HPLC-electrochemical Detection Research Articles

Determination of 8-Oxoguanine in DNA by Gas Chromatography-Mass Spectrometry and HPLC-Electrochemical Detection: Overestimation of the Background Level of the Oxidized Base by the Gas Chromatography-Mass Spectrometry Assay

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Development of an assay for histamine using automated high-performance liquid chromatography with electrochemical detection

Previous studies have measured histamine by derivatization with o-phthaldialdehyde (OPA) and mercaptoethanol (ME), followed by reversed-phase HPLC separation and electrochemical detection. The derivatization product, however, was very unstable. In the present study, inclusion of less polar solvents (e.g., acetonitrile or tetrahydrofuran) in the OPA/ME derivatization reaction produced an OPA/ME-histamine product that was stable for many hours. Changes of the HPLC mobile phase (increasing its ionic strength and pH and including triethylamine) dramatically improved the chromatography and reduced the histamine detection limit to <0.1 pmol. The modified assay was suitable for batchwise manual derivatization of histamine samples followed by their automated analysis by HPLC with an automatic injector.

Determination of the (R)- and (S)-enantiomers of salsolinol and N-methylsalsolinol by use of a chiral high-performance liquid chromatographic column.

A new method for the quantitative determination of the enantiomers of salsolinol and N-methylsalsolinol, biologically important alkaloids, is reported. The enantiomers were completely separated without derivatization, using a cyclodextrin-modified silica gel column with an HPLC-electrochemical detection system. The HPLC conditions were examined for the best resolution. The method was sensitive enough to detect salsolinol and N-methylsalsolinol at a concentration of less than 0.1 pmol per injection. In the product of the Pictet-Spengler reaction of acetaldehyde with dopamine or epinine, almost equimolar (R)- and (S)-enantiomers of salsolinol and N-methylsalsolinol were detected. Preliminary results indicate that the (R)-enantiomer of both isoquinoline derivatives predominate in the human brain.

Quantitative estimation of heterocyclic aromatic amines by ion-exchange chromatography and electrochemical detection

Conditions for the quantitative determination of three heterocyclic aromatic amines (HAAs), produced by heat processing of protein-rich food products, were established for a HPLC-electrochemical detection system. The separation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was achieved on an ion-exchange column with acetonitrile-80 mM phosphate buffer (30:70) mobile phase at pH 5.6. The figures of merit were calculated. Reproducibility gave a relative standard deviation of 1.6–2.4% when measured by peak area. Detection limits (signal-to-noise ratio 3) ranged from 35 pg for MeIQx to 70 pg for MeIQ. The method was applied to the determination of HAAs in a commercial beef extract sample.

407-3 Glutathione Peroxidase Prevents the Inactivation of Nitric Oxide and Restores the Inhibition of Platelet Function by S-Nitrosothiols

The interaction of nitric oxide (NO) with reactive oxygen species in the vasculature can inactivate NO leading to potentially adverse vascular consequences. Glutathione peroxidases (GSH-Px), a family of antioxidant enzymes present at reduced concentrations in plasma and platelets of patients with coronary artery disease, catalyze the reduction of hydrogen peroxide and lipid hydroperoxides (LOOH) by glutathione. Given the role of LOOH in platelet eicosanoid metabolism and their presence in atherosclerotic plaque, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring NO donor, S-nitroso-glutathione (SNO-Glu). Subthreshold inhibitory concentrations of SNO-Glu were added to platelet-rich plasma, and aggregation was induced by arachidonate. The addition of GSHPx (0.2–20 U/ml) to this system led to a dose-dependent inhibition of platelet aggregation with an IC 50 = 0.6 U/ml GSH-Px (p &lt; 0.05 by ANOVA). Superoxide dismutase (0.1–200 U/ml), catalase (0.1–200 U/ml), or GSH-Px without SNO-Glu did not alter platelet aggregation responses. The addi ion of GSH-Px to a subthreshold inhibitory concentration of sodium nitroprusside also did not affect platelet aggregation responses. LOOH increased platelet aggregation in the presence of SNO-Glu, an effect reversed by GSH-Px. Levels of cGMP were measured after platelets were incubated with SNO-Glu, exogenous LOOH, and GSH-Px. SNO-Glu alone increased cGMP levels, and this effect was attenuated by LOOH but restored by the addition of GSH-Px. GSH-Px activity was equivalent with either SNO-Glu or glutathione as cosubstrate. Incubation of SNO-Glu with GSH-Px led to a 48.5% decrease in the concentration of SNO-Glu as determined by HPLC-electrochemical detection. Incubation of SNO-Glu with albumin in the presence of GSH-Px led to increased formation of S-nitroso-albumin, a prevalent reservoir of EDRF in plasma. These results show that GSH-Px, at physiologically relevant concentrations, has a potent effect on NO-induced inhibition of platelet aggregation and that this enzyme may have two functions: (i) metabolism of LOOH, thereby preventing its inactivation of NO; and (ii) metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions These findings suggest that GSH-Px, in addition to its antioxidant functions, regulates the availability of NO in the vasculature and possibly alters plateletdependent thrombotic events.

Butadiene monoxide and deoxyguanosine alkylation products at the N7-position.

3,4-Epoxy-1-butene, an active metabolite of 1,3-butadiene, was reacted with guanosine, deoxyguanosine and calf thymus DNA. The products were isolated and positively identified using various spectroscopic techniques. Treatment of calf thymus-DNA with 3,4-epoxy-1-butene yielded two N7-guanine adducts of equal stability. Depurination by neutral hydrolysis showed that 7-(2-hydroxy-3-buten-1-yl)guanine (compound I) was formed in greater quantities compared to its regioisomer 7-(1-hydroxy-3-buten-2-yl)guanine (compound II); spontaneous depurination experiments showed that compound I was released in the highest proportion. The circular dichroism spectral studies with R and S 3,4-epoxy-1-butene revealed that the reaction mechanism at aqueous neutral pH media is more similar to SN2-type rather than SN1-type. The HPLC-electrochemical detection method used to carry out the DNA alkylation study provides a rapid and sensitive quantitation of N7 guanine adducts in biological fluids. This serves as a useful tool for further human biomonitoring experiments.

Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography-mass spectrometry and other methods of quantitation

A brief overview is given of recent developments from our laboratory in the use of immunoaffinity clean-up in the determination of alkyl-DNA adducts. Compound- and group-specific antibodies have been prepared against 7-alkylguanines and 3-alkyladenines. The antibodies were attached to solid supports to make immunoaffinity columns which could then be used to selectively purify either single adducts or groups of adducts prior to quantitation by various methods. In the case of methyl adducts quantitation was achieved by ELISA (3-methyl-adenine, using a monoclonal antibody) and HPLC-electrochemical detection (7-methylguanine). For groups of adducts, quantitation of the individual compounds was effected by gas chromatography-mass spectrometry (3-alkyladenines, using deuterated analogues of each adduct as an internal standard) and HPLCL-fluorescence detection (7-alkylguanines). In all of these cases efficient purification of adducts from urine or DNA hydrolysates could be easily carried out. Using these techniques human exposure to alkylating agents in tobacco smoke and from cancer chemotherapy has been studied.

Histaminergic Modulation of Hippocampal Acetylcholine Release In Vivo

In order to elucidate the modulatory role of the histaminergic neural system in the cholinergic neural system, the acetylcholine release from the CA1-CA3 region in the hippocampus of anesthetized rats was studied by an in vivo microdialysis method coupled with HPLC-electrochemical detection. The mean value for the basal acetylcholine release was 0.98 +/- 0.04 pmol/20 min. The acetylcholine release was increased to 172% of the basal level when an electrical stimulation at 200 microA was applied to the tuberomammillary nucleus. An administration of alpha-fluoromethylhistidine (100 mg/kg i.p.) blocked the electrically evoked release of histamine both from the septal-diagonal band complex and the hippocampus, and abolished the electrically evoked release of acetylcholine from the hippocampus. Zolantidine (5 mg/kg i.p.) attenuated the increase in the electrically stimulated acetylcholine release, but pyrilamine (5 mg/kg i.p.) did not attenuate the increase in the acetylcholine release. These drugs showed no significant effect on the basal acetylcholine release. An administration of (R)-alpha-methylhistamine (5 mg/kg i.p.) caused a decrease in the acetylcholine release to 48.7% of the basal level, whereas thioperamide (5 mg/kg i.p.) caused an increase in the acetylcholine release 60 min after the injection. These results suggest that the histaminergic system may contribute to the modulation of the activity of the septohippocampal cholinergic system, mainly through H2 receptors.

Acetyl CoA generation and N-acetylation of serotonin (5HT) in the mosquito, Aedes togoi

Acetyl CoA synthetase (ACS; EC 6.2.1.1) was studied in the mosquito, Aedes togoi, by a novel assay which coupled the acetyl-CoA generated to p-aminosalicylic acid (ASA). The N-acetylated product was determined by an HPLC-fluorimetric procedure. High ACS activity was observed in the newly-pupated pupae of both sexes and in the adult male mosquito whose activity was five times that of the female. Acetyl CoA-dependent N-acetyltransferase (NAT; EC 2.3.1.5) activity toward serotonin (5HT) was also studied using HPLC-electrochemical detection (HPLC-ECD). A progressive increase in the 5HT-NAT activity was observed from the fourth-instar larvae to the adult mosquito with the latter showing 6-fold higher activity in the head compared to the abdomen-thorax region. Kinetic studies on the pupal enzyme extracts showed that the apparent K m values for 5HT-NAT acetyl CoA were 63 and 66 μM respectively. Tryptamine inhibited 5HT-NAT non-competitively with a K i value of 8 μM.

Development of an HPLC–ICP–MS method for the determination of platinum species from new anti-tumour drugs

The anti-tumour drug cisplatin and some of its more modern analogues has been determined by use of a HPLC–ICP–MS system. The results compare favourably with those obtained by using HPLC–ICP–AES and HPLC–electrochemical detection and are superior to those obtained by HPLC–ETAAS.

O-259 - Development of HPLC-chemi luminescent and HPLC-electrochemical detection methods for histamine and NT-methylhistanine.

O-259 - Development of HPLC-chemi luminescent and HPLC-electrochemical detection methods for histamine and NT-methylhistanine.

Identification of 8-Hydroxyguanine Glycosylase Activity in Mammalian Tissues Using 8-Hydroxyguanine Specific Monoclonal Antibody

Here we report the finding of glycosylase activity in mammalian tissues to remove 8-hydroxyguanine residues from DNA. To detect this activity, a synthetic duplex DNA containing an 8-hydroxyguanine residue was used as the substrate, and before the product was analyzed by HPLC-electrochemical detection (ECD), 8-hydroxyguanine released by the enzymatic action was specifically collected from reactions using immunoaffinity columns of 8-hydroxyguanine specific monoclonal antibody. With the application of immunoaffinity column chromatography, a single peak of 8-hydroxyguanine free from many interfering peaks of unknown origin was clearly demonstrated in HPLC-ECD even with crude tissue extracts used as assay materials. All the tissues of the rats examined showed the activity, suggesting its presence is ubiquitous. The present coupled technique of immunoaffinity column chromatography and HPLC-ECD will be very useful to many future studies of 8-hydroxyguanine glycosylase such as its activity detection, characterization and purification, etc.

High-performance liquid chromatographic determination of ten heterocyclic aromatic amines with electrochemical detection

Conditions for the HPLC-electrochemical detection determination of ten mutagenic heterocyclic amines that can be produced by heat processing of protein-rich food products were established. The use of reversed-phase chromatography and acetonitrile-ammonium acetate (10:90) at pH 4.0 for the separation of 2-amino-3-methylimidazo[4,5- f]quinoline, 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]-quinoxaline (4,8-DiMeIQx) and 2-amino-6-methyldipyrido[1,2- a:3',2'- d]imidazole (Glu-P-1), and of acetonitrile—ammonium acetate (30:70) at pH 6.0 for the separation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine, 3-amino-1-methyl-5H-pyrido[4,3- b]indole (Trp-P-2), 2-amino-3-methyl-9H-pyrido[2,3- b]indole, 1-methyl-9H-pyrido[4,3- b]indole (harman) and 9H-pyrido[4,3- b]indole (norharman) was proposed. The figures of merit were calculated; reproducibility gave a relative standard deviation of 1.6–4.6% when measured by peak area. Detection limits (signal-to-noise ratio 2:1) ranged from 0.2 ng for Trp-P-2 to 3.4 ng for 4,8-DiMeIQx. The method was applied to the determination of some of these compounds in a commercial beef extract. Levels of 5.1 ng g −1 MeIQx, 5.8 ng g −1 MeIQ and 15.5 ng g −1 of Glu-P-1 were found.

Dopamine as trace amine in the dorsal root ganglia

It has been shown that in the chick dorsal root ganglion (DRG) about 8% of neurons, belonging to both the A and B classes of sensory neurons exhibit a clear dopamine immunoreactivity. In the present study are reported the results of measurements, by mean of HPLC-electrochemical detection (HPLC-ED), of DA and of the DA metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the rat DRG and their central nerves. Very low levels of DA, about 10 folds lower than the levels found in the dorsal horn of the spinal cord, were found in the DRG. However the levels of DOPAC and HVA were approximately equivalent to the levels found in the cord. The immunocytochemical study performed in parallel has shown that some dopaminergic-immunoreactive fibers in the DRG are located around the blood vessels. Few dopamine-immunoreactive sensory neurons were identified in the DRG and immunoreactive fibers, not linked to blood vessels, were identified in the dorsal root nerves. The present work indicates that there is a dopaminergic innervation of the blood vessels in the rat DRG but that dopamine may also be, as in the chick, a transmitter of primary afferent fibers.

Interleukin-1 beta augments release of norepinephrine, dopamine, and serotonin in the rat anterior hypothalamus

We investigated the effects of interleukin-1 beta (IL-1 beta), administered directly into the rat anterior hypothalamus (AHY), on monoamine release in the same region by using a brain microdialysis technique and an HPLC-electrochemical detection system. First, to study the local effects of IL-1 beta, we used a microdialysis probe equipped with a microinjection tube for administering IL-1 beta in the same region into which the probe had been inserted. IL-1 beta (1 ng) injected directly into the AHY elicited release of norepinephrine (NE), dopamine (DA), and 5-HT, as well as increases in their metabolites, 4-hydroxy-3-methoxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, in the AHY. Vehicle alone exerted no effect on monoamine release. Although the elevated levels of NE and DA persisted for more than 6 hr after injection of IL-1 beta, the elevated levels of 5-HT were transient. Second, in order to investigate whether this effect of IL-1 beta is a direct action in the AHY, we performed in vitro experiments using hypothalamus slices. IL-1 beta (0.1 and 1 nM) increased the levels of each monoamine released from hypothalamic slices in a dose-dependent manner. These findings suggest that IL-1 beta acts directly on the hypothalamus to induce release of NE, DA, and 5-HT. Third, the roles of prostaglandins (PGs) in NE release in the AHY elicited by direct injection of IL-1 beta were examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Modelling Cortical Cataractogenesis 13. Early Effects on Lens ATP/ADP and Glutathione in the Streptozotocin Rat Model of the Diabetic Cataract

A possible contribution to cell toxicity in the diabetic lens due to early ATP loss is not well characterized prior to the appearance of vacuoles in the lens. Changes in lens ATP levels at longer periods of hyperglycaemia (6-8 weeks) have been reported. We used [31P]NMR analysis of lens extracts at three time periods. comparing diabetic to concurrent control groups at 1, 2 and 4 weeks of hyperglycaemia. With this design, significant alterations (> 10%) in the ATP/ADP ratio can be monitored. NMR analysis revealed a decreased ATP/ADP ratio at all time periods, averaging a 38% decrease. Luminescent determination of ATP levels indicates that this decrease is mainly caused by a decrease of 25% in ATP concentration. The early loss of GSH was large and not accompanied by an appearance of GSSG, as monitored by HPLC electrochemical detection. A 1-week experiment with animals receiving daily insulin treatment was carried out to control for effects of STZ on the lens. This treatment resulted in normal lens GSH levels and a near normal [31P]NMR profile.

Inhibition of 8-hydroxydeoxyguanosine formation by curcumin in mouse fibroblast cells.

Curcumin, a pigment responsible for the yellow color of curry, has been shown to be an anti-inflammation agent, an antioxidant and an antipromoter. 8-Hydroxydeoxyguanosine (8-OH-dG), an oxidized nucleoside, may be responsible for a genetic event of tumor promotion in carcinogenesis. 8-OH-dG can be detected selectively and sensitively at the fmol level by HPLC-electrochemical detection at an applied potential of +0.8 V versus Ag/AgCl. Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, induces lipid peroxidation and 8-OH-dG formation. Curcumin can strongly scavenge the hydroxyl radical (OH.) to prevent 8-OH-dG formation from dG (deoxyguanosine) in vitro and reduce the production of PMA-induced lipid peroxidation and 8-OH-dG in mouse fibroblast cells. These results suggest that curcumin inhibits the PMA-induced tumor promotion by functioning as an OH. radical scavenger to prevent 8-OH-dG formation within the DNA molecule.

Application of Square Wave Voltammetry for Electrochemical Detection in Gradient Elution HPLC

Abstract The utility of square-wave voltammetry with a conventionally sized indicator electrode for use in the detection of gradient elution HPLC applications is demonstrated. A redesigned electrochemical detector cell, based upon a type of wall-jet design, provides more efficient potential control and allows the voltammetric detection. The minimum detectable quantity and linear dynamic range for the new detector design were found to be 13pg and 5 orders of magnitude, respectively, comparable to other electrochemical detectors. In the separation of a test mixture containing hydroquinone. bromohydroquinone, catechol, resorcinol, and phenylhydroquinone baseline resolution of all five components is obtained in 12 minutes using a nonlinear solvent gradient. Importantly, no change in the chromatographic baseline is observed during the course of the separation. Gradient separations of the extracts of fennel seeds and mainstream cigarette smoke are also demonstrated with no apparent change in the baseline.

Background and radiation-induced 8-hydroxy-2'-deoxyguanosine in gamma-irradiated Escherichia coli.

The DNA base damage product, 8-hydroxy-2'-deoxyguanosine (7-hydro-8-oxo-2'-deoxyguanosine, or 8-OHdG) was measured in 2'-deoxyribonucleoside digests of DNA from irradiated E. coli or model DNA using HPLC separation and electrochemical detection (LCED). The LCED technique enabled the specific quantitation of 25 fmol of 8-OHdG (signal: noise ratio = 4:1) among a variety of DNA base lesions within the sample. A radiation dose-yield relationship for 8-OHdG was observable in cells that were rapidly lysed after irradiation using a chloroform-saturated, sarkosyl and EDTA solution at 4 degrees C (Tilby and Loverock 1983). The observed yield of 8-OHdG was 7.05 +/- 0.27 pmol J-1 and was against a background of 120 8-OHdG per E. coli genome. This yield translates to an average of 12 additional 8-OHdG lesions per genome at the mean lethal dose. The low radiochemical yield and high background suggests that factors other than the toxicity of 8-OHdG per se--such as its participation in clusters of base lesions (e.g. Ward 1985)--must contribute significantly to cell killing. Although exposure of DNA to phenol mixtures has often been reported to increase 8-OHdG, we found that a more significant effect of phenol mixtures was the potentiation of the radiochemical yields of 8-OHdG in model DNA from 5.26 +/- 0.48 to 158 +/- 12.2 nmol J-1. Other factors, such as the exposure of dried 2'-deoxyribonucleoside digests to ambient humidity in the presence of buffer components, were shown to contribute more significantly to the background of 8-OHdG.

Acetyl-L-carnitine releases dopamine in rat corpus striatum: an in vivo microdialysis study

The effect of acetyl-L-carnitine, a compound reported to be beneficial for senile patients, on the release of dopamine (DA) from the striatum was studied by using in vivo brain dialysis in anesthetized rats coupled with HPLC-electrochemical detection. Striatal infusion of acetyl-L-carnitine increased the efflux of DA with no apparent changes in efflux of DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). The DA-releasing effect of acetyl-L-carnitine was concentration- and Ca 2+-dependent, and was abolished by ω-conotoxin fraction GVIA and tetrodotoxin, inhibitors of the voltage-dependent Ca 2+ and Na + channels, respectively. Nomifensine, an inhibitor of DA reuptake did not alter the DA-releasing property of acetyl-L-carnitine. DA released from the striatum by acetyl-L-carnitine was decreased by reserpine pretreatment whereas the d-amphetamine-evoked DA outflow was not affected. In contrast to acetyl-L-carnitine, d-amphetamine reduced the extracellular concentrations of DOPAC and HVA. We conclude from the present data that acetyl-L-carnitine evokes DA release from the vesicular pools of the nigrostriatal dopaminergic neurons by a Ca 2+-dependent, exocytotic process.