Quantitative determination of urinary N3-methyladenine by isotope-dilution LC–MS/MS with automated solid-phase extraction

Hu, Chiung‐Wen; Lin, Bo-Huei; Chao, Mu‐Rong

doi:10.1016/j.ijms.2010.04.003

Cited by 17 publications

(5 citation statements)

References 27 publications

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“…The urinary concentrations of 8-oxoGua and 8-oxodGuo were determined using a validated online SPE LC-MS/MS method as described by Hu et al The urinary concentrations of N3-MeA and N7-MeG were also measured using two different online SPE LC-MS/MS methods. , Representative online SPE LC-MS/MS chromatograms of 8-oxoGua, 8-oxodGuo, N7-MeG, and N3-MeA in urine are shown in Supporting Information, Figures S1–S3. All of the urine samples were also analyzed for creatinine using a validated HPLC-UV assay…”

Section: Methodsmentioning

confidence: 99%

Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions

Chao¹,

Hsu

Liu³

et al. 2015

Chem. Res. Toxicol.

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Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was <15%. The mean relative recoveries of 3-NTYR and NHPA in urine were 89-98% and 90-98%, respectively. We further applied this method to 65 urinary samples from healthy subjects. Urinary samples were also analyzed for N-nitrosodimethylamine (NDMA) as well as oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine (N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPA levels were measured at concentrations of 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels were highly correlated with each other and with 8-oxoGua and 8-oxodGuo. Our findings demonstrated that a relationship exists between oxidative and nitrative stress. However, 3-NTYR and NHPA were correlated with N7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may not be a representative biomarker of N-nitroso compounds that are induced by RNS.

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Section: Methodsmentioning

confidence: 99%

Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions

Chao¹,

Hsu

Liu³

et al. 2015

Chem. Res. Toxicol.

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“…Urinary N7-meG and N3-meA concentrations were determined, respectively, using two previously described methods of online SPE LC-MS/MS. , The detection limits of methods were 8.0 (4.8 fmol) and 35 pg/mL (11.7 fmol) on-column for N7-meG and N3-meA, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…This method was further applied to investigate the possible associations between urinary 5-meC (or 5-medC) and the methylated as well as oxidized DNA lesions. Urinary methylated DNA lesions including N7-meG and N3-methyladenine (N3-meA) and oxidized lesions including 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxodG were determined, respectively, using our previously reported online SPE LC-MS/MS methods. − …”

Section: Introductionmentioning

confidence: 99%

Direct Analysis of 5-Methylcytosine and 5-Methyl-2′-deoxycytidine in Human Urine by Isotope Dilution LC-MS/MS: Correlations with N-Methylated Purines and Oxidized DNA Lesions

Hu¹,

Liu²,

Li³

et al. 2012

Chem. Res. Toxicol.

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Recent evidence suggests that active DNA demethylation involves base excision repair (BER) and nucleotide excision repair (NER) pathways. We hypothesized that the resulting excision products could be further excreted and present in urine. A highly specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was first developed for simultaneously measuring urinary 5-methylcytosine (5-meC) and 5-methyl-2'-deoxycytidine (5-medC). With the use of isotope internal standards and online solid-phase extraction (SPE), the detection limits of 5-meC and 5-medC were estimated to be 1.2 and 0.3 pg, respectively. This method was applied to measure urinary samples of 376 healthy males. Urinary samples were also measured for methylated and oxidized DNA lesions, namely, N7-methylguanine (N7-meG), N3-methyladenine (N3-meA), 8-oxo-7,8-dihydroguanine (8-oxoGua), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using reported online SPE LC-MS/MS methods. Results showed that mean urinary levels of 5-meC and 5-medC were 28.4 ± 14.3 and 7.04 ± 7.2 ng/mg creatinine, respectively, supporting the possible presence of DNA demethylation through BER and NER mechanisms. Urinary levels of 5-meC were significantly positively correlated with N7-meG, N3-meA, and 8-oxodG. Good correlations between 5-meC and methylated and oxidized DNA lesions may have implied the underlying linkage between genetic (DNA lesions) and epigenetic (DNA methylation) alterations derived from exogenous exposure and/or from endogenous cellular processes in human and require further investigation.

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“…generated by 4-aminobiphenyl (10) and malondialdehyde (11). In the urine of smokers, N3-methyladenine (12)(13)(14)(15), N3-ethyladenine (12) and 1,N 6 -ethenoadenine and 3,N 4 -ethenocytosine adducts (16) derived from lipid peroxidation products were found.…”

Section: Introductionmentioning

confidence: 99%

Smoking-related O4-ethylthymidine formation in human lung tissue and comparisons with bulky DNA adducts

et al. 2011

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Tobacco smoke contains many alkylating agents that can react with DNA to produce O(4)-ethylthymidine (O(4)-etT) and several other types of promutagenic base modifications. Our aims were (i) to confirm results of a pilot study (Godschalk, R., Nair, J., Schooten, F. J., Risch, A., Drings, P., Kayser, K., Dienemann, H. and Bartsch, H. (2002) Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking. Carcinogenesis, 23, 2081-2086) on the formation of O(4)-etT in smokers' lung; (ii) to explore associations between levels of O(4)-etT and smoking status and (iii) to investigate whether a correlation exists between levels of O(4)-etT and bulky (polycyclic aromatic hydrocarbons-derived) DNA adducts. Archived DNA samples originated from histologically normal peripheral lung tissues of 64 Hungarian lung cancer patients, who underwent lung resection. O(4)-etT was determined by an immunoenriched (32)P-postlabelling-high-performance liquid chromatography method. Levels of bulky DNA adducts were determined by the nuclease P1 adduct-enriched (32)P-postlabelling. O(4)-etT levels ranged from 0.01 to 3.91 adducts/10(8) thymidines. In the combined group of subjects who smoked until surgery or gave up smoking at most 1 year before it, the mean level of O(4)-etT was 1.7-fold (P = 0.015) and of bulky DNA adducts 2.2-fold (P < 0.0001) higher than in long-term ex-smokers (LES) and never-smokers (NS) combined. We found no significant correlation between the individual levels of the two DNA adduct types. No dose-response was detected between O(4)-etT formation and smoking dose. In one-third of LES, O(4)-etT levels were above the 2.0-fold mean level of adducts found in NS, indicating its high persistence. Our results confirm the smoking-related formation of O(4)-etT in human lung DNA that should be explored as biomarker. Its long persistence in target tissue implicates a role of this potentially miscoding lesion in tobacco smoking-associated cancers.

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Quantitative determination of urinary N3-methyladenine by isotope-dilution LC–MS/MS with automated solid-phase extraction

Cited by 17 publications

References 27 publications

Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions

Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions

Direct Analysis of 5-Methylcytosine and 5-Methyl-2′-deoxycytidine in Human Urine by Isotope Dilution LC-MS/MS: Correlations with N-Methylated Purines and Oxidized DNA Lesions

Smoking-related O4-ethylthymidine formation in human lung tissue and comparisons with bulky DNA adducts

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