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Accepted Manuscript Title: Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS Authors: Danni Wu, Baodong Liu, Junfa Yin, Tian Xu, Shuli Zhao, Qun Xu, Xi Chen, Hailin Wang PII: DOI: Reference:
S1570-0232(17)30729-8 http://dx.doi.org/10.1016/j.jchromb.2017.08.033 CHROMB 20766
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
23-4-2017 28-7-2017 23-8-2017
Please cite this article as: Danni Wu, Baodong Liu, Junfa Yin, Tian Xu, Shuli Zhao, Qun Xu, Xi Chen, Hailin Wang, Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.08.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS Danni Wu1,5, Baodong Liu1, Junfa Yin1, Tian Xu1, Shuli Zhao2, Qun Xu3, Xi Chen4 and Hailin Wang1,5*
1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China 2. China National Environmental Monitoring Center, Beijing, 100012, China 3. Institute of Basis Medical Sciences, Chinese Academy of Medical Sciences, School of Basis Medicine, Peking Union Medical College, Beijing, 100005, China 4. Institute of Environmental Health & Related Products Safety, Chinese Center for Disease Control and Prevention, Beijing, 102206, China 5. University of Chinese Academy of Sciences, Beijing, 100049, China
*
Corresponding Author: Prof. Hailin Wang,
Telephone/Fax:+86-10-62849600, E-mail:[email protected]
Highlights
An UHPLC-MS/MS method was developed for accurate detection of 8-OHdG in leukocyte. 8-OHdG in leukocyte but urine can indicate oxidative DNA damage. Higher levels of leukocyte 8-OHdG were found in cancer patients over health control.
Abstract 8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC-MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1×100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [15N5]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0-100 nM with R2 = 0.999. The accuracy for spiked samples was 90.9% - 94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC-MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). Keywords: Ultra-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS); 8-hydroxydeoxyguanosine (8-OHdG); peripheral blood leukocyte; oxidative DNA damage
1. Introduction Organisms often live with abundant endogenous or exogenous reactive oxygen species (ROS). ROS can be generated from multiple biochemical reactions during various physiological processes such as substance and energy metabolism [1, 2], but also caused by the exposure to exogenous toxic chemicals and pollutants, including tobacco smoke, heavy metal and polycyclic aromatic hydrocarbons [3-5]. Thus, a multitude of oxidative DNA damages are induced by ROS attacking DNA molecules, including DNA bases and sugar modifications, covalent crosslinking, and single- and double-stranded breaks [6, 7]. Among these lesions, 8-OHdG is the predominant and most abundant oxidative product formed in nuclear and mitochondrial DNA. Although numerous DNA enzymatic repair and non-enzymatic antioxidant defenses in organisms to maintain genomic stability, the unrepaired 8-OHdG in DNA can result in G:T transversion and further initiate carcinogenesis [8, 9]. It has proposed that oxidative damage to DNA induced several diseases, including aging [10], cancer [11, 12], neurodegenerative diseases [13], diabetes [14], cardiomyopathy [15] and cardiovascular or infectious diseases [16]. Therefore, 8-OHdG is not only an indicator of endogenous oxidative DNA damage, but also useful for early diagnosis and assessment of high-risk individuals [17]. Peripheral blood leukocytes are the most commonly used cells to assess human disease states [18]. Leukocyte 8-OHdG is an indicator of oxidative stress, as well as impaired metabolism and mitochondrial dysfunction [13], which is related to cancer progression. Moreover, 8-OHdG in leukocyte genomic DNA is associated with mutation and cancer risk, which hardly affected by tumor necrosis and inflammatory [19]. On the other hand, an increase of 8-OHdG may not only because of a rise in oxidative DNA damage, but also because of a decline in the repair rate. A quantitative deficit in DNA repair system may promote an accumulation of 8-OHdG in leukocyte DNA, while the excreted 8-OHdG in urine and serum may have little change [20]. Consequently, 8-OHdG in leukocyte DNA is more accurate and sensitive than serum and urinary 8-OHdG. However, the 8-OHdG content in peripheral blood leukocyte
DNA was only reported in a few of papers [21, 22], which probably due to the problems of sample collection and storage, DNA extraction and separation of hydrolytic enzymes. Although 8-OHdG in urine has been well studied [23], because it is noninvasive and technically less involved, the level of 8-OHdG in peripheral blood leukocytes probably reflect a long term response to oxidative stress. Additionally, urinary creatinine concentration is required to normalize urine volume for urinary 8-OHdG calibration [16]. A variety of biochemical and chemical assays have been developed for 8-OHdG analysis, including enzyme-linked immunosorbent assay (ELISA) [24], 32P post-labeling [25], capillary electrophoresis-ultraviolet detection (CE-UV) [26], high-performance liquid chromatography-electrochemical detection (HPLC-ECD) [27], gas chromatography-mass spectrometry (GC-MS) [28], liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [29-31] and liquid chromatography-single mass spectrometry (HPLC-MS) [32, 33]. HPLC-ECD is frequently utilized, however, complicated HPLC system with column-switching [34] or SPE [35] is necessary to eliminate possible interference from various sample matrix. For GC-MS, the 8-OHdG derivatization is essential to provide adequate vaporation to enter gas phase before analysis, which might lead into possible “artificial” oxidation of nucleosides [36, 37]. Mass spectrometry detection is currently the best approach, with the possibility of using isotopically labeled internal standards that can correct for biased MS response during work-up [36]. The main advantage of HPLC-MS/MS in detection of 8-OHdG is the increase in sensitivity compared with HPLC-MS, since the use of multiple reaction monitoring (MRM) mode leads to a considerable reduction in the background ions-caused noise [38]. The chromatographic method for measuring 8-OHdG in DNA requires chemical hydrolysis or enzymatic digestion following extraction from cells [39]. To reduce artifactual oxidation, a three-enzyme cascade capillary monolithic bioreactor was fabricated for rapid digestion of genomic DNA into single nucleosides [40]. By the use of the immobilized enzymes, the collected products were directly injected into HPLC-MS/MS for detection without separation process. As a result, HPLC-MS/MS is
a powerful technique that has exhibited good selectivity and sensitivity for 8-OHdG analysis. In this study, a robust and sensitive UHPLC-MS/MS method was developed for 8-OHdG analysis in leukocyte DNA. Of note, [15N5]-8-OHdG was used as internal standard (IS). High sensitivity, good precision and reproducibility were validated. The proposed method was further applied to analyze 8-OHdG in healthy volunteers and cancer patients (n = 121).
2. Materials and methods 2.1 Chemicals and Reagents 8-OHdG standard and deferoxamine mesylate (DFO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Wizard® Genomic DNA Purification Kit(Promega, Madison, WI) was used for DNA extraction. Acetic acid was purchased from Fluka Analytical (Steinheim, Germany). DNase I and calf intestinal phosphatase (CIP) were obtained from New England Biolabs (MA, USA). The HPLC grade methanol was purchased from Fisher Scientific Inc. (Waltham, USA).All other chemicals used were of highest purity and purchased from Sigma-Aldrich. An ELGA WaterLab water purification system was used to provide 18.2MΩ ultrapure water throughout the experiments.
2.2 UHPLC-MS/MS Conditions Prior to 8-OHdG analysis, 50 fmol of [15N5]-8-OHdG stable isotope internal standard was added to 5 µg DNA enzymatic digestions. The ultra-performance liquid chromatography was carried out on an Agilent 1290 HPLC system equipped with G6410B triple quadrupole mass spectrometer. Electrospray ionization was set in positive ion mode, and the data was acquired in MRM mode. The column used was a Zorbax Eclipse Plus C18 (2.1×100 mm，1.8 μm) obtained from Agilent, and a guard column Zorbax Eclipse Plus C18 Narrow Bore Guard Column (2.1×12.5 mm) was used to protect the analytical column. The mobile phase consisted of 92% elution A
(ultrapure water containing 0.1% acetic acid) and 8% elution B (methanol). The flow rate was 250 µl/min. The column temperature was kept at 25ºC. 2.3 Sample Collection Total 121 blood samples were collected from the hospital, including 46 cancer patients (28 males and 18 females) and 75 healthy volunteers (36 males and 39 females). All the subjects were aged form 18 to 75. Blood samples were withdrawn into EDTA-2Na-containingvessels, and immediately frozen at -20ºC. The study was approved by the Local Ethics Committee.
2.4 DNA isolation and digestion An aliquot of 600 μl whole blood was subjected to Wizard® Genomic DNA Purification Kit following the provided protocol of the manufacturer. Briefly, 800 μl of the cell lysis solution was added to lyses the red blood cells, followed by lysis of the white blood cells and their nuclei in nuclei lysis solution. 1.5 μl RNase solution was added to each sample, and incubated at 37ºC for 15 minutes. The cellular proteins were then removed by addition of 200 μl protein precipitation solution. Finally, the genomic DNA was concentrated and desalted by isopropanol precipitation. Next, the isolated DNA (5 µg) was digested by 1.0 U DNase I, 0.005 U snake venom phosphodiesteraseand 2.0 U calf intestinal phosphatase at 37ºC overnight. 100 µM DFO was also added to reduce artifactual formation of 8-OHdG during digestion process. Then, the enzymes were removed by ultrafiltration tubes with molecule weight cutoff at 3KDa. 2.5 Method validation Linearity was assessed by calibration curve. A series of standard 8-OHdG samples at concentrations of 1, 2, 5, 10, 50 and 100 nM were spiked with 50 fmol of [15N5]-8-OHdG IS. Calibration curve was established by plotting the peak area ratios of 8-OHdG to IS (Y-axis) versus the concentrations (X-axis), and square regression coefficient (R2) was applied to evaluate the linear regression equation. The limit of detection (signal-to-noise ≥ 3) and limit of quantification (signal-to-noise ≥ 10) were
measured on successive aqueous dilutions of standard 8-OHdG solution.The accuracy and precision were evaluated using a digested DNA sample spiked with standard 8-OHdG (low, middle and high concentration) for three replicate samples at each level. Recovery was calculated according to the following equation: Recovery (%) = [(final concentration-original concentration)/ spiked concentration] × 100%. Relative standard deviation (RSD) was used to assess the precision.
2.6 Measurement of urinary 8-OHdG by ELISA Urine samples were thawed and centrifuged 2,000g at 4ºC for 10 min. Urinary 8-OHdG was measured using an ELISA kit (New 8-OHdG Check, ELISA kit, JaICA, Japan). Calibration curve and data analysis were conducted according to the manufacturer’s instructions. Urinary creatinine was determined by the Creatinine Assay Kit (Abcam, Cambridge, UK). Results were expressed as nanogramme 8-OHdG per milligramme of creatinine. 2.7 Statistical analysis MS data acquisition and quantitative processing were accomplished using Qualitative Analysis, Version B.03.01 (Agilent Technologies, USA). Arithmetic means and standard deviation were calculated for 8-OHdG/105dG ratios in each leukocyte DNA sample. All analyses were performed using the Statistical Package of SPSS 17. T test was used to assess the relationship of 8-OHdG in different groups and p＜0.05 was considered statistically significant.
3. Results and Discussion 3.1 Development of UHPLC-MS/MS method for detection of leukocyte DNA 8-OHdG There is considerable evidence that damage to DNA by oxygen radicals and other reactive species occurs in vivo [41]. Such damage is thought to make a significant contribution to the aging-related development of cancer [42]. It is now widely accepted that the level of 8-OHdG is a biomarker of oxidatively generated DNA damage [43]. The 8-OHdG ion was detected by ESI-MS/MS after the separation through a
reversed-phased C18 column. As previously reported, in positive ion mode, 8-OHdG exhibited more sensitive response over that in negative ion mode [44, 45]. Thus, the positive ion mode in ESI was utilized for subsequent analysis. Moreover, acid addition in mobile phase would also favor 8-OHdG sensitive analysis through enhancing 8-OHdG ionization by protons capture in positive ion mode and improving peak shape. Thus, formic and acetic acid were tested and compared in UHPLC-MS/MS analysis. As shown in Fig.1A, 0.1% acetic acid solution as elution A exhibited about 1.6 fold higher response over that of 0.1% formic acid for 10 nM 8-OHdG standard analysis. Therefore, 0.1% acetic acid was added for 8-OHdG analysis. Stable isotope labeled internal standard [15N5]-8-OHdG was used to overcome sample matrix effects by eliminating the ion suppression and enhancement role of salts and biomolecules in real samples. In the positive mode, a hydrogen atom would transfer from the sugar moiety in the precursor ion [M+H]+, resulted in the N-glycoside bond cleavage. Therefore, the precursor→product ion pairs of m/z 284.1→168.1 for 8-OHdG and m/z 289.1→173.1 for [15N5]-8-OHdG were used as MRM transitions (Table 1) [29]. Fig.1B shows a representative chromatogram of dG, 8-OHdG and IS in MRM mode from a cancer patient’s leukocyte DNA. It should be noted that dG is partly oxidized to 8-OHdG during the ionization process, because a m/z 284.1→168.1 transition is detected at the retention time corresponding to dG, consistent with the previously reported assays [46]. Such an oxidation process emphasizes the need to separate 8-OHdG to dG. With UHPLC high efficient separation, dG was base-line separated from 8-OHdG or [15N5]-8-OHdG, with reproducible retention time at 1.85 ± 0.03 min and 2.54 ± 0.02 min, respectively. Finally, the optimized conditions for 8-OHdG analysis in UHPLC-MS/MS were as follows: an Agilent Zorbax Eclipse Plus C18 (2.1×100 mm，1.8 μm) column at a flow rate of 250 µl/min with the temperature maintained at 25ºC. Sample injection volume is 5 µl. The mobile phase consists of 0.1% acetic acid and methanol with an isocratic flow of 8% methanol for 10 min. The ESI source was operated in positive ion mode, monitoring m/z 284.1→168.1 for 8-OHdG, 289.1→173.1 for [15N5]-8-OHdG and the corresponding m/z 268.1→110.1 for dG.
The nitrogen gas drying temperature was set at 300°C and introduced into the capillary at a flow rate 9.0 l/min, with capillary voltage set at 3360 V, and the collision energy was fixed at 15 eV.
3.2 Method validation Fig. 2A shows typical MRM chromatograms of a blank leukocyte DNA sample and a blank leukocyte DNA sample spiked with 5 nM 8-OHdG standard from a cancer patient. As shown in the chromatogram, there are no obvious endogenous interferences at the retention time of 2.54 ± 0.2 min under the described conditions. 8-OHdG standards at a series of concentrations from 1 to 100 nM were subjected for UHPLC-MS/MS analysis with [15N5]-8-OHdG as calibrating internal standard. As shown in Fig.2B, good linearity with high coefficient (R2 > 0.99) has been acquired, with regression formula of y=0.117x−0.014, where y and x referred to the peak area ratio of 8-OHdG/[15N5]-8-OHdG and 8-OHdG concentration, respectively. The dynamic range for 8-OHdG is over 2 orders of magnitude (1 - 100 nM). The limit of detection (LOD) for 8-OHdG is measured as 0.30 nM and the acquired limit of quantification (LOQ) is 1.0 nM, which is sufficient for the subsequent application study. The accuracy and precision of the proposed method were further evaluated with 8-OHdG standards spiked at 1, 10 and 50 nM. As summarized in Table 2, the recovery of 8-OHdG is in the range of 90.9% -94.8%. The precision is 0.9%-3.7% (RSD), indicating the reliability and reproducibility of the proposed method. The addition of acetic acid in mobile phase is helpful to increase the detection sensitivity by enhancing 8-OHdG ionization. Using stable isotopically labelled internal standard for calibration and quantification allows for a better accuracy. Another factor that must be taken into account is that procedures such as phenol extraction and nuclease digestion of DNA could conceivably result in artifactual oxidation. The Wizard® Genomic DNA Purification Kit was used to minimize oxidation of DNA during extraction. The major advantage of this kit is the absence of the “Phenol/Chloroform” extraction of proteins, resulting in a shorter “hands on” time
because the number of actions is decreased and lower artifactual production of oxidized bases [36]. The addition of 100 µM DFO is used to protect DNA from substantial oxidation and to minimize the artifactual formation of 8-OHdG during hydrolysis [47]. Moreover, the base line noise and occurrence of co-eluting peaks were eliminated, improving the selectivity of this method. Therefore, our UHPLC-MS/MS method with excellent features is desired for 8-OHdG level in genomic DNA analysis from blood samples.
3.3 Detection of 8-OHdG in human peripheral blood and urine samples Under the selected conditions, we applied this method on screening the 8-OHdG of DNA samples in peripheral blood from 46 cancer patients and 75 healthy volunteers. The 8-OHdG content varies significantly among different people. Fig.3 shows chromatograms of 8-OHdG monitoring at m/z 284.1→168.1 for 6 subjects, of which trace 1-3 were from healthy volunteers and trace 4-6 from cancer patients. Result indicate that the cancer patients had increased levels of leukocyte 8-OHdG as compared with healthy control. The levels of 8-OHdGfor 121 subjects were summarized in Fig.4, which is 10.50 ± 5.87 per 105 dG in health control and is 16.81 ± 8.97 per 105 dG (p<0.05) in cancer patients. The average 8-OHdG level in leukocyte DNA determined in our study is comparable to the reported levels (11.8 ± 5.9 per 105dG) [48]. Result suggests that the mean level of leukocyte 8-OHdG is significantly higher in cancer patients than that in health control. This is consistent with the result of a previous study which showed significantly higher serum 8-OHdG levels in the thyroid carcinoma group compared with the health control [49]. We further determined 8-OHdG in urine samples collected from the same subjects. Totally 121 urine samples were collected, in which 75 from healthy volunteers and 46 from cancer patients, correspondingly. Fig. 5 shows the levels of urinary 8-OHdG in these 121 samples. The urinary 8-OHdG exhibits a wide range of inter-individual variations, from 0.45 to 120.44 ng/mg creatinine. The median 8-OHdG concentration is 11.17 ± 0.45 ng/mg creatinine for health control and 13.74 ±
0.72 ng/mg creatinine for cancer patients, respectively. Interestingly, 25 of 75 healthy volunteers exhibited higher urinary 8-OHdG levels than the mean level of cancer patients, but their leukocyte 8-OHdG levels were significantly lower than that of cancer patients. This inconformity is not well understood, and is probably explained by inter-individual variations, such as gender [50], body mass index (BMI) [51], smoking, alcohol consumption, physical activity and other environmental factors [52-54]. Our result suggests that the 8-OHdG in leukocyte might be more suitable for evaluating the association of oxidatively generated DNA damage with cancer.
4. Conclusion In summary, we described a UHPLC-MS/MS method for quantitative detection of 8-OHdG in blood leukocyte DNA by using stable-isotope-labeled 8-OHdG as an internal standard. The addition of acetic acid in mobile phase significantly enhanced the sensitivity of ESI-MS/MS detection. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress better than that in urine. We applied this method to analyze 8-OHdG in leukocyte DNA from 121 human subjects. Result suggests that the level of leukocyte 8-OHdG of the cancer patients group is significantly higher than that of the health control. As 8-OHdG is considered as a good biomarker for risk assessment of various cancers and degenerative diseases, this analytical method is also promising to serve as a prognostic tool for these diseases.
Acknowledgement This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0203102 and 2016YFC0900301), the National Natural Science Foundation of China (21375142, 21321004, and 21435008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030000), the special fund for public benefit from Ministry of Environmental Protection of China (201309045), and the Key Research Program of Frontier Sciences, CAS.
REFERANCE [1] G. Loschen, A. Azzi, L. Flohe, Mitochondrial H2O2 formation: relationship with energy conservation, FEBS letters, 33 (1973) 84-87. [2] V.G. Grivennikova, A.D. Vinogradov, Mitochondrial production of reactive oxygen species, Biochemistry. Biokhimiia, 78 (2013) 1490-1511. [3] S.P. Faux, T. Tai, D. Thorne, Y. Xu, D. Breheny, M. Gaca, The role of oxidative stress in the biological responses of lung epithelial cells to cigarette smoke, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals, 14 Suppl 1 (2009) 90-96. [4] J.M. Mates, J.A. Segura, F.J. Alonso, J. Marquez, Roles of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated mechanisms, Free radical biology & medicine, 49 (2010) 1328-1341. [5] T.M. Penning, S.T. Ohnishi, T. Ohnishi, R.G. Harvey, Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase, Chem Res Toxicol, 9 (1996) 84-92. [6] M. Dizdaroglu, Oxidative damage to DNA in mammalian chromatin, Mutation research, 275 (1992) 331-342. [7] J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J.P. Pouget, J.L. Ravanat, S. Sauvaigo, Hydroxyl radicals and DNA base damage, Mutation research, 424 (1999) 9-21. [8] K.C. Cheng, D.S. Cahill, H. Kasai, S. Nishimura, L.A. Loeb, 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions, Journal of Biological Chemistry, 267 (1992) 166-172. [9] S. Shibutani, M. Takeshita, A.P. Grollman, Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG, Nature, 349 (1991) 431-434. [10] Z. Radak, I. Boldogh, 8-Oxo-7,8-dihydroguanine: links to gene expression, aging, and defense against oxidative stress, Free radical biology & medicine, 49 (2010) 587-596. [11] H. Sova, A. Jukkola-Vuorinen, U. Puistola, S. Kauppila, P. Karihtala, 8-Hydroxydeoxyguanosine: a new potential independent prognostic factor in breast cancer, British Journal of Cancer, 102 (2010) 1018-1023. [12] J. Sheridan, L.M. Wang, M. Tosetto, K. Sheahan, J. Hyland, D. Fennelly, D. O'Donoghue, H. Mulcahy, J. O'Sullivan, Nuclear oxidative damage correlates with poor survival in colorectal cancer, British Journal of Cancer, 100 (2009) 381-388. [13] J.D. Long, W.R. Matson, A.R. Juhl, B.R. Leavitt, J.S. Paulsen, 8OHdG as a marker for Huntington disease progression, Neurobiology of disease, 46 (2012) 625-634. [14] S. Nakanishi, G. Suzuki, Y. Kusunoki, K. Yamane, G. Egusa, N. Kohno, Increasing of oxidative stress from mitochondria in type 2 diabetic patients, Diabetes/metabolism research and reviews, 20 (2004) 399-404. [15] S. Loft, H.E. Poulsen, Cancer risk and oxidative DNA damage in man, J Mol Med (Berl), 74 (1996) 297-312. [16] L.L. Wu, C.C. Chiou, P.Y. Chang, J.T. Wu, Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics, Clinica chimica acta; international journal of clinical chemistry, 339 (2004) 1-9. [17] M. Pylväs-Eerola, P. Karihtala, U. Puistola, Preoperative serum 8-hydroxydeoxyguanosine is associated with chemoresistance and is a powerful prognostic factor in endometrioid-type epithelial
ovarian cancer, BMC Cancer, 15 (2015) 493. [18] A. Di Francesco, B. Arosio, A. Falconi, M.V. Micioni Di Bonaventura, M. Karimi, D. Mari, M. Casati, M. Maccarrone, C. D'Addario, Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells, Brain, behavior, and immunity, 45 (2015) 139-144. [19] Y.C. Chen, C.M. Chen, J.L. Liu, S.T. Chen, M.L. Cheng, D.T. Chiu, Oxidative markers in spontaneous intracerebral hemorrhage: leukocyte 8-hydroxy-2'-deoxyguanosine as an independent predictor of the 30-day outcome, Journal of neurosurgery, 115 (2011) 1184-1190. [20] P. Mecocci, M.C. Polidori, T. Ingegni, A. Cherubini, F. Chionne, R. Cecchetti, U. Senin, Oxidative damage to DNA in lymphocytes from AD patients, Neurology, 51 (1998) 1014-1017. [21] B. Marczynski, P. Rozynek, T. Kraus, S. Schlosser, H.J. Raithel, X. Baur, Levels of 8-hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany, Mutation research, 468 (2000) 195-202. [22] B. Marczynski, P. Rozynek, H.J. Elliehausen, M. Korn, X. Baur, Detection of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in white blood cells of workers occupationally exposed to styrene, Archives of toxicology, 71 (1997) 496-500. [23] S. Loft, K. Vistisen, M. Ewertz, A. Tjonneland, K. Overvad, H.E. Poulsen, Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index, Carcinogenesis, 13 (1992) 2241-2247. [24] R. Yoshida, Y. Ogawa, H. Kasai, Urinary 8-Oxo-7,8-dihydro-2′-deoxyguanosine Values Measured by an ELISA Correlated Well with Measurements by High-Performance Liquid Chromatography with Electrochemical Detection, Cancer Epidemiology Biomarkers & Prevention, 11 (2002) 1076-1081. [25] L.J. Lu, F. Tasaka, J.A. Hokanson, K. Kohda, Detection of 8-hydroxy-2'-deoxyguanosine in deoxyribonucleic acid by the 32P-postlabeling method, Chemical & pharmaceutical bulletin, 39 (1991) 1880-1882. [26] V. Kvasnicova, E. Samcova, A. Jursova, I. Jelinek, Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection, Journal of Chromatography. A, 985 (2003) 513-517. [27] H. Kasai, A New Automated Method to Analyze Urinary 8-Hydroxydeoxyguanosine by a High-performance Liquid Chromatography-Electrochemical Detector System, Journal of Radiation Research, 44 (2003) 185-189. [28] M. Dizdaroglu, [1] Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry,
Methods in enzymology, Academic Press1994, pp. 3-16.
[29] A. Weimann, D. Belling, H.E. Poulsen, Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography–electrospray tandem mass spectrometry, Nucleic Acids Research, 30 (2002) e7-e7. [30] J.L. Ravanat, B. Duretz, A. Guiller, T. Douki, J. Cadet, Isotope dilution high-performance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in biological samples, Journal of Chromatography. B, Biomedical sciences and applications, 715 (1998) 349-356. [31] J. Serrano, C.M. Palmeira, K.B. Wallace, D.W. Kuehl, Determination of 8-hydroxydeoxyguanosine in biological tissue by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry, Rapid communications in mass spectrometry : RCM, 10 (1996) 1789-1791. [32] M. Hayakawa, K. Torii, S. Sugiyama, M. Tanaka, T. Ozawa, Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm, Biochemical and biophysical
research communications, 179 (1991) 1023-1029. [33] M. Hayakawa, T. Ogawa, S. Sugiyama, M. Tanaka, T. Ozawa, Massive conversion of guanosine to 8-hydroxy-guanosine in mouse liver mitochondrial DNA by administration of azidothymidine, Biochemical and biophysical research communications, 176 (1991) 87-93. [34] E.M. Park, M.K. Shigenaga, P. Degan, T.S. Korn, J.W. Kitzler, C.M. Wehr, P. Kolachana, B.N. Ames, Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column, Proceedings of the National Academy of Sciences of the United States of America, 89 (1992) 3375-3379. [35] H. Kasai, A new automated method to analyze urinary 8-hydroxydeoxyguanosine by a high-performance liquid chromatography-electrochemical detector system, J Radiat Res, 44 (2003) 185-189. [36] G. Guetens, G. De Boeck, M. Highley, A.T. van Oosterom, E.A. de Bruijn, Oxidative DNA damage: biological significance and methods of analysis, Critical reviews in clinical laboratory sciences, 39 (2002) 331-457. [37] M.C. Peoples, H.T. Karnes, Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 827 (2005) 5-15. [38] A. Weimann, D. Belling, H.E. Poulsen, Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry, Free radical biology & medicine, 30 (2001) 757-764. [39] J. Cadet, M. Weinfeld, Detecting DNA damage, Analytical chemistry, 65 (1993) 675A-682A. [40] J. Yin, T. Xu, N. Zhang, H. Wang, Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides, Analytical chemistry, 88 (2016) 7730-7737. [41] E.R. Bongarzone, J.M. Pasquini, E.F. Soto, Oxidative damage to proteins and lipids of CNS myelin produced by in vitro generated reactive oxygen species, Journal of neuroscience research, 41 (1995) 213-221. [42] B. Halliwell, Can oxidative DNA damage be used as a biomarker of cancer risk in humans? Problems, resolutions and preliminary results from nutritional supplementation studies, Free radical research, 29 (1998) 469-486. [43] S. Loft, H.E. Poulsen, Markers of oxidative damage to DNA: antioxidants and molecular damage, Methods in enzymology, 300 (1999) 166-184. [44] T. Renner, T. Fechner, G. Scherer, Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2’-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection, Journal of Chromatography B Biomedical Sciences & Applications, 738 (2000) 311-317. [45] A. Weimann, D. Belling, H.E. Poulsen, Measurement of 8-oxo-2’-deoxyguanosine and 8-oxo-2’-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry, Free radical biology & medicine, 30 (2001) 757-764. [46] M. Dizdaroglu, P. Jaruga, H. Rodriguez, Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry, Nucleic Acids Res, 29 (2001) E12. [47] C.M. Gedik, A. Collins, Establishing the background level of base oxidation in human lymphocyte
DNA: results of an interlaboratory validation study, FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 19 (2005) 82-84. [48] S. Bashir, G. Harris, M.A. Denman, D.R. Blake, P.G. Winyard, Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases, Annals of the rheumatic diseases, 52 (1993) 659-666. [49] S. Tabur, S.N. Aksoy, H. Korkmaz, M. Ozkaya, N. Aksoy, E. Akarsu, Investigation of the role of 8-OHdG and oxidative stress in papillary thyroid carcinoma, Tumour biology : the journal of the International Society for Oncodevelopmental. Biology and Medicine, 36 (2015) 2667-2674. [50] A. Topic, D. Francuski, B. Markovic, M. Stankovic, S. Dobrivojevic, S. Drca, D. Radojkovic, Gender-related reference intervals of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine determined by liquid chromatography-tandem mass spectrometry in Serbian population, Clinical Biochemistry, 46 (2013) 321-326. [51] C. Protano, R. Andreoli, A. Mutti, S. Petti, M. Vitali, Biomarkers of oxidative stress to nucleic acids: background levels and effects of body mass index and life-style factors in an urban paediatric population, Science of the Total Environment, 500-501 (2014) 44-51. [52] Toraason, M., 8 Hydroxydeoxyguanosine as a biomarker of workplace exposures. Biomarkers,4(1999) 3-26. [53]Foksinski, M., et al., Cellular level of 8-oxo-2'-deoxyguanosine in DNA does not correlate with urinary excretion of the modified base/nucleoside. Acta Biochim Pol,50(2003)549-553. [54] Pilger, A. and H.W. Rudiger, 8-Hydroxy-2'-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures. Int Arch Occup Environ Health, 80(2006)1-15.
Figure Legends Fig.1 A) Extracted ion chromatograms at m/z 284.1→168.1 show the effect of mobile phase additives on peak shape and retention time after injection of standard 8-OHdG (10nM). Peaks were overlaid on the same time-axis, and normalized to equal height on the Y-axis. B) MRM chromatograms for dG, 8-OHdG and [15N5]-8-OHdG in a peripheral blood sample treated according to the experimental protocol. Fig.2 A) Specificity: representative chromatograms of 8-OHdG in a blank leukocyte DNA sample and a blank leukocyte DNA sample spiked with 5 nM 8-OHdG standard. B) Linearity: chromatograms of 1, 2, 5, 10,50 and 100nM 8-OHdG standard solution. Calibration curve for 8-OHdG was obtained by plotting the peak area ratio of analyte peak and IS peak against the concentration. Fig.3 MRM chromatograms (m/z 284.1→168.1) for 10nM standard 8-OHdG compared with that isolated from peripheral blood samples. Samples 1-3 were from healthy volunteers, 4-6 from cancer patients. Fig.4 8-OHdG levels in the peripheral leukocyte DNA of healthy donors and cancer patients. Significant differences between cancer patients and healthy control (p< 0.05) Fig. 5 Urinary 8-OHdG levels of health control and cancer patients. No significant differences between cancer patients and healthy control (p > 0.05)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1. MRM transitions of the analytes and their retention time. Compound MRM Transitions Retention Time(min) 8-OHdG 284.1→168.1 2.54±0.02 15 N5-8-OHdG 289.1→173.1 2.54±0.02 dG 268.1→110.1 1.85±0.03
Table 2. Accuracy and precision of the UPLC-MS/MS method for the determination of 8-OHdG level in leukocyte DNA. Spiked Detected Average Accuracy Precision Concentration Concentration Concentration (%) (%) (nM) (nM) (nM) 4.72 1 90.9 3.7 5.01 4.81 4.69 10
13.07 13.03 13.55
13.22
93.2
2.2
50
50.82 51.38 51.71
51.30
94.8
0.92
S1570-0232(17)30729-8 http://dx.doi.org/10.1016/j.jchromb.2017.08.033 CHROMB 20766
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
23-4-2017 28-7-2017 23-8-2017
Please cite this article as: Danni Wu, Baodong Liu, Junfa Yin, Tian Xu, Shuli Zhao, Qun Xu, Xi Chen, Hailin Wang, Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.08.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of 8-Hydroxydeoxyguanosine (8-OHdG) as a Biomarker of Oxidative Damage in Peripheral Leukocyte DNA by UHPLC-MS/MS Danni Wu1,5, Baodong Liu1, Junfa Yin1, Tian Xu1, Shuli Zhao2, Qun Xu3, Xi Chen4 and Hailin Wang1,5*
1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China 2. China National Environmental Monitoring Center, Beijing, 100012, China 3. Institute of Basis Medical Sciences, Chinese Academy of Medical Sciences, School of Basis Medicine, Peking Union Medical College, Beijing, 100005, China 4. Institute of Environmental Health & Related Products Safety, Chinese Center for Disease Control and Prevention, Beijing, 102206, China 5. University of Chinese Academy of Sciences, Beijing, 100049, China
*
Corresponding Author: Prof. Hailin Wang,
Telephone/Fax:+86-10-62849600, E-mail:[email protected]
Highlights
An UHPLC-MS/MS method was developed for accurate detection of 8-OHdG in leukocyte. 8-OHdG in leukocyte but urine can indicate oxidative DNA damage. Higher levels of leukocyte 8-OHdG were found in cancer patients over health control.
Abstract 8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC-MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1×100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [15N5]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0-100 nM with R2 = 0.999. The accuracy for spiked samples was 90.9% - 94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC-MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). Keywords: Ultra-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS); 8-hydroxydeoxyguanosine (8-OHdG); peripheral blood leukocyte; oxidative DNA damage
1. Introduction Organisms often live with abundant endogenous or exogenous reactive oxygen species (ROS). ROS can be generated from multiple biochemical reactions during various physiological processes such as substance and energy metabolism [1, 2], but also caused by the exposure to exogenous toxic chemicals and pollutants, including tobacco smoke, heavy metal and polycyclic aromatic hydrocarbons [3-5]. Thus, a multitude of oxidative DNA damages are induced by ROS attacking DNA molecules, including DNA bases and sugar modifications, covalent crosslinking, and single- and double-stranded breaks [6, 7]. Among these lesions, 8-OHdG is the predominant and most abundant oxidative product formed in nuclear and mitochondrial DNA. Although numerous DNA enzymatic repair and non-enzymatic antioxidant defenses in organisms to maintain genomic stability, the unrepaired 8-OHdG in DNA can result in G:T transversion and further initiate carcinogenesis [8, 9]. It has proposed that oxidative damage to DNA induced several diseases, including aging [10], cancer [11, 12], neurodegenerative diseases [13], diabetes [14], cardiomyopathy [15] and cardiovascular or infectious diseases [16]. Therefore, 8-OHdG is not only an indicator of endogenous oxidative DNA damage, but also useful for early diagnosis and assessment of high-risk individuals [17]. Peripheral blood leukocytes are the most commonly used cells to assess human disease states [18]. Leukocyte 8-OHdG is an indicator of oxidative stress, as well as impaired metabolism and mitochondrial dysfunction [13], which is related to cancer progression. Moreover, 8-OHdG in leukocyte genomic DNA is associated with mutation and cancer risk, which hardly affected by tumor necrosis and inflammatory [19]. On the other hand, an increase of 8-OHdG may not only because of a rise in oxidative DNA damage, but also because of a decline in the repair rate. A quantitative deficit in DNA repair system may promote an accumulation of 8-OHdG in leukocyte DNA, while the excreted 8-OHdG in urine and serum may have little change [20]. Consequently, 8-OHdG in leukocyte DNA is more accurate and sensitive than serum and urinary 8-OHdG. However, the 8-OHdG content in peripheral blood leukocyte
DNA was only reported in a few of papers [21, 22], which probably due to the problems of sample collection and storage, DNA extraction and separation of hydrolytic enzymes. Although 8-OHdG in urine has been well studied [23], because it is noninvasive and technically less involved, the level of 8-OHdG in peripheral blood leukocytes probably reflect a long term response to oxidative stress. Additionally, urinary creatinine concentration is required to normalize urine volume for urinary 8-OHdG calibration [16]. A variety of biochemical and chemical assays have been developed for 8-OHdG analysis, including enzyme-linked immunosorbent assay (ELISA) [24], 32P post-labeling [25], capillary electrophoresis-ultraviolet detection (CE-UV) [26], high-performance liquid chromatography-electrochemical detection (HPLC-ECD) [27], gas chromatography-mass spectrometry (GC-MS) [28], liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [29-31] and liquid chromatography-single mass spectrometry (HPLC-MS) [32, 33]. HPLC-ECD is frequently utilized, however, complicated HPLC system with column-switching [34] or SPE [35] is necessary to eliminate possible interference from various sample matrix. For GC-MS, the 8-OHdG derivatization is essential to provide adequate vaporation to enter gas phase before analysis, which might lead into possible “artificial” oxidation of nucleosides [36, 37]. Mass spectrometry detection is currently the best approach, with the possibility of using isotopically labeled internal standards that can correct for biased MS response during work-up [36]. The main advantage of HPLC-MS/MS in detection of 8-OHdG is the increase in sensitivity compared with HPLC-MS, since the use of multiple reaction monitoring (MRM) mode leads to a considerable reduction in the background ions-caused noise [38]. The chromatographic method for measuring 8-OHdG in DNA requires chemical hydrolysis or enzymatic digestion following extraction from cells [39]. To reduce artifactual oxidation, a three-enzyme cascade capillary monolithic bioreactor was fabricated for rapid digestion of genomic DNA into single nucleosides [40]. By the use of the immobilized enzymes, the collected products were directly injected into HPLC-MS/MS for detection without separation process. As a result, HPLC-MS/MS is
a powerful technique that has exhibited good selectivity and sensitivity for 8-OHdG analysis. In this study, a robust and sensitive UHPLC-MS/MS method was developed for 8-OHdG analysis in leukocyte DNA. Of note, [15N5]-8-OHdG was used as internal standard (IS). High sensitivity, good precision and reproducibility were validated. The proposed method was further applied to analyze 8-OHdG in healthy volunteers and cancer patients (n = 121).
2. Materials and methods 2.1 Chemicals and Reagents 8-OHdG standard and deferoxamine mesylate (DFO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Wizard® Genomic DNA Purification Kit(Promega, Madison, WI) was used for DNA extraction. Acetic acid was purchased from Fluka Analytical (Steinheim, Germany). DNase I and calf intestinal phosphatase (CIP) were obtained from New England Biolabs (MA, USA). The HPLC grade methanol was purchased from Fisher Scientific Inc. (Waltham, USA).All other chemicals used were of highest purity and purchased from Sigma-Aldrich. An ELGA WaterLab water purification system was used to provide 18.2MΩ ultrapure water throughout the experiments.
2.2 UHPLC-MS/MS Conditions Prior to 8-OHdG analysis, 50 fmol of [15N5]-8-OHdG stable isotope internal standard was added to 5 µg DNA enzymatic digestions. The ultra-performance liquid chromatography was carried out on an Agilent 1290 HPLC system equipped with G6410B triple quadrupole mass spectrometer. Electrospray ionization was set in positive ion mode, and the data was acquired in MRM mode. The column used was a Zorbax Eclipse Plus C18 (2.1×100 mm，1.8 μm) obtained from Agilent, and a guard column Zorbax Eclipse Plus C18 Narrow Bore Guard Column (2.1×12.5 mm) was used to protect the analytical column. The mobile phase consisted of 92% elution A
(ultrapure water containing 0.1% acetic acid) and 8% elution B (methanol). The flow rate was 250 µl/min. The column temperature was kept at 25ºC. 2.3 Sample Collection Total 121 blood samples were collected from the hospital, including 46 cancer patients (28 males and 18 females) and 75 healthy volunteers (36 males and 39 females). All the subjects were aged form 18 to 75. Blood samples were withdrawn into EDTA-2Na-containingvessels, and immediately frozen at -20ºC. The study was approved by the Local Ethics Committee.
2.4 DNA isolation and digestion An aliquot of 600 μl whole blood was subjected to Wizard® Genomic DNA Purification Kit following the provided protocol of the manufacturer. Briefly, 800 μl of the cell lysis solution was added to lyses the red blood cells, followed by lysis of the white blood cells and their nuclei in nuclei lysis solution. 1.5 μl RNase solution was added to each sample, and incubated at 37ºC for 15 minutes. The cellular proteins were then removed by addition of 200 μl protein precipitation solution. Finally, the genomic DNA was concentrated and desalted by isopropanol precipitation. Next, the isolated DNA (5 µg) was digested by 1.0 U DNase I, 0.005 U snake venom phosphodiesteraseand 2.0 U calf intestinal phosphatase at 37ºC overnight. 100 µM DFO was also added to reduce artifactual formation of 8-OHdG during digestion process. Then, the enzymes were removed by ultrafiltration tubes with molecule weight cutoff at 3KDa. 2.5 Method validation Linearity was assessed by calibration curve. A series of standard 8-OHdG samples at concentrations of 1, 2, 5, 10, 50 and 100 nM were spiked with 50 fmol of [15N5]-8-OHdG IS. Calibration curve was established by plotting the peak area ratios of 8-OHdG to IS (Y-axis) versus the concentrations (X-axis), and square regression coefficient (R2) was applied to evaluate the linear regression equation. The limit of detection (signal-to-noise ≥ 3) and limit of quantification (signal-to-noise ≥ 10) were
measured on successive aqueous dilutions of standard 8-OHdG solution.The accuracy and precision were evaluated using a digested DNA sample spiked with standard 8-OHdG (low, middle and high concentration) for three replicate samples at each level. Recovery was calculated according to the following equation: Recovery (%) = [(final concentration-original concentration)/ spiked concentration] × 100%. Relative standard deviation (RSD) was used to assess the precision.
2.6 Measurement of urinary 8-OHdG by ELISA Urine samples were thawed and centrifuged 2,000g at 4ºC for 10 min. Urinary 8-OHdG was measured using an ELISA kit (New 8-OHdG Check, ELISA kit, JaICA, Japan). Calibration curve and data analysis were conducted according to the manufacturer’s instructions. Urinary creatinine was determined by the Creatinine Assay Kit (Abcam, Cambridge, UK). Results were expressed as nanogramme 8-OHdG per milligramme of creatinine. 2.7 Statistical analysis MS data acquisition and quantitative processing were accomplished using Qualitative Analysis, Version B.03.01 (Agilent Technologies, USA). Arithmetic means and standard deviation were calculated for 8-OHdG/105dG ratios in each leukocyte DNA sample. All analyses were performed using the Statistical Package of SPSS 17. T test was used to assess the relationship of 8-OHdG in different groups and p＜0.05 was considered statistically significant.
3. Results and Discussion 3.1 Development of UHPLC-MS/MS method for detection of leukocyte DNA 8-OHdG There is considerable evidence that damage to DNA by oxygen radicals and other reactive species occurs in vivo [41]. Such damage is thought to make a significant contribution to the aging-related development of cancer [42]. It is now widely accepted that the level of 8-OHdG is a biomarker of oxidatively generated DNA damage [43]. The 8-OHdG ion was detected by ESI-MS/MS after the separation through a
reversed-phased C18 column. As previously reported, in positive ion mode, 8-OHdG exhibited more sensitive response over that in negative ion mode [44, 45]. Thus, the positive ion mode in ESI was utilized for subsequent analysis. Moreover, acid addition in mobile phase would also favor 8-OHdG sensitive analysis through enhancing 8-OHdG ionization by protons capture in positive ion mode and improving peak shape. Thus, formic and acetic acid were tested and compared in UHPLC-MS/MS analysis. As shown in Fig.1A, 0.1% acetic acid solution as elution A exhibited about 1.6 fold higher response over that of 0.1% formic acid for 10 nM 8-OHdG standard analysis. Therefore, 0.1% acetic acid was added for 8-OHdG analysis. Stable isotope labeled internal standard [15N5]-8-OHdG was used to overcome sample matrix effects by eliminating the ion suppression and enhancement role of salts and biomolecules in real samples. In the positive mode, a hydrogen atom would transfer from the sugar moiety in the precursor ion [M+H]+, resulted in the N-glycoside bond cleavage. Therefore, the precursor→product ion pairs of m/z 284.1→168.1 for 8-OHdG and m/z 289.1→173.1 for [15N5]-8-OHdG were used as MRM transitions (Table 1) [29]. Fig.1B shows a representative chromatogram of dG, 8-OHdG and IS in MRM mode from a cancer patient’s leukocyte DNA. It should be noted that dG is partly oxidized to 8-OHdG during the ionization process, because a m/z 284.1→168.1 transition is detected at the retention time corresponding to dG, consistent with the previously reported assays [46]. Such an oxidation process emphasizes the need to separate 8-OHdG to dG. With UHPLC high efficient separation, dG was base-line separated from 8-OHdG or [15N5]-8-OHdG, with reproducible retention time at 1.85 ± 0.03 min and 2.54 ± 0.02 min, respectively. Finally, the optimized conditions for 8-OHdG analysis in UHPLC-MS/MS were as follows: an Agilent Zorbax Eclipse Plus C18 (2.1×100 mm，1.8 μm) column at a flow rate of 250 µl/min with the temperature maintained at 25ºC. Sample injection volume is 5 µl. The mobile phase consists of 0.1% acetic acid and methanol with an isocratic flow of 8% methanol for 10 min. The ESI source was operated in positive ion mode, monitoring m/z 284.1→168.1 for 8-OHdG, 289.1→173.1 for [15N5]-8-OHdG and the corresponding m/z 268.1→110.1 for dG.
The nitrogen gas drying temperature was set at 300°C and introduced into the capillary at a flow rate 9.0 l/min, with capillary voltage set at 3360 V, and the collision energy was fixed at 15 eV.
3.2 Method validation Fig. 2A shows typical MRM chromatograms of a blank leukocyte DNA sample and a blank leukocyte DNA sample spiked with 5 nM 8-OHdG standard from a cancer patient. As shown in the chromatogram, there are no obvious endogenous interferences at the retention time of 2.54 ± 0.2 min under the described conditions. 8-OHdG standards at a series of concentrations from 1 to 100 nM were subjected for UHPLC-MS/MS analysis with [15N5]-8-OHdG as calibrating internal standard. As shown in Fig.2B, good linearity with high coefficient (R2 > 0.99) has been acquired, with regression formula of y=0.117x−0.014, where y and x referred to the peak area ratio of 8-OHdG/[15N5]-8-OHdG and 8-OHdG concentration, respectively. The dynamic range for 8-OHdG is over 2 orders of magnitude (1 - 100 nM). The limit of detection (LOD) for 8-OHdG is measured as 0.30 nM and the acquired limit of quantification (LOQ) is 1.0 nM, which is sufficient for the subsequent application study. The accuracy and precision of the proposed method were further evaluated with 8-OHdG standards spiked at 1, 10 and 50 nM. As summarized in Table 2, the recovery of 8-OHdG is in the range of 90.9% -94.8%. The precision is 0.9%-3.7% (RSD), indicating the reliability and reproducibility of the proposed method. The addition of acetic acid in mobile phase is helpful to increase the detection sensitivity by enhancing 8-OHdG ionization. Using stable isotopically labelled internal standard for calibration and quantification allows for a better accuracy. Another factor that must be taken into account is that procedures such as phenol extraction and nuclease digestion of DNA could conceivably result in artifactual oxidation. The Wizard® Genomic DNA Purification Kit was used to minimize oxidation of DNA during extraction. The major advantage of this kit is the absence of the “Phenol/Chloroform” extraction of proteins, resulting in a shorter “hands on” time
because the number of actions is decreased and lower artifactual production of oxidized bases [36]. The addition of 100 µM DFO is used to protect DNA from substantial oxidation and to minimize the artifactual formation of 8-OHdG during hydrolysis [47]. Moreover, the base line noise and occurrence of co-eluting peaks were eliminated, improving the selectivity of this method. Therefore, our UHPLC-MS/MS method with excellent features is desired for 8-OHdG level in genomic DNA analysis from blood samples.
3.3 Detection of 8-OHdG in human peripheral blood and urine samples Under the selected conditions, we applied this method on screening the 8-OHdG of DNA samples in peripheral blood from 46 cancer patients and 75 healthy volunteers. The 8-OHdG content varies significantly among different people. Fig.3 shows chromatograms of 8-OHdG monitoring at m/z 284.1→168.1 for 6 subjects, of which trace 1-3 were from healthy volunteers and trace 4-6 from cancer patients. Result indicate that the cancer patients had increased levels of leukocyte 8-OHdG as compared with healthy control. The levels of 8-OHdGfor 121 subjects were summarized in Fig.4, which is 10.50 ± 5.87 per 105 dG in health control and is 16.81 ± 8.97 per 105 dG (p<0.05) in cancer patients. The average 8-OHdG level in leukocyte DNA determined in our study is comparable to the reported levels (11.8 ± 5.9 per 105dG) [48]. Result suggests that the mean level of leukocyte 8-OHdG is significantly higher in cancer patients than that in health control. This is consistent with the result of a previous study which showed significantly higher serum 8-OHdG levels in the thyroid carcinoma group compared with the health control [49]. We further determined 8-OHdG in urine samples collected from the same subjects. Totally 121 urine samples were collected, in which 75 from healthy volunteers and 46 from cancer patients, correspondingly. Fig. 5 shows the levels of urinary 8-OHdG in these 121 samples. The urinary 8-OHdG exhibits a wide range of inter-individual variations, from 0.45 to 120.44 ng/mg creatinine. The median 8-OHdG concentration is 11.17 ± 0.45 ng/mg creatinine for health control and 13.74 ±
0.72 ng/mg creatinine for cancer patients, respectively. Interestingly, 25 of 75 healthy volunteers exhibited higher urinary 8-OHdG levels than the mean level of cancer patients, but their leukocyte 8-OHdG levels were significantly lower than that of cancer patients. This inconformity is not well understood, and is probably explained by inter-individual variations, such as gender [50], body mass index (BMI) [51], smoking, alcohol consumption, physical activity and other environmental factors [52-54]. Our result suggests that the 8-OHdG in leukocyte might be more suitable for evaluating the association of oxidatively generated DNA damage with cancer.
4. Conclusion In summary, we described a UHPLC-MS/MS method for quantitative detection of 8-OHdG in blood leukocyte DNA by using stable-isotope-labeled 8-OHdG as an internal standard. The addition of acetic acid in mobile phase significantly enhanced the sensitivity of ESI-MS/MS detection. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress better than that in urine. We applied this method to analyze 8-OHdG in leukocyte DNA from 121 human subjects. Result suggests that the level of leukocyte 8-OHdG of the cancer patients group is significantly higher than that of the health control. As 8-OHdG is considered as a good biomarker for risk assessment of various cancers and degenerative diseases, this analytical method is also promising to serve as a prognostic tool for these diseases.
Acknowledgement This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0203102 and 2016YFC0900301), the National Natural Science Foundation of China (21375142, 21321004, and 21435008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030000), the special fund for public benefit from Ministry of Environmental Protection of China (201309045), and the Key Research Program of Frontier Sciences, CAS.
REFERANCE [1] G. Loschen, A. Azzi, L. Flohe, Mitochondrial H2O2 formation: relationship with energy conservation, FEBS letters, 33 (1973) 84-87. [2] V.G. Grivennikova, A.D. Vinogradov, Mitochondrial production of reactive oxygen species, Biochemistry. Biokhimiia, 78 (2013) 1490-1511. [3] S.P. Faux, T. Tai, D. Thorne, Y. Xu, D. Breheny, M. Gaca, The role of oxidative stress in the biological responses of lung epithelial cells to cigarette smoke, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals, 14 Suppl 1 (2009) 90-96. [4] J.M. Mates, J.A. Segura, F.J. Alonso, J. Marquez, Roles of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated mechanisms, Free radical biology & medicine, 49 (2010) 1328-1341. [5] T.M. Penning, S.T. Ohnishi, T. Ohnishi, R.G. Harvey, Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase, Chem Res Toxicol, 9 (1996) 84-92. [6] M. Dizdaroglu, Oxidative damage to DNA in mammalian chromatin, Mutation research, 275 (1992) 331-342. [7] J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J.P. Pouget, J.L. Ravanat, S. Sauvaigo, Hydroxyl radicals and DNA base damage, Mutation research, 424 (1999) 9-21. [8] K.C. Cheng, D.S. Cahill, H. Kasai, S. Nishimura, L.A. Loeb, 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions, Journal of Biological Chemistry, 267 (1992) 166-172. [9] S. Shibutani, M. Takeshita, A.P. Grollman, Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG, Nature, 349 (1991) 431-434. [10] Z. Radak, I. Boldogh, 8-Oxo-7,8-dihydroguanine: links to gene expression, aging, and defense against oxidative stress, Free radical biology & medicine, 49 (2010) 587-596. [11] H. Sova, A. Jukkola-Vuorinen, U. Puistola, S. Kauppila, P. Karihtala, 8-Hydroxydeoxyguanosine: a new potential independent prognostic factor in breast cancer, British Journal of Cancer, 102 (2010) 1018-1023. [12] J. Sheridan, L.M. Wang, M. Tosetto, K. Sheahan, J. Hyland, D. Fennelly, D. O'Donoghue, H. Mulcahy, J. O'Sullivan, Nuclear oxidative damage correlates with poor survival in colorectal cancer, British Journal of Cancer, 100 (2009) 381-388. [13] J.D. Long, W.R. Matson, A.R. Juhl, B.R. Leavitt, J.S. Paulsen, 8OHdG as a marker for Huntington disease progression, Neurobiology of disease, 46 (2012) 625-634. [14] S. Nakanishi, G. Suzuki, Y. Kusunoki, K. Yamane, G. Egusa, N. Kohno, Increasing of oxidative stress from mitochondria in type 2 diabetic patients, Diabetes/metabolism research and reviews, 20 (2004) 399-404. [15] S. Loft, H.E. Poulsen, Cancer risk and oxidative DNA damage in man, J Mol Med (Berl), 74 (1996) 297-312. [16] L.L. Wu, C.C. Chiou, P.Y. Chang, J.T. Wu, Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics, Clinica chimica acta; international journal of clinical chemistry, 339 (2004) 1-9. [17] M. Pylväs-Eerola, P. Karihtala, U. Puistola, Preoperative serum 8-hydroxydeoxyguanosine is associated with chemoresistance and is a powerful prognostic factor in endometrioid-type epithelial
ovarian cancer, BMC Cancer, 15 (2015) 493. [18] A. Di Francesco, B. Arosio, A. Falconi, M.V. Micioni Di Bonaventura, M. Karimi, D. Mari, M. Casati, M. Maccarrone, C. D'Addario, Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells, Brain, behavior, and immunity, 45 (2015) 139-144. [19] Y.C. Chen, C.M. Chen, J.L. Liu, S.T. Chen, M.L. Cheng, D.T. Chiu, Oxidative markers in spontaneous intracerebral hemorrhage: leukocyte 8-hydroxy-2'-deoxyguanosine as an independent predictor of the 30-day outcome, Journal of neurosurgery, 115 (2011) 1184-1190. [20] P. Mecocci, M.C. Polidori, T. Ingegni, A. Cherubini, F. Chionne, R. Cecchetti, U. Senin, Oxidative damage to DNA in lymphocytes from AD patients, Neurology, 51 (1998) 1014-1017. [21] B. Marczynski, P. Rozynek, T. Kraus, S. Schlosser, H.J. Raithel, X. Baur, Levels of 8-hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany, Mutation research, 468 (2000) 195-202. [22] B. Marczynski, P. Rozynek, H.J. Elliehausen, M. Korn, X. Baur, Detection of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in white blood cells of workers occupationally exposed to styrene, Archives of toxicology, 71 (1997) 496-500. [23] S. Loft, K. Vistisen, M. Ewertz, A. Tjonneland, K. Overvad, H.E. Poulsen, Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index, Carcinogenesis, 13 (1992) 2241-2247. [24] R. Yoshida, Y. Ogawa, H. Kasai, Urinary 8-Oxo-7,8-dihydro-2′-deoxyguanosine Values Measured by an ELISA Correlated Well with Measurements by High-Performance Liquid Chromatography with Electrochemical Detection, Cancer Epidemiology Biomarkers & Prevention, 11 (2002) 1076-1081. [25] L.J. Lu, F. Tasaka, J.A. Hokanson, K. Kohda, Detection of 8-hydroxy-2'-deoxyguanosine in deoxyribonucleic acid by the 32P-postlabeling method, Chemical & pharmaceutical bulletin, 39 (1991) 1880-1882. [26] V. Kvasnicova, E. Samcova, A. Jursova, I. Jelinek, Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection, Journal of Chromatography. A, 985 (2003) 513-517. [27] H. Kasai, A New Automated Method to Analyze Urinary 8-Hydroxydeoxyguanosine by a High-performance Liquid Chromatography-Electrochemical Detector System, Journal of Radiation Research, 44 (2003) 185-189. [28] M. Dizdaroglu, [1] Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry,
Methods in enzymology, Academic Press1994, pp. 3-16.
[29] A. Weimann, D. Belling, H.E. Poulsen, Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography–electrospray tandem mass spectrometry, Nucleic Acids Research, 30 (2002) e7-e7. [30] J.L. Ravanat, B. Duretz, A. Guiller, T. Douki, J. Cadet, Isotope dilution high-performance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in biological samples, Journal of Chromatography. B, Biomedical sciences and applications, 715 (1998) 349-356. [31] J. Serrano, C.M. Palmeira, K.B. Wallace, D.W. Kuehl, Determination of 8-hydroxydeoxyguanosine in biological tissue by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry, Rapid communications in mass spectrometry : RCM, 10 (1996) 1789-1791. [32] M. Hayakawa, K. Torii, S. Sugiyama, M. Tanaka, T. Ozawa, Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm, Biochemical and biophysical
research communications, 179 (1991) 1023-1029. [33] M. Hayakawa, T. Ogawa, S. Sugiyama, M. Tanaka, T. Ozawa, Massive conversion of guanosine to 8-hydroxy-guanosine in mouse liver mitochondrial DNA by administration of azidothymidine, Biochemical and biophysical research communications, 176 (1991) 87-93. [34] E.M. Park, M.K. Shigenaga, P. Degan, T.S. Korn, J.W. Kitzler, C.M. Wehr, P. Kolachana, B.N. Ames, Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column, Proceedings of the National Academy of Sciences of the United States of America, 89 (1992) 3375-3379. [35] H. Kasai, A new automated method to analyze urinary 8-hydroxydeoxyguanosine by a high-performance liquid chromatography-electrochemical detector system, J Radiat Res, 44 (2003) 185-189. [36] G. Guetens, G. De Boeck, M. Highley, A.T. van Oosterom, E.A. de Bruijn, Oxidative DNA damage: biological significance and methods of analysis, Critical reviews in clinical laboratory sciences, 39 (2002) 331-457. [37] M.C. Peoples, H.T. Karnes, Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 827 (2005) 5-15. [38] A. Weimann, D. Belling, H.E. Poulsen, Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry, Free radical biology & medicine, 30 (2001) 757-764. [39] J. Cadet, M. Weinfeld, Detecting DNA damage, Analytical chemistry, 65 (1993) 675A-682A. [40] J. Yin, T. Xu, N. Zhang, H. Wang, Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides, Analytical chemistry, 88 (2016) 7730-7737. [41] E.R. Bongarzone, J.M. Pasquini, E.F. Soto, Oxidative damage to proteins and lipids of CNS myelin produced by in vitro generated reactive oxygen species, Journal of neuroscience research, 41 (1995) 213-221. [42] B. Halliwell, Can oxidative DNA damage be used as a biomarker of cancer risk in humans? Problems, resolutions and preliminary results from nutritional supplementation studies, Free radical research, 29 (1998) 469-486. [43] S. Loft, H.E. Poulsen, Markers of oxidative damage to DNA: antioxidants and molecular damage, Methods in enzymology, 300 (1999) 166-184. [44] T. Renner, T. Fechner, G. Scherer, Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2’-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection, Journal of Chromatography B Biomedical Sciences & Applications, 738 (2000) 311-317. [45] A. Weimann, D. Belling, H.E. Poulsen, Measurement of 8-oxo-2’-deoxyguanosine and 8-oxo-2’-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry, Free radical biology & medicine, 30 (2001) 757-764. [46] M. Dizdaroglu, P. Jaruga, H. Rodriguez, Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry, Nucleic Acids Res, 29 (2001) E12. [47] C.M. Gedik, A. Collins, Establishing the background level of base oxidation in human lymphocyte
DNA: results of an interlaboratory validation study, FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 19 (2005) 82-84. [48] S. Bashir, G. Harris, M.A. Denman, D.R. Blake, P.G. Winyard, Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases, Annals of the rheumatic diseases, 52 (1993) 659-666. [49] S. Tabur, S.N. Aksoy, H. Korkmaz, M. Ozkaya, N. Aksoy, E. Akarsu, Investigation of the role of 8-OHdG and oxidative stress in papillary thyroid carcinoma, Tumour biology : the journal of the International Society for Oncodevelopmental. Biology and Medicine, 36 (2015) 2667-2674. [50] A. Topic, D. Francuski, B. Markovic, M. Stankovic, S. Dobrivojevic, S. Drca, D. Radojkovic, Gender-related reference intervals of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine determined by liquid chromatography-tandem mass spectrometry in Serbian population, Clinical Biochemistry, 46 (2013) 321-326. [51] C. Protano, R. Andreoli, A. Mutti, S. Petti, M. Vitali, Biomarkers of oxidative stress to nucleic acids: background levels and effects of body mass index and life-style factors in an urban paediatric population, Science of the Total Environment, 500-501 (2014) 44-51. [52] Toraason, M., 8 Hydroxydeoxyguanosine as a biomarker of workplace exposures. Biomarkers,4(1999) 3-26. [53]Foksinski, M., et al., Cellular level of 8-oxo-2'-deoxyguanosine in DNA does not correlate with urinary excretion of the modified base/nucleoside. Acta Biochim Pol,50(2003)549-553. [54] Pilger, A. and H.W. Rudiger, 8-Hydroxy-2'-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures. Int Arch Occup Environ Health, 80(2006)1-15.
Figure Legends Fig.1 A) Extracted ion chromatograms at m/z 284.1→168.1 show the effect of mobile phase additives on peak shape and retention time after injection of standard 8-OHdG (10nM). Peaks were overlaid on the same time-axis, and normalized to equal height on the Y-axis. B) MRM chromatograms for dG, 8-OHdG and [15N5]-8-OHdG in a peripheral blood sample treated according to the experimental protocol. Fig.2 A) Specificity: representative chromatograms of 8-OHdG in a blank leukocyte DNA sample and a blank leukocyte DNA sample spiked with 5 nM 8-OHdG standard. B) Linearity: chromatograms of 1, 2, 5, 10,50 and 100nM 8-OHdG standard solution. Calibration curve for 8-OHdG was obtained by plotting the peak area ratio of analyte peak and IS peak against the concentration. Fig.3 MRM chromatograms (m/z 284.1→168.1) for 10nM standard 8-OHdG compared with that isolated from peripheral blood samples. Samples 1-3 were from healthy volunteers, 4-6 from cancer patients. Fig.4 8-OHdG levels in the peripheral leukocyte DNA of healthy donors and cancer patients. Significant differences between cancer patients and healthy control (p< 0.05) Fig. 5 Urinary 8-OHdG levels of health control and cancer patients. No significant differences between cancer patients and healthy control (p > 0.05)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1. MRM transitions of the analytes and their retention time. Compound MRM Transitions Retention Time(min) 8-OHdG 284.1→168.1 2.54±0.02 15 N5-8-OHdG 289.1→173.1 2.54±0.02 dG 268.1→110.1 1.85±0.03
Table 2. Accuracy and precision of the UPLC-MS/MS method for the determination of 8-OHdG level in leukocyte DNA. Spiked Detected Average Accuracy Precision Concentration Concentration Concentration (%) (%) (nM) (nM) (nM) 4.72 1 90.9 3.7 5.01 4.81 4.69 10
13.07 13.03 13.55
13.22
93.2
2.2
50
50.82 51.38 51.71
51.30
94.8
0.92