978 resultados para HPLC-ECD


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Für die Krebsentstehung sind nach heutigen Vorstellungen Mutationen in bestimmten Genen somatischer Zellen verantwortlich, die ihrerseits durch chemische Modifikationen der DNA (DNA-Schäden) verursacht werden. Von besonderem Interesse als DNA-schädigende Agentien sind reaktive Sauerstoffspezies (ROS), die endogen - wie z.B. in der Lipidperoxidation oder mitochondrialen Atmungskette - oder exogen - z.B. durch Arzneimittel oder andere aus der Umwelt stammende Chemikalien - gebildet werden können. Dem Schutz der DNA vor dieser Schädigung und ihren Folgen dienen antioxidative Systeme und DNA-Reparatur, deren Effizienz von verschiedenen Genen der Zelle abhängig ist. Ziel dieser Arbeit war die Untersuchung des Einflusses bestimmter Faktoren auf die Bildung, Inhibierung (antioxidative Wirkung) und Reparatur oxidativer DNA-Schäden. Als Beispiel für exogene Stoffe, die zur Bildung oxidativer DNA-Schäden führen, wurden die Gyrasehemmer Ofloxacin und Norfloxacin nach Bestrahlung mit UV-360 untersucht. Unter Verwendung spezifischer DNA-Reparatur-endonukleasen als Sonden (Fpg-Protein, Endonuklease III, Endonuklease IV, Exonuklease III, T4 Endonuklease V) wurden spezifisch DNA-Modifikationen an zellfreier und zellulärer DNA quantifiziert. Es zeigte sich, daß es sich im Falle des Ofloxacins bei der ultimal mit der DNA reagierenden reaktiven Spezies um Hydroxylradikale handelt, während durch Norfloxacin Singulettsauerstoff entsteht. Durch Zusatz verschiedener Antioxidantien konnten diese Ergebnisse bestätigt werden. Im Gegensatz zu Hydroxylradikalen ist über die DNA-Schäden und Mutationen durch Alkoxyl-Radikale, die endogen bei der Lipidperoxidation entstehen, nichts bekannt. Die Substanz BCBT ([4-[(tert-Butyldioxy)carbonyl]benzyl]-triethyl-ammonium-chlorid) generiert Alkoxyl-Radikale. BCBT + UV-360 induzierte vorwiegend Fpg-sensitive Modifikationen, die durch Bestimmung mittels HPLC/ECD zu 83 ± 18% als 8-Hydroxyguanin identifiziert wurden. Der Vergleich des Einflusses der Zusätze tert-Butanol und Natriumazid auf die Schädigung mit BCBT mit anderen reaktiven Spezies zeigte klar, daß die Schädigung hier über Alkoxyl-Radikale verläuft. Die Analyse und Sequenzierung der durch BCBT induzierten Mutanten zeigte, daß vorwiegend GC->TA Transversionen gebildet werden, die auf 8-oxoG zurückzuführen sind. Auf der Ebene der Antioxidantien wurde der Einfluß des zelleigenen Glutathions untersucht. Mit der Depletion des Glutathion-Gehalts durch Vorbehandlung mit L-Buthionin-SR-sulfoximin (BSO) ging eine Erhöhung des Steady-State-Spiegels in den untersuchten AS52 Zellen einher. Selen ist ein wichtiges Spurenelement und u.a. Bestandteil von Glutathionperoxidasen (GPx). Um die Bedeutung von Selen bei der oxidativer DNA-Modifikationen bzw. deren Inhibierung zu untersuchen, wurden Natriumselenit und Ebselen eingesetzt. Ab einer Konzentration von 50 µM trat durch Natriumselenit eine Erhöhung des Steady-State-Spiegels auf (prooxidativer Effekt). Parallel mit der oxidativen Schädigung der DNA ist eine Erhöhung des zellulären Gehalts an Glutathion feststellbar. Ebselen bedingte in Konzentrationen bis 200 µM keine Erhöhung des Steady-State-Spiegels oder des Gehalts an reduziertem Glutathion. Beide Substanzen waren in der Lage, die durch sichtbares Licht und UV-B-Strahlung induzierten Einzelstrangbrüche zu reduzieren. Mikrokerne, die durch Schädigung mit den Agentien Kaliumbromat, Ro 19-8022 + Licht und Wasserstoffperoxid entstanden, waren ebenso durch Natriumselenit und Ebselen reduzierbar. In dieser Arbeit konnte ferner gezeigt werden, daß p53 keinen Einfluß auf die Reparatur UV-induzierter Modifikationen (Nukleotidexzisionsreparatur) und die Reparatur Fpg-sensitiver Modifikationen (Basenexzisionsreparatur) hat.

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The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

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High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

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An HPLC with SPE method has been developed for analysis of constituents in rat blood after oral administration of the extract of Acanthopanax senticosus (ASE). The plasma sample was prepared by SPE method equipped with Oasis HLB cartridge (3cc, 60 mg). The analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mmx150 mm, 5 microm) with the gradient elution of solvent A (ACN) and solvent B (0.1% aqueous phosphoric acid, v/v) and the detection wavelength was set at 270 nm. The calibration curve was linear over the range of 0.156-15.625 microg/mL. The LOD was 60 ng/mL. The intraday precision was less than 5.80%, and the interday precision was less than 6.0%. The recovery was (87.30 +/- 1.73)%. As a result, 19 constituents were detected in rat plasma after oral administration of the ASE, including 11 original compounds in ASE and eight metabolites, and three of the metabolites originated from syringin in ASE. Six constituents were identified by comparing with the corresponding reference compounds.

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Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

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A new method has been developed for the quantification of 2-hydroxyethylated cysteine resulting as adduct in blood proteins after human exposure to ethylene oxide, by reversed-phase HPLC with fluorometric detection. The specific adduct is analysed in albumin and in globin. After isolation of albumin and globin from blood, acid hydrolysis of the protein and precolumn derivatisation of the digest with 9-fluorenylmethoxycarbonylchloride, the levels of derivatised S-hydroxyethylcysteine are analysed by RP-HPLC and fluorescence detection, with a detection limit of 8 nmol/g protein. Background levels of S-hydroxyethylcysteine were quantified in both albumin and globin, under special consideration of the glutathione transferase GSTT1 and GSTM1 polymorphisms. GSTT1 polymorphism had a marked influence on the physiological background alkylation of cysteine. While S-hydroxyethylcysteine levels in "non-conjugators" were between 15 and 50 nmol/g albumin, "low conjugators" displayed levels between 8 and 21 nmol/g albumin, and "high conjugators" did not show levels above the detection limit. The human GSTM1 polymorphism had no apparent effect on background levels of blood protein 2-hydroxyethylation.

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A combined data matrix consisting of high performance liquid chromatography–diode array detector (HPLC–DAD) and inductively coupled plasma-mass spectrometry (ICP-MS) measurements of samples from the plant roots of the Cortex moutan (CM), produced much better classification and prediction results in comparison with those obtained from either of the individual data sets. The HPLC peaks (organic components) of the CM samples, and the ICP-MS measurements (trace metal elements) were investigated with the use of principal component analysis (PCA) and the linear discriminant analysis (LDA) methods of data analysis; essentially, qualitative results suggested that discrimination of the CM samples from three different provinces was possible with the combined matrix producing best results. Another three methods, K-nearest neighbor (KNN), back-propagation artificial neural network (BP-ANN) and least squares support vector machines (LS-SVM) were applied for the classification and prediction of the samples. Again, the combined data matrix analyzed by the KNN method produced best results (100% correct; prediction set data). Additionally, multiple linear regression (MLR) was utilized to explore any relationship between the organic constituents and the metal elements of the CM samples; the extracted linear regression equations showed that the essential metals as well as some metallic pollutants were related to the organic compounds on the basis of their concentrations

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Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.