Evaluation of enzyme-linked immunosorbent assay and liquid chromatography–tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Quantitative analysis of two opioid peptides in plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

Quantitative analysis of two opioid peptides, DSLET [(d-Ser²)Leu-enkephalin-Thr⁶] and Met-enkephalin-Arg-Gly-Leu, was performed using microbore liquid chromatography interfaced to electrospray ionization tandem mass spectrometry. Validation of the methodology was demonstrated for each peptide in plasma. Quantitative analyses were performed through the use of a deuterium labelled peptide analog as an internal standard. Linearity was observed for the analysis of DSLET (5–1000 ng/ml) and Met-enkephalin-Arg-Gly-Leu (1–1000 ng/ml) in plasma with a limit of detection of 0.25 ng/ml for Met-enkephalin-Arg-Gly-Leu and 1.0 ng/ml for DSLET. In general, the observed concentrations showed good reproducibility with coefficients of variation of within 15%. In the concentration range studied, only 0.5 ml of plasma was required for optimal detection of Met-enkephalin-Arg-Gly-Leu and 0.25 ml for DSLET. Application of this method was demonstrated by studying the disposition of DSLET in a rat. DSLET administered to a rat exhibited a short half-life and a high clearance value.     

Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography–electrospray ionization tandem mass spectrometry

Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53–7.09% and 0.40–2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.     

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r² > 0.997, n = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n = 5) and inter-day (n = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Inter-laboratory Validation of Procedures for Measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2′-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Optimized analytical method for cyclosporin A by high-performance liquid chromatography–electrospray ionization mass spectrometry

The Micromass Platform LCZ mass detector parameters were optimized for simultaneous recording of the protonated (CsA∼H⁺), sodium adduct (CsA∼Na⁺) and potassium adduct (CsA∼K⁺) of cyclosporin A eluted from a Symmetry Shield RP8 column. The optimized procedure allows a precise analysis of CsA in whole blood or serum without removal of salts prior to analysis. The ratio of the three forms of CsA varied depending on the assay condition and the types of specimens being analyzed. The summation of three ionic forms of CsA detected by LC–ESI-MS is a reliable and simple method to assess CsA concentration in the blood.     

Characterization of the triacylglycerol profile in marine diatoms by ultra performance liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry

Diatoms are considered to have great potential as new biofuel sources because they can effectively accumulate triacylglycerols (TAGs). Detailed structure information of TAG in diatoms is much needed not only for the assessment of biofuel quality such as fatty acid chain length and unsaturation degree but also for the tracing of biosynthetic precursors because the biosynthesis of TAG is typically completed by utilizing the diacylglycerol acyltransferase in the cytoplasm. In this report, a comprehensive characterization of TAGs in marine diatoms was performed using ultra performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Many types of major TAGs were identified for the first time in these diatoms: 12 TAGs in Chaetoceros debilis, 9 TAGs in Phaeodactylum tricornutum Bohlin, 16 TAGs in Nitzschia closterium f. minutissima, 16 TAGs in Thalassiosira weissflogii, 13 TAGs in Thalassiosira sp., 16 TAGs in Stephanodiscus asteaea and 7 TAGs in Skeletonema costatum. Semi-quantification of TAGs in these diatoms was also carried out, and it was found that the contents of individual TAGs ranged from 0.5?±?0.1 to 217.9?±?8.1 nmol mg^?1 total lipids. In addition, the total lipid contents in diatoms ranged from 143.6?±?16.3 to 201.1?±?16.3 mg g^?1 dry microalgae and the total TAG contents ranged from 36.8?±?9.5 to 793.2?±?54.4 nmol mg^?1 total lipids. By comparative analysis of the compositions and concentrations of major TAGs in the seven algal strains, N. closterium f. minutissima with high abundance of TAGs containing the most monounsaturated fatty acids (mainly palmitoleic acid) was considered as one of the most promising diatom strains for microalgal biofuel production. Additionally, based on the information of sn-2 fatty acid obtained (mainly C16 in the sn-2 position), we propose the hypothesis that TAGs in diatoms are mainly derived from lipids in chloroplasts through the prokaryotic biosynthesis pathway, including monogalactosyldiacylglycerol and digalactosyldiacylglycerol.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography–mass spectrometry

We first detected glutathionyl hemoglobin (Hb) β-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography–mass spectrometry. The ratio of glutathionyl Hb β-chain to total β-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.     

Harmonising measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and urine

Abstract

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2′-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2′-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Determination of methocarbamol concentration in human plasma by high performance liquid chromatography–tandem mass spectrometry

A simple and reproducible high performance liquid chromatography–tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150–12,000 ng/mL with good intraday assay and interday assay precision (CV% < 10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Quantification of immature and mature collagen crosslinks by liquid chromatography–electrospray ionization mass spectrometry in connective tissues

We describe a novel high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) method for the simultaneous quantification of enzymatic immature (dihydroxylysinonorleucine DHLNL, hydroxylysinonorleucine HLNL) and mature (pyridinoline PYD, deoxypyridinoline DPD) collagen crosslinks in connective tissues. The crosslinks were separated on a C18 Atlantis^® T3 reversed-phase column with heptafluorobutyric acid (HFBA) as volatile ion-pairing reagent in an acetonitrile–water mobile phase. Detection was carried out by electrospray ionization mass spectrometry in a positive ion mode with selected ion recording (SIR). This method is more sensitive and selective than ion exchange chromatography with post-column ninhydrin detection which is the reference method used for the simultaneous quantification of collagen enzymatic divalent and trivalent crosslinks. The intra and inter-day precision errors were less than 3.4 and 7.7%, respectively for DHLNL, 3.5 and 5.9%, respectively for HLNL, 4.0 and 5.2%, respectively for PYD, 8.2 and 10.7%, respectively for DPD. This novel technique should be useful to quantify simultaneously DHLNL, HLNL, PYD and DPD in connective tissues and to evaluate the maturation of collagen by determination of the ratio between immature and mature enzymatic crosslinks.     

Urinary analysis of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: Study of 8-oxo-7,8-dihydroguanine stability

A highly sensitive quantitative method based on LC-MS/MS was developed to simultaneously and directly measure 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in urine. It was found that 8-oxoGua could be artifactually generated from 8-oxodGuo during the ionization process by both in-source thermolysis and collisionally induced dissociation. Our method applied a two-stage wash procedure in the online solid-phase extraction system that not only eliminated ion suppression but also prevented artifactual interference with 8-oxoGua by 8-oxodGuo by eluting the analytes individually. With the use of isotope internal standards, the detection limits of 8-oxoGua and 8-oxodGuo were estimated to be 30 and 3.5 fmol, respectively. The 8-oxoGua stability under common storage conditions was first investigated. Dissolved 8-oxoGua in NaOH (pH 12) was quite fragile and stable for only < 1 day at room temperature. When pH and temperature were reduced, the 8-oxoGua stability at ? 20°C was significantly increased to ～ 87 days in water (pH ～ 7) and ～ 112 days when diluted in 5% methanol. This method was further applied to measure urinary samples of healthy subjects. A molar ratio of 8-oxoGua to 8-oxodGuo of ～ 4.6 was found, supporting the hypothesis that oxidatively damaged DNA is primarily repaired by the base excision repair pathway.     

Leukocyte 8-oxo-7,8-dihydro-2′-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane—an iron chelator—was proven to decrease free radical production and anthracycline cardiotoxicity.

In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) relative to 2′-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry.

Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3?μM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant ( ).

This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.