Facts and artifacts in the measurement of oxidative base damage to DNA

This short survey is aimed at critically evaluating the main available methods for measuring oxidative base damage within cellular DNA. Emphasis is placed on separative methods which are currently widely applied. These mostly concern high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with sensitive detection techniques such as electrochemistry (EC) and mass spectrometry (MS). In addition, the comparison is extended to 32p-postlabeling methods, immunoassays and measurement of two main classes of oxidative DNA damage within isolated cells. It may be concluded that the HPLC-electrochemical detection (ECD) method, even if restricted to the measurement of only a few electroactive oxidized bases and nucleosides, is the simplest and safest available method at the moment. In contrast, the more versatile GC-MS method, which requires a HPLC pre-purification step in order to prevent artifactual oxidation of overwhelming normal bases to occur during derivatization, is more tedious and its sensitivity may be questionable. Alternative simpler procedures of background prevention for the GC-MS assay, which, however, remain to be validated, include low-temperature for derivatization and addition of antioxidants to the silylating reagents. Interestingly, similar levels of 8-oxo-7,8-dihydroguanine were found in cellular DNA using HPLC-ECD, HPLC-MS/MS and HPLC/32P-postlabeling methods. However, it should be noted that the level of cellular 8-oxodGuo, thus determined, is on average basis 10-fold higher than that was inferred for more indirect measurement involving the use of DNA repair enzymes with methods on isolated cells. Further efforts should be made to resolve this apparent discrepancy. In addition, the question of the biological validation of the non-invasive measurement of oxidized bases and nucleosides in urine is addressed.     

Recent aspects of oxidative DNA damage: guanine lesions,measurement and substrate specificity of DNA repair glycosylases

This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.     

Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 10⁶?C10⁸ normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2?C3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC?CMS/MS) is the most accurate and versatile approach whereas HPLC?Celectrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC?CMS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.     

Simultaneous measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine by HPLC-MS/MS

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry.     

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

Oxidative DNA damage, linked pathogenically to a variety of diseases such as cancer and ageing, can be investigated by measuring specific DNA repair products in urine. Within the last decade, since it was established that such products were excreted into urine, progress in their analysis in urine has been limited. Guanine is the DNA base most prone to oxidation. We present a method for determination of the urinary 8-hydroxylated species of guanine, based on direct injection of urine onto a high-performance liquid chromatography (HPLC)–tandem mass spectrometry system. The analysis covers the 8-hydroxylated base, ribonucleoside and deoxynucleoside, and the corresponding non-oxidised species. Without pre-treatment of urine the detection limits for the nucleobases are ~2 nM (50 fmol injected) and for the nucleosides ~0.5 nM (12.5 fmol injected). Previously, liquid chromatography of the nucleobases has been problematic but is made possible by low-temperature reverse-phase C18 chromatography, a method that increases retention on the column. In the case of the nucleosides, retention was almost total and provides a means for on-column concentration of larger urine samples and controlled high peak gradient elution. The total excretion of 8-hydroylated guanine species was 212 nmol/24 h. The oxidised base accounted for 64%, the ribonucleoside for 23% and the deoxynucleoside for 13%, indicating substantial oxidation of RNA in humans. In rat urine, excretion of the oxidised base was more dominant, the percentages of the oxidised base, ribonucleoside and deoxynucleosides being 89, 8 and 3%. This finding is at odds with previous reports using immunoaffinity pre-purification and HPLC–electrochemical detection analysis. The developed method now makes it possible to measure oxidative nucleic acid stress to both RNA and DNA in epidemiological and intervention settings, and our findings indicate a substantial RNA oxidation in addition to DNA oxidation. The small volume needed also makes the method applicable to small experimental animals.     

Oxidative damage to DNA: formation, measurement and biochemical features

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents including UVA and ionizing radiations. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.     

Room temperature derivatization of 5-hydroxy-2'- deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine for analysis by GC/MS

Hydroxylated DNA basesare one type of oxygen free radical-induced damage to DNA. Such damage has been implicated in the process of carcinogenesis, and the levels of hydroxylated DNA bases may serve as a marker of cancer risk in humans. Measurement of oxidative DNA damage can be hampered by the ease with which artifactual oxidative DNA damage can be induced via sample processing. In this report we describe convenient room temperature derivatization and stability of 5-hydroxy-2'-deoxycytidine (5-OHdCyd) and 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) using GC/MS analysis. The derivatization reagent was N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchloro-silane:acetonitrile, 2:1. This method avoids use of acid and is much milder than previously reported derivatization conditions which typically involve heating above 100C for at least 20 min. Although heating has been reported to be problematic, the calculated levels of 5-OHdCyd and 5-OHmdU in enzymatically-hydrolysed calf thymus DNA were very similar in our hands with and without heating the sample for 20 min. As an example of the technique, comparison of 5-OHdCyd and 5-OHmdU levels in calf thymus DNA indicated relatively higher endogenous levels of 5-OHdCyd. In DNA treated with hydrogen peroxide and ferric chloride, however, the levels of 5-OHmdU increased much more than that of 5-OHdCyd. In addition to these hydroxylated derivatives of deoxycytidine and thymidine, the method also appears to work well with 8-oxoguanine, 4,6-diamino-5-(formylamino)pyrimidine, and 5-methyl-2'-deoxycytidine. This method may therefore be useful with a variety of modified DNA bases and nucleosides.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans

Persistent oxidative stress and excess lipid peroxidation (LPO), induced by inflammatory processes, impaired metal storage, and/or dietary imbalance, cause accumulations and massive DNA damage. This massive DNA damage, along with deregulation of cell homeostasis, leads to malignant diseases. Reactive aldehydes produced by LPO, such as 4-hydroxy-2-nonenal, malondialdehyde, acrolein, and crotonaldehyde, react directly with DNA bases or generate bifunctional intermediates which form exocyclic DNA adducts. Modification of DNA bases by these electrophiles, yielding promutagenic exocyclic adducts, is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. Ultrasensitive detection methods have facilitated studies of the concentrations of promutagenic DNA adducts in human tissues, white blood cells, and urine, where they are excreted as modified nucleosides and bases. Thus, immunoaffinity-(32)P-postlabeling, high-performance liquid chromatography-electrochemical detection, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, immunoslotblot assay, and immunohistochemistry have made it possible to detect background concentrations of adducts arising from endogenous LPO products in vivo and studies of their role in carcinogenesis. These background adduct levels in asymptomatic human tissues occur in the order of 1 adduct/10(8) and in organs affected by cancer-prone inflammatory diseases these can be 1 or 2 orders of magnitude higher. In this review, we critically discuss the accuracy of the available methods and their validation and summarize studies in which measurement of exocyclic adducts suggested new mechanisms of cancer causation, providing potential biomarkers for cancer risk assessment in humans with cancer-prone diseases.     

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.     

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.     

A chemically synthesized peptoid-based drag-tag enhances free-solution DNA sequencing by capillary electrophoresis

We report a capillary-based DNA sequencing read length of 100 bases in 16 min using end-labeled free-solution conjugate electrophoresis (FSCE) with a monodisperse poly-N-substituted glycine (polypeptoid) as a synthetic drag-tag. FSCE enabled rapid separation of single-stranded (ss) DNA sequencing fragments with single-base resolution without the need for a viscous DNA separation matrix. Protein-based drag-tags previously used for FSCE sequencing, for example, streptavidin, are heterogeneous in molar mass (polydisperse); the resultant band-broadening can make it difficult to obtain the single-base resolution necessary for DNA sequencing. In this study, we synthesized and HPLC-purified a 70mer poly-N-(methoxyethyl)glycine (NMEG) drag-tag with a molar mass of - 11 kDa. The NMEG monomers that comprise this peptoid drag-tag are interesting for bioanalytical applications, because the methoxyethyl side chain's chemical structure is reminiscent of the basic monomer unit of polyethylene glycol, a highly biocompatible commercially available polymer, which, however, is not available in monodisperse preparation at an - 11 kDa molar mass. This is the first report of ssDNA separation and of four-color, base-by-base DNA sequencing by FSCE through the use of a chemically synthesized drag-tag. These results show that high-molar mass, chemically synthesized drag-tags based on the polyNMEG structure, if obtained in monodisperse preparation, would serve as ideal drag-tags and could help FSCE reach the commercially relevant read lengths of 100 bases or more.     

Analysis of purine and pyrimidine bases,ribonucleosides, and ribonucleotides by high-pressure liquid chromatography

A high-pressure liquid chromatographic method has been developed for the separation and quantitation of purine and pyrimidine bases, ribonucleosides, and ribonucleotides. The procedure is carried out on a 1.8 × 700-mm column packed with Aminex-A-25 anion-exchange resin. The column is eluted with a linear gradient of ammonium chloride. The elution buffer contained borate also to complex the sugar phosphates and ethanol to improve the separation of bases and nucleosides. The analysis is completed in about 160 min. The potential application of this method for the quantitation of acid-soluble metabolites in fibroblasts is described.     

Permanganate oxidation of nucleic acid components: a reinvestigation.

KMnO4 consumption in solution by nucleoside added at an excess was measured to explore any possible oxidative interactions between these molecules. Thymidine, 5-methyldeoxycytidine and deoxycytidine rapidly decomposed KMnO4 with the pseudo-first-order kinetics (thymidine greater than 5-methyl-deoxycytidine greater than deoxycytidine), while deoxyguanosine showed only a very slow consumption and deoxyadenosine did not decompose KMnO4 at all under the conditions employed (0.16 mM KMnO4, 0.80 mM nucleoside, at 27 degrees C and pH 4.3, 7.4 and 8.6, up to 60 min). UV absorption spectra of KMnO4-treated nucleosides also indicated modification of the pyrimidine nucleosides but not of the purine nucleosides. These results are consistent with the original report of Hayatsu and Ukita published in 1967 on the KMnO4 oxidation of nucleosides, and provide difficulty in understanding the mechanism of recently reported formation of 8-hydroxypurines on treatment of DNA with KMnO4.     

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel

The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.     

Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo

Oxidative modification of genetic material has been implicated as a factor in carcinogenesis, particularly during promotion and progression, and therefore there is a need for sensitive detection of oxidized DNA bases. We developed a method that can be applied to DNA isolated from any source and used to simultaneously quantify oxidized nucleosides without a need to prelabel the DNA or use destructive hydrolytic procedures. This method is based on: (a) enzymatic DNA digestion; (b) HPLC separation of the resultant nucleosides; (c) acetylation of the oxidized nucleosides with [3H]Ac2O (acetic anhydride); (d) removal of the radioactive debris; and (e) quantitative analysis of tritiated nucleoside acetates by HPLC. Enzymatic DNA digestion was optimized using DNase I in the presence of Mg2+ (pH 7), followed by nuclease P1 in the presence of Zn2+ (pH 5.1) and alkaline phosphatase (pH 7.5). Analysis of DNA oxidized with H2O2 in the presence of Fe2+/EDTA for 30 min showed that the levels of 8-OHdG (8-hydroxy-2'-deoxyguanosine) were increased 2.7-fold, HMdU (5-hydroxymethyl-2'-deoxyuridine) 3.15-fold, and FdU (5-formyl-2'-deoxyuridine) 2.5-fold. Although the (-)-isomer of cis-dTG (cis-thymidine glycol) was enhanced 2.3 times, the (+)-isomer remained virtually unchanged. Analysis of DNA isolated from epidermal cells of mice treated in vivo with the tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) showed 4.8-, 2.7-, and 8.7-fold increases in the levels of total cis-dTG, 8-OHdG, and HMdU, respectively, and of some unknown DNA oxidation products. These results prove applicability of the 3H-postlabeling method to the analysis of DNA (and potentially RNA) isolated from many sources, including animals and humans.     

Assay for the determination of urinary 8-hydroxy-2′-deoxyguanosine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2′-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2′-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2′-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.     

Commentary the Measurement of Oxidative Damage to DNA by HPLC and GC/MS Techniques

Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2′-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a “routine” marker of oxidative DNA damage in vivo.     

High-performance liquid chromatographic method with electrochemical detection for the analysis of O6-methylguanine

An improved system consisting of a combination of high-performance liquid chromatographic methods with electrochemical detection for the separation and analysis of the DNA adduct O⁶-methylguanine (O6MG) has been developed. This adduct is produced by the interaction of methylating agents with DNA and induces mispairing in the DNA of the target cells. A good separation of modified from unmodified bases is first achieved with an HPLC system using a Partisil 10 SCX column and a salt gradient. A second HPLC step with electrochemical detection and a C18 column is used for farther separation and quantitation of O⁶-methylguanine. This method shows a linear response up to 15 pg of O6MG tested. The lowest amount detected was 0.5 pg of O6MG and is highly reproducible. This method is useful to study DNA damage as a product of cellular metabolism and its effects on the process of carcinogenesis.     

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.