Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry.

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N=3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 +/- 11.0 (mean +/- S.D.) in 10(5) tyrosine and 4.4 +/- 3.9 (mean +/- S.D.) in 10(3) tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R(2) = 0.55, p = 0.0065, n=23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.

Investigation of DNA-protein cross-link formation between lysozyme and oxanine by mass spectrometry.

Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion product originating from the guanine base through exposure to nitric oxide, nitrous acid, or N-nitrosoindoles. Oxanine was found to mediate formation of DNA-protein cross-links (DPCs) in the cell extract. We have previously characterized two DNA-protein cross-links from the reaction between Oxa and glutathione: namely, the thioester and the amide. In this study, lysozyme was used to study site-specific modification on protein by Oxa moieties in DNA. With the aid of nanoLC coupled with nanospray ionization tandem mass spectrometry, addition of Oxa was found at Lys13, Lys97, Lys116, Ser85, and Ser86 of lysozyme when it was treated with 2'-deoxyoxanosine (dOxo). Furthermore, incubation of lysozyme with Oxa-containing calf thymus DNA, produced by treating DNA with nitrous acid, led to lysozyme modification at Lys116, Ser85, and Ser86. Interestingly, none of the cysteine residues was modified by dOxo, in contrast with our previous findings that dOxo reacted with oxidized glutathione disulfide, forming the thioester. This might be due to the half-life of the dOxo-derived thioester being 2.2 days at the pH of incubation. Furthermore, the sites of modifications on lysozyme are in good agreement with the solvent accessibility of the residues. Since repair of Oxa-derived DPCs has not been extensively investigated, these results suggest that these stable DPCs might represent important forms of cellular damage caused by reactive nitrogen species involved in inflammationrelated diseases.

Association between cigarette smoking and urinary excretion of 1,N2-ethenoguanine measured by isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry.

Levels of the promutagenic 1,N2-ethenoguanine (1,N2-epsilonGua), an etheno DNA adduct derived mainly from lipid peroxidation, in experimental animals are associated with risk of cancer formation. Since 1,N2-epsilonGua can be repaired by human glycosylases, it is possible to use it as a biomarker for cancer risk assessment in humans. In the present study, a highly sensitive and specific stable isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) was developed for accurate quantification of 1,N2-epsilonGua in human urine. The sample pretreatment involved a consecutive strong cation exchange solid-phase extraction (SPE) and reversed phase SPE chromatography. The pretreated sample was analyzed by LC-ESI/MS/MS under multiple reaction monitoring mode (MRM) using a triple quadrupole mass spectrometer. The detection limit of 1,N2-epsilonGua using this LC-ESI/MS/MS assay was 1.0 pg (5.8 fmol) injected on-column. Levels of urine samples collected from healthy volunteers were found to range from 0 to 199 pg/mL, and levels as low as 5.0 pg/mL (29 pM) could be accurately quantified. After adjusting for creatinine levels and body weight, an statistically significant association was observed between urinary levels of 1,N2-epsilonGua and cigarette smoking (p = 0.0006). This highly specific and sensitive assay should be valuable in measuring urinary 1,N2-epsilonGua as a potential noninvasive biomarker for oxidative DNA damage.

Detection and quantification of 1,N6-ethenoadenine in human urine by stable isotope dilution capillary gas chromatography/negative ion chemical ionization/mass spectrometry.

1,N(6)-Ethenoadenine (epsilonAde) is a promutagenic lesion detected in tissue DNA; it has been shown that epsilonAde can be repaired by human DNA glycosylases, and it is expected to be excreted in urine. In this paper, we present for the first time detection and accurate quantification of epsilonAde in human urine samples by a highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS). Analysis by GC/NICI/MS includes adduct enrichment by a solid phase extraction column, followed by electrophore labeling and postderivatization cleanup. Using selective ion monitoring mode, the assay allows quantification of 0.5 pg of epsilonAde in as little as 0.1 mL of the urine sample, which is equivalent to corresponding concentration quantification limit of 31 pM. Using this assay, concentrations of epsilonAde in the 24 h urine samples of 23 healthy individuals were determined, which ranged from 0 to 124 pg/mL. After we adjusted for creatinine, a statistically significant correlation was found between epsilonAde excretion and cigarette smoking in males (p = 0.03). Thus, this stable isotope dilution GC/NICI/MS assay offers a sensitive and accurate quantification of urinary epsilonAde as a potential biomarker for oxidative damage of DNA and repair.

Effect of cigarette smoking on urinary 3,N4-ethenocytosine levels measured by gas chromatography/mass spectrometry.

Etheno DNA adducts are DNA damages derived from exogenous carcinogens as well as endogenous lipid peroxidation and oxidative stress. Elevated levels of etheno DNA adducts were found in cancer-prone tissues and blood samples, suggesting that these promutagenic lesions correlate with risk of cancers. We previously reported the detection of 3,N4-ethenocytosine (epsilon Cyt) in the urine samples of two smokers using the isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay (Chen et al., 2001, Chem. Res. Toxicol. 14, 1612-1619). Since smokers are found to have elevated levels of lipid peroxidation and oxidative stress, we examined the association between urinary epsilon Cyt levels with cigarette smoking. Among the 23 samples analyzed, the average concentration of urinary epsilon Cyt in smokers was significantly higher than that of nonsmokers, 2.65 +/- 4.0 versus 0.61 +/- 0.90 ng/kg/g creatinine (p= 0.03). Albeit the number of subjects is limited, the results indicate that the measurement of epsilon Cyt in human urine may provide a useful noninvasive biomarker for oxidative DNA damage and cancer chemoprevention studies.