette smoke contains liver carcinogens such as N-nitrosodimethylamine, and there are other mechanistic pathways as well [1]. In the study reported here, we focus on the possible role of endogenous nitrosation and advanced glycation endproducts in DNA damage in smokers as compared to non-smokers. Endogenous nitrosation has been clearly demonstrated in cigarette smokers by analysis of urinary N-nitrosoproline and, as discussed below, relevant products formed by endogenous nitrosation induce liver cancer in rats [2].</p><p>In a previous study, we detected the DNA adduct 7-(2'-carboxyethyl)guanine (7–2'-CEG, 7, Scheme 1), as its methyl ester, in all 24 human liver samples analyzed, with a mean of 373 ± 320 fmol/μmol G (74.6 adducts per 109 nucleotides and a range of 17 – 1189 fmol/μmol G), but information on cigarette smoking in that study was unavailable [3]. Therefore, in the present study we have extended this research by analyzing leukocyte DNA from confirmed current cigarette smokers and non-smokers participating in an observational study.</p><p>One likely source of 7–2'-CEG was N-nitrosodihydrouracil (NDHU, 4, Scheme 1), a potent hepatocarcinogen in rats [4], which produced 7–2'-CEG in hydrolysates of rat hepatic DNA [5]. A plausible source of human exposure to NDHU is endogenous nitrosation of dihydrouracil (DHU, 2, Scheme 1). DHU is formed from uracil (1) as a normal intermediate in mammalian metabolism, in a reaction catalyzed by dihydropyrimidine dehydrogenase [6, 7]. DHU is excreted by healthy humans in urine, with a range of about 2–10 mg/day, and is also present in plasma [6–9]. The in vivo nitrosation of DHU was established by treatment of rats with DHU plus NaNO2 resulting in the formation of NDHU and significant increases of hepatic 7–2'-CEG [10]. As shown in Scheme 1, NDHU can be hydrolyzed in vivo giving N-nitroso-β-ureidopropionic acid (N-β-UPA, 5) which is further converted to the alkylating intermediate 2-carboxyethyldiazonium ion (6), which reacts with DNA to produce 7–2'-CEG [10]. Treatment of rats with NaNO2 plus β-UPA (3), a hydrolysis product of DHU (2), also resulted in significant increases of 7–2'-CEG in hepatic DNA [10]. Thus, in this study, we hypothesized that endogenous nitrosation of DHU or β-UPA in cigarette smokers could result in increased levels of 7–2'-CEG in leukocyte DNA.</p><p>Acrylic acid is another potential source of 7–2'-CEG [10]. It is a metabolite of acrolein, which is found in high concentrations in cigarette smoke [11]. Mercapturic acid metabolites of acrolein are 4–5 times higher in smokers than in non-smokers [12]. Although acrylic acid reacts only slowly with DNA in vitro to form 7–2'-CEG, we observed significant increases in this adduct in rats treated for 4 weeks with acrylic acid in the drinking water at concentrations of 1565 or 3130 ppm [10, 13].</p><p>Collectively, these results suggested that smokers could have higher levels of 7–2'-CEG than non-smokers due to endogenous nitrosation of DHU or β-UPA, or by reaction of acrylic acid with DNA. Therefore, we developed a liquid chromatograpy-nanoelectrospray Ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method for 7–2'-CEG, as its methyl ester, in leukocyte DNA from smokers and non-smokers. This analysis produced an unexpected result – substantially higher levels of N2-(1'-carboxyethyl)guanine (N2-1'-CEG), a DNA adduct formed from methyl glyoxal, as summarized in Scheme 2.</p><p>N2-1'-CEdG is a weakly mutagenic DNA adduct associated with advanced glycation end products which are involved in multiple disease states and can be affected by cigarette smoking [14–16]. Methyl glyoxal is the most potent glycation agent in the body and may play a role in cancer development [17–19]. Methyl glyoxal also has been reported as a component of tobacco smoke but quantitative data are lacking [20].</p><p>In this study, we have used LC-NSI-HRMS/MS to analyze 7–2'-CEG and N2-1'-CEG (which was formed from N2-1'-CEdG during analysis), as their methyl esters, in leukocyte DNA from smokers and non-smokers. We were mindful of the possibility of artefact formation in the analysis of N2-1'-CEG and took special precautions using aminoguanidine as a scavenger and purified deoxyguanosine (dG) as a monitor for artefact formation [21].</p><!><p>Blood was collected from smokers and non-smokers participating in an observational study carried out in Washington, DC and Minneapolis, MN. The study was approved by the Georgetown University Institutional Review Board and the University of Minnesota Institutional Review Board. The study had a repeated measures design to characterize three groups: current smokers, former smokers, and never smokers, at monthly intervals over 12 weeks. Volunteers were recruited from prior studies; local print, radio and internet advertisements; and snowball sampling. Samples from current smokers and never smokers were used here.</p><p>Eligible participants included adults (age 18–70 years) in good physical and mental health as evidenced by a medical history with no unstable medical conditions. Current smokers were required to have a stable smoking pattern of at least ten cigarettes per day and a smoking history of at least ten years. Never smokers were required to have smoked 100 cigarettes or less in their lifetime. Participants were excluded if they reported consuming >21 drinks per week or there was an active infection such as influenza, cold, or respiratory infection; current oral cavity or respiratory disease, such as cancer, chronic obstructive pulmonary disease, radiation therapy to the lung, or general anesthesia within six months; uncontrolled hypertension; treatment with nicotine replacement therapy within six months; treatment with antidepressants or drugs used for smoking cessation within six months; a psychiatric disorder or any other reason that precluded understanding of the informed consent; a pregnancy, plans to become pregnant in the next three months or currently breastfeeding; a reported clinical diagnosis of an immune system disease such as lupus, multiple sclerosis, rheumatoid arthritis, hepatitis or HIV; a reported diagnosis of cancer, past or present, excluding non-melanoma skin cancers; or a reported diagnosis of diabetes, kidney, or liver disease. In addition, smokers were excluded if they reported using any combination of other tobacco products, such as cigars, pipes or smokeless tobacco, more than three times per week in the past six months or marijuana use in the past six months. Never smokers were excluded if they used other tobacco products, such as cigars, pipes, smokeless tobacco, or marijuana in the last five years.</p><p>Participants attended an orientation visit and four clinical visits approximately four weeks apart (baseline, 4, 8, and 12 weeks). In addition to other biologic samples, a fasting blood sample (eight hours) was collected at each visit. Blood components were separated and stored in a −70⸰C freezer.</p><p>Leukocytes were isolated as described previously [22].</p><!><p>[15N]DNA was prepared according to Gouw et al [23]. 7-2′-CEG and [15N5]7-2′-CEG were synthesized as described previously [3]. N2-1′-CEdG and [15N513C10]N2-1′-CEdG were prepared essentially according to the methods of Synold et al [21] with modifications. Briefly, a solution of 50 mg of dG or 1 mg of [15N513C10]dG in 1ml of 1M phosphate buffer (pH 7) was incubated with 100 mg or 2 mg of dihydroxyacetone, respectively, at 70 0C for 24 h. The final products, N2-1'-CEdG and its corresponding isotopically labeled version [15N513C10]N2-1'-CEdG, were isolated and purified by HPLC using a Waters 510 instrument with a Shimadzu SPD-10A UV detector operated at 254 nm. A 250 × 4.6 mm, 5 μm, Supelco C18 column (Sigma-Aldrich, St. Louis, MO) was eluted with 95% 15 mM NH4OAc and 5% CH3CN at a flow rate of 1 ml/min. 1HNMR of N2-1'-CEdG (500 MHz, DMSO-d6) was as follows: δ 7.85 (s, 1H, 8-CH), 7.16 (d, J = 5.9 Hz, 1H, 2-NH), 6.13 (t, J = 7.1 Hz, 1H, dG-1'-CH), 4.35 (h, J = 2.6 Hz, 1H, dG-3'-CH), 3.98 (q, J = 6.7 Hz, 1H, 1'-CH(Me)COOH), 3.81 (qd, J = 4.8, 2.9, 2.0 Hz, 1H, dG-4'-CH), 3.57 (dd, J = 11.8, 5.1 Hz, 1H, dG-5'a-CH2), 3.48 (dd, J = 11.9, 5.1 Hz, 1H, dG-5'b-CH2), 2.66 (dt, J = 13.6, 6.8 Hz, 1H, dG-2'a-CH2), 2.19 (ddd, J = 12.4, 6.0, 2.7 Hz, 1H, dG-2'b-CH2), 1.32 (d, J = 6.9 Hz, 3H, 2'-CH3). MS of the standards was carried out on an Agilent ion trap instrument (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 1100 Capillary HPLC system equipped with a 150 × 0.5 mm, 5 μm, C18 Agilent Zorbax column. The column flow was 10 μL/min and a linear gradient of CH3CN in 5 mM NH4OAc was used with CH3CN increasing from 15% to 75% in 30 min, held at 75% for 5 min, followed by a 14 min re-equilibration at 15% CH3CN. The ESI source conditions were as follows: negative ion mode; capillary 3.1kv; nebulizer 10psi, dry gas 4 l/min, dry temperature 250 °C, skim −40v, cap exit −120v. The analysis of N2-1'-CEdG gave m/z 338 [M+H]+, m/z 294 [M+H –COO] + and m/z 178 [M+H -COO, dR] + while the analysis of [15N513C10]N2-1'-CEdG gave m/z 353 [M+H]+and m/z 308.9 [M+H –COO] + and m/z 187.8 [M+H -COO, -[13C5]dR] +.</p><p>Puregene DNA purification solutions, proteinase K and RNase A were procured from Qiagen (Valencia,CA).</p><p>Calf thymus DNA was purchased from Worthington Biochemical (Lakewood,NJ), 10K Amicom centrifuge filters were from Merck Millipore Ltd (Tullagreen, Carrigtwohill Co Cork IRL), and Oasis MCX 1 cc 30 mg LP extraction cartridges were procured from Waters (Milford, MA). Strata-X cartridges (33 μm, 30 mg/1 mL) were obtained from Phenomenex (Torrance, CA). Alkaline phosphatase was purchased from Roche Diagnostic Corp. (Indianapolis, IN). All other chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO).</p><!><p>DNA was isolated and purified from human buffy coat samples by following the Gentra Puregene protocol (Qiagen, Valencia, CA) with minor modifications. Briefly, buffy coat was treated with red blood cell lysis solution and then centrifuged. The supernatant was discarded and the cells were treated with cell lysis solution containing aminoguanidine (10 mM). After the samples were treated with RNase A and proteins were precipitated, DNA was precipitated with 100% 2-propanol, dried and then stored at −20 °C in sealed vials.</p><!><p>Enzymatic hydrolysis of DNA was performed by dissolving DNA (10–50 μg) in 0.4 mL of 10 mM PIPES/5 mM MgCl2 buffer (pH 7.0), 10 mM aminoguanidine, containing 130 fmol of [15N5]7–2'-CEG and 724 fmol of [15N513C10]N2-1'-CEdG. The solution was incubated for 70 min at 37 °C in the presence of 50 units of DNase I (type II, bovine pancreas), 0.002 units of phosphodiesterase I (type II, Crotalus adamanteus venom), and 25 units of alkaline phosphatase (calf intestine). The hydrolysate was filtered using 10K Amicon centrifuge filters to remove enzymes and residual proteins, and a 10 μL aliquot was set aside for dG quantitation. The hydrolysate was then purified using a mixed mode cation exchange (MCX Vac RC, 30 mg/1ml, Waters) cartridge. The cartridge was conditioned with 3 × 1 mL of CH3OH and 1 mL of 2% H3PO4. The sample was acidified with 4 μL of 86% H3PO4, loaded onto the cartridge, and washed with 1 mL of 0.1% H3PO4 and 1 mL of CH3OH. The analytes were eluted with 1 mL of 3% NH4OH in CH3OH. This fraction was collected and evaporated to dryness on a SpeedVac. Freshly prepared 10% CH3COCl in CH3OH (0.4 mL) was added to the dried vials, and the mixture was heated for 1 h at 50 °C and then concentrated to dryness. The residue was dissolved in 1 mL of H2O and purified using a Strata-X (33 μm, 30 mg/1 mL, Phenomenex) solid-phase extraction cartridge. The cartridge was conditioned with 3 × 1 mL of CH3OH and 1 mL of H2O. The sample was loaded on the cartridge which was washed with 3 × 1 mL of H2O and 1 mL of 10% CH3OH. The analytes were eluted with 1 mL of 80% CH3OH and the collected fraction was evaporated to dryness. The residue was dissolved in 50 μL of 5 mM NH4OAc buffer and 2 μL aliquots were analyzed by LC-NSI-HRMS/MS using a Dionex RSLCNano UPLC and a LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, Waltham, MA). Separation was performed with a nanoflow column (75 μm ID, 15 cm length, 10 μm orifice) created by hand-packing a commercially available fused-silica emitter (New Objective, Woburn MA) with 5 μm Luna C18 bonded separation media (Phenomenex, Torrance, CA). The column was eluted at 800 nL/min with 5% CH3CN in 5 mM NH4OAc for the first 5 min, then the flow was changed to 300 nL/min with a 15 min linear gradient from 15% to 45% CH3CN, followed by a 2 min hold at 90% CH3CN to wash the column before returning to the initial conditions. A 5 μl injection loop was used with injection volumes of 2 μL, and the injection valve was switched to the load position at 5 min to take the injection loop out of the flow path to minimize the delay time.</p><p>Positive mode ionization was performed using a Nanoflex nanospray ionization source (Thermo Scientific) with a voltage of 2.3 kV and an ion tube temperature of 350 °C. DNA adducts were quantified by HRMS/MS analysis of 7–2'-CEG and N2-1'-CEG methyl esters by monitoring transitions of m/z 238.1 → m/z 152.0565 and m/z 238.1 → m/z 178.0723, respectively, and transitions m/z 243.1 → m/z 157.0416 and m/z 248.1 → m/z 188.0743 for the internal standards [15N5]7–2′-CEG and [15N513C10]N2-1'-CEG, respectively. The analysis was performed by generating extracted ion chromatograms of the fragment ions at 5 ppm tolerance utilizing the Orbitrap detector. Fragmentation was performed using the HCD collision cell with 1 amu isolation width, collision energy of 50% and a resolution setting of 30,000 (at 400 amu) with an actual resolution of approximately 55,000 (at fragmentation m/z values). A full scan event was also performed over m/z 100 – 500 amu at a resolution setting of 30,000 to monitor background ion signal.</p><p>Quantitation of dG was performed using an Agilent 1100 capillary flow instrument with a diode array UV detector (Agilent Technologies, Palo Alto, CA) operated at 254 nm with a 250 × 0.5mm, 5 μm Luna C18 column (Phenomenex, Torrance, CA) eluted with 5mM NH4OAc/MeOH with a 5% to 20% MeOH gradient in 20 min.</p><!><p>To determine the accuracy of the quantitation of the adducts by LC-NSI-HRMS/MS and validate the assay, various amounts of 7–2'-CEG (5, 10, 20, 40, 80 fmol) were spiked into 50 μg of calf thymus DNA in PIPES buffer along with 260 fmol of [15N5]7–2'-CEG as internal standard. Three spiked samples at each concentration level were prepared and analyzed by LC-NSI-HRMS/MS following the sample work-up protocol described above. The concentration was calculated using a calibration curve constructed by varying the concentration of 7–2'-CEG (0.08 – 0.64 fmol on-column) and keeping the concentration of [15N5]7–2'-CEG constant (1 fmol on-column). Calf thymus DNA had high background levels of N2-1'-CEdG; therefore, in order to evaluate the accuracy and precision of the N2-1'-CEdG quantitation, bacterial [15N]DNA, with lower background levels of the adduct, was used for this experiment. Aliquots of 20 μg of [15N]DNA in PIPES buffer were spiked with 100, 200, 400, 800, 1600 fmol of N2-1'-CEdG and 1325 fmol [15N513C10] N2-1'-CEdG as internal standard, and subjected to the sample preparation procedures described above, prior to LC-NSI-HRMS/MS analysis. The concentration in each sample was calculated using a calibration curve constructed by varying the amounts of N2-1'-CEdG (2.5–30 fmol on-column) and keeping the concentration of [15N513C10]N2-1'-CEdG (30 fmol on-column) constant. Each concentration point of the calibration curve was dried and reconstituted in freshly prepared 10% CH3COCl in CH3OH (0.4 mL). The mixture was heated for 1 h at 50 °C and then concentrated to dryness. The residue was dissolved in 100 μL of H2O, to result in the corresponding guanine adducts. Background levels of both adducts in the accuracy experiment were determined by analysis of triplicate samples of calf thymus DNA or [15N]DNA and subtracted from each amount detected in the spiked samples.</p><!><p>Two sample t tests were performed to compare the adduct levels between smokers and nonsmokers, and between males and females. Statistical significance was set at p < 0.05.</p><!><p>The analytical method is outlined in Scheme 3. The internal standards for this analysis were [15N5]7–2'-CEG and [13C1015N5]N2-1'-CEdG. The DNA was hydrolyzed enzymatically and the adducts were enriched by solid phase extraction on a mixed mode cation exchange column. The fraction containing the adducts was esterified with acetyl chloride and methanol to produce the methyl esters of the guanine adducts as illustrated in Scheme 4. This step was efficient and necessary based on our previously validated analysis of 7–2'-CEG [3]. During the esterification, N2-1'-CEdG is converted to N2-1'-CEG, and, after further purification, both analytes were detected by LC-NSI-HRMS/MS as their methyl esters.</p><p>Tests of this method for artefactual formation of 7–2'-CEG and N2-1'-CEG were performed using pure dG and commercially purchased buffy coat. Aminoguanidine, 10 mM, was added as scavenger at various steps of the protocol and its influence on the adduct levels was evaluated. Addition of aminoguanidine prior to the extraction of DNA from buffy coat and during the enzyme hydrolysis of DNA resulted in a reduction of the levels of N2-1'-CEG by more than 95% while it did not affect the levels of 7–2'-CEG, as summarized in Table 1. Thus, aminoguanidine was used in the analytical protocol to scavenge methyl glyoxal during the DNA isolation and hydrolysis steps.</p><p>Typical LC-NSI-HRMS/MS chromatograms obtained upon analysis of a buffy coat DNA sample from a smoker are shown in Figure 1. Clear and convincing chromatograms were obtained for both analytes, as their methyl esters, monitoring the transitions m/z 238.1 [M + H]+ → m/z 152.0565 [BH]+ for 7–2'-CEG and m/z 238.1 [M + H]+ → m/z 178.0723 [M + H – C2H4O2]+ for N2-1'-CEG.</p><p>Accuracy and precision were determined by analysis of various amounts of N2-1'-CEdG and a constant amount of the internal standard added to [15N]DNA in the presence of 10 mM aminoguanidine,. A similar experiment was performed by adding known amounts of 7–2'-CEG and constant amounts of its internal standard to calf thymus DNA. As shown in Figure 2, a linear relationship was observed between added and measured 7–2'-CEG, and added N2-1'-CEdG and measured N2-1'-CEG, and the measured amounts agreed with the added amounts, as summarized in Table 2. For the N2-1'-CEG analysis, the average accuracy was 107% and precision was CV = 6%. The analysis of 7–2'-CEG was artefact free, and its accuracy and precision were consistent with what has been previously established using essentially the same analytical method as described here, with the exception that low resolution LC-ESI-MS/MS was used instead of LC-NSI-HRMS/MS [3].</p><p>Leukocyte DNA samples from 20 cigarette smokers (12 male) and 20 non-smokers (7 male) were analyzed. Current smoking status was confirmed by exhaled CO. Leukocyte DNA was isolated and hydrolyzed enzymatically, and the final fraction containing both adducts was analyzed by LC-NSI-HRMS/MS. All samples were positive for both DNA adducts. There was no significant difference in the adduct levels between smokers and non-smokers, or between males and females in each group. Levels of 7–2'-CEG (mean ± S.D.) were 0.6 ± 0.2 pmol/μmol dG in smokers and 0.5 ± 0.2 pmol/μmol dG in nonsmokers (p = 0.26), while those of N2-1'-CEG were 5.4 ± 1.9 pmol/μmol dG in smokers and 5.6 ± 2 pmol/μmol dG in non-smokers (p = 0.43). These results are presented in Figure 3.</p><!><p>The initial goal of this study was to quantify 7–2'-CEG in leukocyte DNA of confirmed cigarette smokers and non-smokers using a method which we had previously validated and extended to LC-NSI-HRMS/MS analysis. Previous studies have shown that nitrosation of orally administered proline takes place in smokers, presumably occurring in the stomach after inhalation of nitrogen oxides from smoke that are converted to nitrosating agents. We achieved our initial goal, but the results did not support our hypothesis that endogenous formation of NDHU or N-β-UPA occurs more readily in smokers than in non-smokers. There was no indication of a difference in 7–2'-CEG levels between smokers and non-smokers, suggesting that this DNA adduct, which was also found in all human hepatic DNA samples, is an endogenous adduct not related to cigarette smoking; its origins remain to be determined.</p><p>During the analysis, however, we observed much higher levels of N2-1'-CEG than its isomer 7–2'-CEG in leukocyte DNA. Previous studies have demonstrated that N2-1'-CEdG could be formed as an artefact from methyl glyoxal present in reagents [21, 24]. We applied the methodology used by Synold et al and Thornalley et al to control artefact formation with aminoguanidine, which scavenges methyl glyoxal [21, 24]. This resulted in a 35-fold decrease in leukocyte levels of N2-1'-CEG, as shown in Table 1, while not affecting levels of 7–2'-CEG, which is not derived from methyl glyoxal. With artefact formation inhibited, we were able to determine that levels of N2-1'-CEG in leukocyte DNA of smokers and non-smokers also did not differ. The amounts found - 5.4 ± 1.9 pmol/μmol dG in smokers and 5.6 ± 2 pmol/μmol dG in non-smokers – were approximately 10-fold higher than those of 7–2'-CEG, and similar to values reported previously using low resolution LC-MS/MS methods. Synold et al reported levels of N2-1'-CEdG ranging from 0.27 – 1.23 pmol/μmol dG in a human breast tumor and adjacent normal tissue [21]. Thornally et al reported 1.03 ± 0.38 N2-1'-CEdG adducts per 106 nucleotides in leukocytes from 3 healthy humans [24].</p><p>Although we did not find differences in levels of either DNA adduct in leukocyte DNA of smokers vs. non-smokers, we did successfully establish the first LC-NSI-HRMS/MS method for quantitation of N2-1'-CEG in DNA. This method, when combined with insights from the previous MS/MS methods noted above, is a state of the art technique for reliable measurement of this potentially important endogenous DNA adduct. The accuracy, precision, and applicability of this method to human leukocyte DNA were established in our study. Advanced glycation end products such as methyl glyoxal are implicated in a number of human diseases including type 2 diabetes, cardiovascular disease, cancer, and others [18]. The method reported here promises to be useful in further elucidation of the role of N2-1'-CEG in disease etiology related to methyl glyoxal.</p><p>This study confirms and extends our previous results on the presence of 7–2'-CEG in human DNA. The levels found here in leukocyte DNA, 0.6 ± 0.2 pmol/μmol dG in smokers and 0.5 ± 0.2 pmol/μmol dG in nonsmokers, were quite similar to that found in human hepatic DNA, 0.37 pmol/umol Gua, and to the levels found in rat hepatic DNA after treatment with only NaNO2 or DHU, 0.50 and 0.45 pmol/umol Gua, respectively [3, 10]. These results suggest that there is a steady state of 7–2'-CEG in human and rat DNA, resulting from endogenous processes which include formation, spontaneous loss, and repair of this adduct. While we have presented evidence that endogenous nitrosation of DHU or β-UPA as well as direct reaction of acrylic acid with DNA are plausible mechanisms as determined in laboratory studies with rats [10], further studies are required to investigate the source and mechanism of in vivo formation of 7–2'-CEG in human DNA.</p><p>DHU levels could have a significant effect on the yield of NDHU formed by endogenous nitrosation. A recent study has examined the effect of food consumption on uracil and DHU plasma levels as a biomarker for dihydropyrimidine dehydrogenase activity in humans [25]. This is important for individualizing fluoropyrimidine anticancer therapy. This randomized, cross over study of 16 healthy volunteers showed that DHU levels were influenced by food intake, with mean plasma levels of 147.0 ± 36.4 ng/ml in the fasting state and 85.7 ± 22.1 ng/ml (P<0.001) in the fed state. Timing of food consumption was not taken into account in the present study and could have influenced our results. DHU levels in the human stomach do not seem to have been reported.</p><p>In summary, we describe a new LC-NSI-HRMS/MS method for quantitation of N2-1'-CEG and 7–2'-CEG in human leukocyte DNA. This method will be useful for future studies investigating DNA adduct formation from methyl glyoxal. Quantitation of N2-1'-CEG and 7–2'-CEG in leukocyte DNA of smokers and non-smokers did not show any differences in the levels of these DNA adducts, indicating that cigarette smoking is not involved in their formation.</p>