Monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.     

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.     

超高效液相色谱-串联质谱法测定氧化损伤标志物8-羟基脱氧鸟苷

发展了一种超高效液相色谱-串联质谱法(UPLC-MS/MS)检测脱氧核糖核酸(DNA)分子中8-羟基脱氧鸟苷(8-OHdG)的方法。DNA分子在酶解过程中,脱氧鸟苷(dG)易被氧化形成8-OHdG,从而使得8-OHdG的检测结果不准确。通过加入甲磺酸去铁铵作为保护剂,有效地避免了酶解过程造成的dG氧化。酶解液通过超滤膜(截留相对分子质量为3 000的分子)处理,有效去除大量蛋白分子后,直接进行UPLC-MS/MS测定。采用外标法定量,在17.6～1 400fmol范围内,8-OHdG的峰面积与其物质的量具有良好的线性关系,相关系数为0.999 8。利用本方法测定了小牛胸腺DNA中8-OHdG的含量(用比值8-OHdG/106dG表示)为12.9±2.35,与前人报道的检测结果一致。本方法也可以应用于评价各种氧化因素引起的DNA氧化损伤。     

One-electron oxidation of the guanine moiety of 2'-deoxyguanosine: influence of 8-oxo-7,8-dihydro-2'-deoxyguanosine

The influence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) on riboflavin and UVA-mediated one-electron oxidation of an aqueous aerated solution of 2'-deoxyguanosine (dGuo) has been studied. Using labeled experiments, we have demonstrated that, despite not being able to detect significant amounts of 8-oxodGuo upon one-electron oxidation of dGuo, 8-oxodGuo is indeed produced but is further rapidly degraded to oxidized nucleosides. Evidence is provided showing that an efficient electron transfer reaction from 8-oxodGuo to the guanine radical cation or rather its deprotonated form occurs, giving rise to the specific decomposition of 8-oxodGuo together with the restitution of dGuo. It could be concluded that 8-oxodGuo efficiently protects dGuo from decomposition by the one-electron oxidation reaction.     

Simultaneous electrochemical sensing of ascorbic acid,dopamine and uric acid at anodized nanocrystalline graphite-like pyrolytic carbon film electrode

Nanocrystalline graphite-like pyrolytic carbon film (PCF) electrode fabricated by a non-catalytic chemical vapor deposition (CVD) process was used for the simultaneous electrochemical sensing of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The electrode was studied with respect to changes in electrocatalytic activity caused by a simple and fast electrochemical pretreatment. The anodized electrode exhibited excellent performance compared to many chemically modified electrodes in terms of detection limit, linear dynamic range, and sensitivity. Differential pulse voltammetry (DPV) was used for the simultaneous determination of ternary mixtures of DA, AA, and UA. Under optimum conditions, the detection limits were 2.9 μM for AA, 0.04 μM for DA, and 0.03 μM for UA with sensitivities of 0.078, 5.345, and 6.192 A M⁻¹, respectively. The peak separation was 219 mV between AA and DA and 150 mV between DA and UA. No electrode fouling was observed and good reproducibility was obtained in all the experiments. The sensor was successfully applied for the assay of DA in an injectable drug and UA in human urine by using standard addition method.     

Simultaneous determination of epinephrine, uric acid and xanthine in the presence of ascorbic acid using an ultrathin polymer film of 5-amino-1,3,4-thiadiazole-2-thiol modified electrode

This paper describes the simultaneous determination of epinephrine (EP), uric acid (UA) and xanthine (XN) in the presence of ascorbic acid (AA) using electropolymerized ultrathin film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.2 M phosphate buffer solution (pH 5). Although bare GC electrode resolves the voltammetric signals of AA and XN, it fails to resolve the voltammetric signals of EP and UA in a mixture. However, the p-ATT modified electrode not only separates the voltammetric signals of AA, EP, UA and XN with potential difference of 150, 120 and 400 mV between AA-EP, EP-UA and UA-XN, respectively but also shows higher oxidation current for these molecules. The p-ATT modified electrode exhibits excellent selectivity towards the oxidation of EP, UA and XN in the presence of 40-fold higher concentration of AA. Further, the p-ATT modified electrode was also used for the selective determination of EP in the presence of 40-fold higher concentrations of AA, UA and XN. Using amperometric method, we achieved the lowest detection of 40 nM EP and 60 nM each UA and XN. The amperometric current response was increased linearly with increasing EP concentration in the range of 4.0 × 10⁻⁸ to 4.0 × 10⁻⁵ M and the detection limit was found to be 27 × 10⁻¹¹ M (S/N = 3). The practical application of the present modified electrode was demonstrated by determining the concentration of EP in epinephrine tartrate injection and XN in human urine samples.     

Characterization of 5-hydroxy-8-oxo-7,8-dihydroguanosine in the photosensitized oxidation of 8-oxo-7,8-dihydroguanosine and its rearrangement to spiroiminodihydantoin

The photosensitized oxidation of 2',3',5'-tris-(O-tert-butyldimethylsilyl)-8-oxo-7,8-dihydroguanosine (8-oxoG) with singlet oxygen was studied by low-temperature NMR. A stable intermediate was characterized at -60 degrees C by (13)C, 2D NMR HMBC spectra, and chemical shifts calculated by hybrid Hartree-Fock density functional theory which agreed with the structure 5-hydroperoxy-8-oxo-7,8-dihydroguanosine. Reduction of this intermediate at low temperature afforded the corresponding alcohol, the long-postulated 5-hydroxy-8-oxo-7,8-dihydroguanosine, the last intermediate in the formation of spiroiminodihydantoin. Upon warming to room temperature, this alcohol rearranges to form the spiroiminodihydantoin in good yield within 2 h.     

Combination of nitrogen dioxide radicals with 8-oxo-7,8-dihydroguanine and guanine radicals in DNA: oxidation and nitration end-products

The oxidation and nitration reactions in DNA associated with the combination of nitrogen dioxide radicals with 8-oxo-7,8-dihydroguanine (8-oxoGua) and guanine radicals were explored by kinetic laser spectroscopy and mass spectrometry methods. The oxidation/nitration processes were triggered by photoexcitation of 2-aminopurine (2AP) residues site-specifically positioned in the 2'-deoxyribooligonucleotide 5'-d(CC[2AP]TC[X]CTACC) sequences (X = 8-oxoGua or G), by intense 308 nm excimer laser pulses. The photoionization products, 2AP radicals, rapidly oxidize either 8-oxoGua or G residues positioned within the same oligonucleotide but separated by a TC dinucleotide step on the 3'-side of 2AP. The two-photon ionization of the 2AP residue also generates hydrated electrons that are trapped by nitrate anions thus forming nitrogen dioxide radicals. The combination of nitrogen dioxide radicals with the 8-oxoGua and G radicals occurs with similar rate constants (approximately 4.3 x 10(8) M(-1) s(-1)) in both single- and double-stranded DNA. In the case of 8-oxoGua, the major end-products of this bimolecular radical-radical addition are spiroiminodihydantoin lesions, the products of 8-oxoGua oxidation. Oxygen-18 isotope labeling experiments reveal that the O-atom in the spiroiminodihydantoin lesion originates from water molecules, not from nitrogen dioxide radicals. In contrast, combination of nitrogen dioxide and guanine neutral radicals generated under the same conditions results in the formation of the nitro products, 5-guanidino-4-nitroimidazole and 8-nitroguanine adducts. The mechanistic aspects of the oxidation/nitration processes and their biological implications are discussed.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.     

FORMATION OF 7,8-DIHYDRO-8-OXOGUANINE IN THE 1,2-DIOXETANE-INDUCED OXIDATION OF CALF THYMUS DNA: EVIDENCE FOR PHOTOSENSITIZED DNA DAMAGE BY THERMALLY GENERATED TRIPLET KETONES IN THE DARK

Abstract— Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxymethyl-3,4,4-tri-methyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane, 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxeta-ne (HTMD), 3,3,4,4-tetramethyl-1,2-dioxetane and 3,4,4-trimethyl-1,2-dioxetane (TrMD), which on thermal decomposition generate triplet-excited carbonyl products. To monitor quantitatively the formation of the mutagenic oxidation product 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) of the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of the available guanine) were obtained for HTMD and TrMD. Both were investigated in detail with respect to effects of concentration, time and temperature. The oxidative reactivity of 1,2-dioxetanes was compared with several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-ben-zodioxin-1,4-dipropionate as a chemical source of singlet oxygen. The persistence of 8-oxoGua towards oxidation by HTMD was examined in the reaction with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with oxidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the oxidized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R* and 4S* diastereomers of 9-(2-deoxy-ß-D-erythropentofuranosyl)-4,8-dihydro-4-hydroxy-8-oxoguanine as main oxidation products. Trapping experiments with teft-butanol confirmed that hydroxyl radicals are not involved, whereas the use of the triplet quenchers sodium 9,10-dibromo-anthra-cene-2-sulfonate and 2,3-diazabicyclo[2.2.1]hept-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transfer chemistry). The role of singlet oxygen was tested by means of deuterium isotope effects in D₂O versus H₂O, but no definitive conclusion could be reached in regard to the involvement of ¹0₂ in these oxidations. The present results reveal that 1,2-dioxetanes are efficient DNA oxidants and excellent tools to study photooxidation reactions of DNA in the dark.     

Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2'-deoxyguanosine by high-performance liquid chromatography-electrospray tandem mass spectrometry as a measure of oxidative stress

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and (13)C- and (15)N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.     

Supramolecular Cationic Tetraruthenated Porphyrin Induces Single-Strand Breaks and 8-Oxo-7,8-dihydro-2'-deoxyguanosine Formation in DNA in the Presence of Light

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct-fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μ M ) and light leads to single-strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8-oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near-infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.     

Formation of cyclobutane pyrimidine dimers and 8-oxo-7,8-dihydro-2'-deoxyguanosine in mouse and organ-cultured human skin by irradiation with broadband or with narrowband UVB

Narrowband UVB (NB-UVB) is a newly developed UVB source that, in addition to the previously used broadband UVB (BB-UVB), has been effectively used in phototherapy of various skin diseases. Besides its therapeutic effectiveness, NB-UVB also has some adverse effects that should be evaluated. As with all phototherapies, the photocarcinogenic potential of NB-UVB is the major concern. To assess the carcinogenic potential we measured the DNA damage induced by the two UVB sources because exposure of cells to UVB directly or indirectly induces DNA damage such as cyclobutane pyrimidine dimers (CPD) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. These types of DNA damage cause mutations of oncogenes and tumor suppressor genes, which can lead to photocarcinogenesis. In the present study we measured the yield of CPD and the oxidative DNA damage marker, 8-oxodGuo, in organ-cultured human skin and in mouse skin after exposure to NB-UVB or BB-UVB at therapeutically equivalent doses. We show that a 10-fold higher dose of NB-UVB yields a similar amount of CPD compared with BB-UVB in two types of samples examined. In contrast to CPD, the formation of 8-oxodGuo after irradiation with NB-UVB at a 10-fold higher dose is 1.5-3 times higher than that caused by BB-UVB. These results suggest that although NB-UVB at equivalent erythema-edema doses is not more potent in inducing CPD formation than is BB-UVB, NB-UVB may generate a higher yield of oxidized DNA damage.     

Simultaneous determination of uric acid,xanthine, hypoxanthine and caffeine in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode

This paper describes the fabrication of graphene on glassy carbon electrode (GCE) by electrochemical reduction of graphene oxide (GO) attached through 1,6-hexadiamine on GCE and the simultaneous determination of structurally similar four purine derivatives using the resultant electrochemically reduced GO (ERGO) modified electrode. The electrocatalytic activity of ERGO was investigated toward the oxidation of four important purine derivatives, uric acid (UA), xanthine (XN), hypoxanthine (HXN) and caffeine (CAF) at physiological pH. The modified electrode not only enhanced the oxidation currents of the four purine derivatives but also shifted their oxidation potentials toward less positive potentials in contrast to bare GCE. Further, it successfully separates the voltammetric signals of the four purine derivatives in a mixture and hence used for the simultaneous determination of them. Selective determination of one purine derivative in the presence of low concentrations other three purine derivatives was also realized at the present modified electrode. Using differential pulse voltammetry, detection limits of 8.8 × 10⁻⁸ M, 1.1 × 10⁻⁷ M, 3.2 × 10⁻⁷ M and 4.3 × 10⁻⁷ M were obtained for UA, XN, HXN and CAF, respectively. The practical application of the modified electrode was demonstrated by simultaneously determining the concentrations of UA, XN, HXN and CAF in human blood plasma and urine samples.     

8-oxoguanine and 8-oxodeoxyguanosine Biomarkers of Oxidative DNA Damage: A Review on HPLC–ECD Determination

Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2′-deoxyguanosine (8-oxodG)—the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC–ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC–ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.     

Simultaneous determination of ascorbic acid, dopamine, uric acid and xanthine using a nanostructured polymer film modified electrode

This paper describes the simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and xanthine (XN) using an ultrathin electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon (GC) electrode in 0.20 M phosphate buffer solution (pH 5.0). Bare GC electrode failed to resolve the voltammetric signals of AA, DA, UA and XN in a mixture. On the other hand, the p-ATD modified electrode separated the voltammetric signals of AA, DA, UA and XN with potential differences of 110, 152 and 392 mV between AA-DA, DA-UA and UA-XN, respectively and also enhanced their oxidation peak currents. The modified electrode could sense 5 μM DA and 10 μM each UA and XN even in the presence of 200 μM AA. The oxidation currents were increased from 30 to 300 μM for AA, 5 to 50 μM for DA and 10 to 100 μM for each UA and XN, and the lowest detection limit was found to be 2.01, 0.33, 0.19 and 0.59 μM for AA, DA, UA and XN, respectively (S/N = 3). The practical application of the present modified electrode was demonstrated by the determination of AA, UA and XN in human urine samples.     

Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection (Part II)

A direct, versatile method for the determination of ethyl glucuronide (EtG), a biomarker of ethanol consumption, in urine has been developed using reversed-phase liquid chromatography with pulsed electrochemical detection (PED). EtG and methyl glucuronide (MetG), which serves as an internal standard, are readily separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH allows for the detection of all glucuronides using PED at a gold working electrode. Upon optimization, EtG was found to have a limit of detection of 0.03 μg/mL (7 pmol; 50 μL injection volume) and repeatability at the limit of quantitation of 1.7%R.S.D. (relative standard deviation). Solid-phase extraction (SPE) using an aminopropyl phase was used to remove interferents in urine samples prior to their analysis. Compound recovery following SPE was approximately 50 ± 2%. The forensic utility of this method was further validated by the analysis of 29 post-mortem urine specimens, whose results agreed strongly with certified determinations.     

A quercetin-modified biosensor for amperometric determination of uric acid in the presence of ascorbic acid

The present work reports a quercetin-modified wax-impregnated graphite electrode (Qu/WGE) prepared through an electrochemical oxidation procedure in quercetin-containing phosphate buffer solution (PBS), for the purpose of detecting uric acid (UA) in the presence of ascorbic acid (AA). During modification quercetin was oxidized to the corresponding quinonic structure, and in the blank buffer solution the electrodeposited film exhibits a voltammetric response anticipated for the surface-immobilized quercetin. Retarding effect of the film towards the reaction of anionic species was found; therefore the pH of sample solutions was selected to ensure the analyte in molecular form. At suitable pHs the Qu/WGE shows excellent electrocatalytic effect towards the oxidation of both AA and UA, and separates the voltammetric signal of UA from AA by about 280 mV, allowing simultaneous detection of these two species. A linear relation between the peak current and concentration was obtained for UA in the range of 1-50 μM in the presence of 0.5 mM AA, with a detection limit 1.0 μM (S/N = 3). This sensor was stable, reproducible and outstanding for long-term use.     

Development and application of a novel immunoassay for measuring oxidative DNA damage in the environment

We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic ligand containing multiple 8-oxodGuo residues. This radioimmunoassay (RIA) displays a high affinity for 8-oxodGuo in DNA, with a detection limit of approximately 1 adduct in 10(5) bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methylene blue and exposed to visible light. As an initial application we quantified 8-oxodGuo in dosimeters deployed at increasing depths in the Southern Ocean during the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a faster rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultraviolet-B radiation (UVB) attenuation, whereas the lower attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo: CPD was also found to covary with changes in stratospheric ozone concentrations at Palmer Station. These data demonstrate the usefulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events.     

Simultaneous electrochemical determination of nitrate and nitrite in aqueous solution using Ag-doped zeolite-expanded graphite-epoxy electrode

In this work a new electrochemical sensor based on an Ag-doped zeolite-expanded graphite-epoxy composite electrode (AgZEGE) was evaluated as a novel alternative for the simultaneous quantitative determination of nitrate and nitrite in aqueous solutions. Cyclic voltammetry was used to characterize the electrochemical behavior of the electrode in the presence of individual or mixtures of nitrate and nitrite anions in 0.1 M Na₂SO₄ supporting electrolyte. Linear dependences of current versus nitrate and nitrite concentrations were obtained for the concentration ranges of 1-10 mM for nitrate and 0.1-1 mM for nitrite using cyclic voltammetry (CV), chronoamperometry (CA), and multiple-pulsed amperometry (MPA) procedures. The comparative assessment of the electrochemical behavior of the individual anions and mixtures of anions on this modified electrode allowed determining the working conditions for the simultaneous detection of the nitrite and nitrate anions. Applying MPA allowed enhancement of the sensitivity for direct and indirect nitrate detection and also for nitrite detection. The proposed sensor was applied in tap water samples spiked with known nitrate and nitrite concentrations and the results were in agreement with those obtained by a comparative spectrophotometric method. This work demonstrates that using multiple-pulse amperometry with the Ag-doped zeolite-expanded graphite-epoxy composite electrode provides a real opportunity for the simultaneous detection of nitrite and nitrate in aqueous solutions.