Deoxyguanosine In Human Urine Research Articles

Development and validation of an HPLC-MS/MS method for the simultaneous analysis of volatile organic compound metabolites, hydroxylated polycyclic aromatic hydrocarbons, and 8-hydroxy-2′-deoxyguanosine in human urine

Humans are widely and concurrently exposed to volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs). However, few studies have reported the internal co-exposure levels of these chemicals in occupational and general populations. Specifically, the associations revealed between the urinary levels of metabolites of VOCs (mVOCs), hydroxylated PAHs (OH-PAHs), and oxidative stress biomarkers for humans remain limited. In this study, a method based on solid-phase extraction (SPE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of 22 mVOCs, 12 OH-PAHs, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in human urine samples. The method was validated with all target analyte accuracies and precisions in the range of 76 %–120 % and 1 %–14 % at three levels of spiked urine samples, respectively. The limit of detection (LOD) and limit of quantification (LOQ) of the target analytes were 0.01–0.34 ng/mL and 0.01–7.57 ng/mL, respectively. And the method was applied to measure urinary levels of target analytes from 38 petrochemical workers in Guangzhou, South China. Except for 3-hydroxy-benzo[a]pyrene, all target analytes were detected in the urine samples. The average levels were 0.05–12.6 ng/mL for individual OH-PAHs, 0.20–73620 ng/mL for individual mVOCs, and 1.00 ng/mL for 8-OHdG. Additionally, 3-hydroxy-phenanthrene, 1-hydroxy-pyrene, 6-hydroxy-chrysene, N-acetyl-S-(trichlorovinyl)-L-cysteine, 2-methylhippuric acid, thiodiacetic acid, trans, trans-Muconic acid, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine had statistically significant positive effects on 8-OHdG levels, while 1-hydroxy-naphthalene, 1,2-dihydroxybenzene, and hippuric acid showed a negative effect on 8-OHdG, indicating these metabolites could lead to synergistic or antagonistic oxidative DNA damage. This study provides a robust analytical method that permits a comprehensive assessment of co-exposure to PAHs and VOCs and their potential adverse health effects.

Comparison of an HPLC-MS/MS Method with Multiple Commercial ELISA Kits on the Determination of Levels of 8-oxo-7,8-Dihydro-2'-Deoxyguanosine in Human Urine

Introduction: Analysis of 8-oxodG is usually conducted by either chromatography-based methods or by immunochemical methods commonly used based upon their low cost and high-throughput. However, concern regarding the accuracy of ELISA methods has complicated their use. We directly compare the levels of urinary 8-oxodG obtained by HPLC-MS/MS with three commercially available ELISA kits in this report. Methods: In the current study, a total of 9 human urine samples were analyzed by LC-MS/MS and three commonly used commercial available ELISA kits. Results: We found that urinary 8-oxodG levels analyzed by HPLC-MS/MS [1.4 ± 0.3 nmol/mmol creatinine) were 7.6- to 23.5-fold lower than those detected by ELISA. Overall, the correlations between ELISA and HPLC-MS/MS were poor but were improved after SPE purification for kits from ENZO (P = 0.2817 without SPE; P = 0.0086 with SPE) and Abcam (P = 0.0596 without SPE; P = 0.0473 with SPE). Discussion and conclusion: While we confirmed that SPE purification can improve the correlation between the selected ELISA kits and HPLC-MS/MS, HPLC-MS/MS is still the method of choice to accurately assess the levels of 8-oxodG in human urine.

A novel aptamer-based online magnetic solid phase extraction method for the selective determination of 8-hydroxy-2′-deoxyguanosine in human urine

In this work, an innovative magnetic aptamer adsorbent (Fe3O4-aptamer MNPs) was synthesized for the selective extraction of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Amino-functionalized-Fe3O4 was crosslinked with 8-OHdG aptamer by glutaraldehyde and fixed into a steel stainless tube as the sorbent of magnetic solid phase extraction (MSPE). After selective extraction by the aptamer adsorbent, the adsorbed 8-OHdG was desorbed dynamically and online analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The synthesized sorbent presented outstanding features, including specific selectivity, high enrichment capacity, stability and biocompatibility. Moreover, this proposed MSPE-HPLC-MS can achieve adsorption and desorption operation integration, greatly simplify the analysis process and reduce human errors. When compared with offline MSPE, a sensitivity enhancement of 800 times was obtained for the online method. Some experimental parameters such as the amount of the sorbent, sample flow rate and sample volume, were optimized systematically. Under the optimal conditions, low limit of detection (0.01 ng mL−1, S/N = 3), limit of quantity (0.03 ng mL−1, S/N = 10) and wide linear range with a satisfactory correlation coefficient (R2 ≥ 0.9992) were obtained. And the recoveries of 8-OHdG in the urine samples varied from 82% to 116%. All these results revealed that the method is simple, rapid, selective, sensitive and automated, and it could be expected to become a potential approach for the selective determination of trace 8-OHdG in complex urinary samples.

A comparison of electrochemically pre-treated and spark-platinized carbon fiber microelectrode. Measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in human urine and plasma

A novel method of carbon fiber microelectrode activation using spark discharge was demonstrated and compared to conventional electrochemical pretreatment by potential cycling. The spark discharge was performed at 800 V between the microelectrode connected to positive pole of the power supply and platinum counter electrode. Spark discharge led both to trimming of the fiber tip into conical shape and to the modification of carbon fiber microelectrode with platinum, as proven by scanning electron microscopy and electron dispersive X-ray spectroscopy. After the characterization of electrochemical properties using ferricyanide voltammetry, the activated electrodes were used for electrochemical analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative stress marker. Subnanomolar detection limits (0.55 nmol L−1) in high-performance liquid chromatography were achieved for spark platinized electrodes incorporated into the flow detection cell.

Detection of 1,N(2)-propano-2'-deoxyguanosine in human urine by stable isotope dilution UHPLC-MS/MS analysis.

A sensitive and accurate stable isotope dilution UHPLC-MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8-107.2%, and the precision are about 0.8-3.6% for intraday and 2.8-10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N=3) and quantification (LOQs, S/N=10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC-MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine.

Analysis of 8-hydroxy-2'-deoxyguanosine in human urine by ultra-high performance liquid chromatography mass spectrometer method

To develop an ultra-high performance liquid chromatography mass spectrometer method for the rapid determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human urines. 8-OHdG standard solution with the concentration from 0.01 to 0.1 μg/ml was formulated. The solution was implanted into ion source with a rate of 7 μl/min, the mass-to-charge ratio of parent ion and product ions, and ion mass to charge ratio was identified. The mass spectrum parameters of each Ion pairs, such as DP, EP and EXP, were gradually optimized. The urine sample with a concentration of 10.0 μg/L was detected, and the pH of the sample was adjusted using 1 mol/L ammonium formate and formic acid solution with a volume ratio of 5∶1, 4∶1, 3∶1, 2∶1, and 1∶1. It was tested using three different polarity of SPE, i.e.: HLB, MCX, and MAX. The elution effect of methanol and water mixture with the proportion of 90∶10, 80∶20, 50: 50, 20: 80, and 10: 90 were tested, and then acetonitrile and water mixture with the proportion of 90∶10, 80∶20, 50∶50, 20∶80, 10∶90 were also tested. The standard curve was constructed using the ratio of a standard series application fluid concentration to corresponding compounds quantitative ion liquid concentration of the peak area. The detection limit was determined as 3 times of the signal to noise ratio corresponding to the concentration of 8-OHdG, and the quantitative lower limit was determined as 10 times of the signal to noise ratio corresponding to the concentration of 8-OHdG. The blank urine spiked recovery method was used to evaluate the precision and recovery rate. The mass to charge ratio of parent ion was 284.1 and the product ions was 168.1, 140.1, 123.0, and 112.0, respectively. The collision voltage of quantitative ion-pair 284.1/168.1 was 18 V, the 284.1/140.1 collision voltage was 42 V, the 284.1/123.0 collision voltage was 48 V, and the 284.1/112.0 collision voltage was 53 V. The recovery rate was the highest (87.9%-104.3%) when the pH of urine was adjusted by a 10 ml 1 mol/L ammonium formate solution, 2 ml of formic acid, 88 ml of water are mixed with the sample solution volume ratio of 1∶5, and then purified with 3 ml of methanol and 3 ml water activated HLB extraction column. Within 1.0-100.0 μg/L concentration range, 8-OHdG standard application solution test results showed a good linear relationship. The regression equation was y= 1.25x+0.74, and the correlation coefficient was r=0.999 5. The detection limit was 0.2 μg/L, and the limit of quantification was 0.7 μg/L. The method of recovery rate was in the range of 87.9% to 104.3%, the precision was in the range from 1.5% to 3.7% and inter-assay precision was in the range from 1.6% to 5.4%. The method developed in this study had high sensitivity, good precision and accuracy, and a wide range of testing concentrations.

Analysis of 8-hydroxy-2′-deoxyguanosine in human urine using hydrophilic interaction chromatography with tandem mass spectrometry

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used noninvasive biomarker of oxidative stress. A selective, sensitive and rapid method for determining 8-OHdG in human urine was developed using hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) with electrospray ionization. 8-OHdG and isotopically labeled 8-OHdG (internal standard) were separated on a HILIC column with a mobile phase of 10mM ammonium acetate: acetonitrile (1:9, v/v) within 10min and detected by using a positive electrospray ionization interface under the selected reaction monitoring mode. The detection limits of 8-OHdG (corresponding to a signal-to-noise ratio of 3) for the HILIC-MS/MS system and the conventional method using a reversed-phase column with MS/MS were 1.0 and 26.0fmol/injection, respectively. The proposed method makes it possible to monitor the basal level of urinary 8-OHdG from non-exposed healthy subjects and can be used for large-scale human studies.

A high performance liquid chromatography-electrochemical array method for the measurement of oxidative/nitrative changes in human urine

Oxidative stress has been implicated in various pathologies as well as in environmental pollutant-induced negative health outcomes. In this work we have developed an analytical method to measure oxidative stress markers namely m-, o-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine, and the DNA damage marker 8-hydroxy deoxyguanosine in human urine. The method involves the base hydrolysis of the urine sample followed by solid phase extraction of target analytes using a reverse phase polymeric sorbent column prior to the HPLC-EC analysis. The recovery studies indicated that the overall method recovery for all analytes were >60%. The limit of quantification of all target analytes was <10 nM with a linear range from 2 nM to 150 μM. The applicability of this method is demonstrated by analyzing the above markers in healthy human urine ( n = 10) samples.

Automated method for the direct analysis of 8-oxo-guanosine and 8-oxo-2′-deoxyguanosine in human urine using ultraperformance liquid chromatography and tandem mass spectrometry

The potential use of oxidative stress-induced DNA and RNA damage products as biomarkers is an important aspect of biomedical research. There is a need for assays with high specificity and sensitivity that also can be used in molecular epidemiology studies with a large number of subjects. In addition there is a need for assays that can measure more than one product from DNA oxidation. We present a sensitive, precise, and accurate method for quantitative analysis of the oxidized nucleosides 8-oxoGuo and 8-oxodG in human urine. The assay is based on automated sample handling using a BIOMEK 3000 Workstation, and UPLC-ESI(+)-MS/MS analysis. High specificity is evidenced by the use of qualifier ions for both analytes. The quantification limit in urine samples is 1 nM for both analytes. Accuracy and precision were documented, showing average recoveries of 106.2% (8-oxoGuo) and 106.9% (8-oxodG), and overall precision (within-day and between-days) of 6.1 and 4.4%, respectively.

Evidence of oligonucleotides containing 8-hydroxy-2′-deoxyguanosine in human urine

The product of oxidative damage to DNA, 8-hydroxy-2′-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of < 30–55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.

Quantitative determination of 8-hydroxy-2′-deoxyguanosine in human urine by isotope dilution mass spectrometry: normal levels in hemochromatosis

A previously developed method for quantitative determination of 8-hydroxyguanine by gas chromatography-mass spectrometry was modified to allow measurement of 8-hydroxy-2′-deoxyguanosine in human urine. [4,5,6,8- 13C 4]8-Hydroxy-2′-deoxyguanosine was prepared by enzymatic coupling of [4,5,6,8- 13C 4]8-hydroxyguanine to deoxyribose-1-phosphate. Samples of human urine (2 ml) were spiked with the labeled nucleoside (13 nmol) and subjected to solid phase extraction and reversed phase high performance liquid chromatography. The 8-hydroxy-2′-deoxyguanosine thus isolated was hydrolyzed by treatment with aqueous formic acid, and the resulting 8-hydroxyguanine was converted into its tetrakis-trimethylsilyl derivative and subjected to gas-liquid chromatographic-mass spectrometric analysis. Repeated determinations of 8-hydroxy-2′-deoxyguanosine in pools of urine showed coefficients of variation of 5 and 8% at concentrations of 8-hydroxy-2′-deoxyguanosine equal to 18 and 2 nM, respectively. Determination of 8-hydroxy-2′-deoxyguanosine in samples of urine spiked with different amounts of the unlabeled nucleoside showed a mean recovery of 102%. Application of the analytical method to a group of 11 apparently healthy subjects (mean age, 47 years) showed a mean level of endogenously produced 8-hydroxy-2′-deoxyguanosine equal to 1.33 ± 0.29 μmol/mol creatinine. The level recorded for another group of 15 younger subjects (mean age, 28 years) was somewhat higher, that is, 1.58 ± 0.84 μmol/mol creatinine, corresponding to a 24-h production rate of 8-hydroxy-2′-deoxyguanosine equal to 20.6 ± 11.6 nmol (288 ± 140 pmol/24 h · kg body weight). Hemochromatosis is a hereditary disease characterized by increased absorption of iron from the gastrointestinal tract and deposition of iron in organs. Application of the analytical method to a group of 12 patients with hereditary hemochromatosis who were under treatment with venesections showed a mean level of urinary 8-hydroxy-2′-deoxyguanosine equal to 1.39 ± 0.40 μmol/mol creatinine. This value was not significantly different from those of healthy subjects. The fact that these patients had only slight or moderate iron overload at the time of urinary sample collection may have influenced the urinary levels of 8-hydroxy-2′-deoxyguanosine in the present study.