Hydroxy fatty acids and isoprostanes as markers of lipid peroxidation in brain mitochondria

Hydroxy fatty acids and isoprostanes as markers of lipid peroxidation in brain mitochondria

(3121 311 El SPONTANEOUS INTERMEMBRANE TRANSFER OF CHOLESTEROL HYDROPEROXIDES Andrew Vila and Albert W. Girotti, Biochemistry Dept., Medical College ...

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311 El SPONTANEOUS INTERMEMBRANE TRANSFER OF CHOLESTEROL HYDROPEROXIDES Andrew Vila and Albert W. Girotti, Biochemistry Dept., Medical College of Wisconsin, Milwaukee, Wi, 53226

MEASUREMENT OF 80X0-2i-DEOXYGUANOSINE AND 80X0-2i-DEOXYADENOSINE IN URINE BY HPLC-MASS SPECTROMETRY. Allnn We’ arm nnd Henrik En husen Paulsen. Deportment ?f Clinical

Lipid hydroperoxides (LOOHs) can be generated in cells when unsaturated lipids are degraded under conditions of oxidative stress. If LOOHs escape reductive detoxification by glutathioneredox-active metal ions may dependent selenoperoxidases, catalyze l-electron LOOH reduction and chain peroxidation, thus exacerbating damage to cell membranes. Because LOOHs have long lifetimes relative to free radical precursors/products, intermembrane transfer (similar to that of unoxidized lipids) may be possible, and this may jeopardize acceptor membranes. We have begun testing this hypothesis, using photoperoxidized erythrocyte ghosts as cholesterol hydroperoxide (ChOOH) donors and small unilamellar liposomes, e.g. DMPC/Ch (9:1 mol/mol) ChOOH material consisted mainly of 5a-OOH (a as acceptors. singlet oxygen adduct) and a small amount of 7c&OOH (possibly from 5a-OOH rearrangement). Time-dependent transfer of ChOOH vs. Ch at 37” C was determined, using HPTLC with A typical phosphorimaging to analyze liposomal extracts. experiment in which the starting ChOOH/Ch mol ratio in ghosts was -0.05 showed that the initial transfer rate of ChOOH was These findings - l× greater than that of parent Ch. support the notion that intermembrane transfer can contribute to LOOH cytotoxicity. (Supported by NIH Grant CA72630)

Oxidation of DNA from endogenous and exogenous sources has been advocated as an important pathophysiological mechanism for cancer, ageing and some rare diseases. The chemistry of DNA, base and nucleoside oxidation is well described with more than 100 modification products identified. The in vivo demonstration of DNA oxidation is more controversial. The true values of modified bases/nucleosides in tissue is debated and artificial oxidation during DNA extraction and purification has been suggested. Likewise, some of the methodologies to estimate oxidative DNA lesions, particularly GC-MS and 32-l’-postlabelling, can produce artificial oxidation. For estimation of oxidative DNA lesions HPLC coupled with tandem mass spectrometry appears the best alternative method for estimating the true values of DNA oxidation products and at the same time presents fast sample preparation and high throughput from automatisation. A method based on high




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Leu Weiner. Esther Roth, Yehuda Mazur and Israel Silman The Weizmann Institute of Science. 76100 Rehovot, Israel The natural product, hypericin (Hy), is a photosensitive polycyclic arqmatic dione compound, which has been widely investigated due to Its virucidal and anti-tumor roperties. Although it has been P suggested that singlet oxygen( 0,) or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins. We here demonstrate that Hy binds to a molten globule (MG) species of acetylcholinesterase (AChE), but not to the corresponding native enzyme preparation. Irradiation with visible light causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no crosslinking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the cross-linking, suggesting that ‘0, is responsible for the phenomenon. This agrees with our observation,using spin-traps , that mainly 0, IS produced by the complex of Hy with the MG of AChE. Cross-linking is enhanced in the presence of liposomes to which the MG of AChE is quantitatively adsorbed. MG species are believed to be intermediates in both protein folding and assembly. and in membrane translocation. Thus Hy m?y serve as a valuable tool for trapping such intermediates. This nught also explain its therapeutic effectiveness towards virusinfected or tumor cells.



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performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the determination of S-Hydroxy-2i-deoxyguanosine (80HdG) and 8-Hydroxy-2i-deoxyadenosine ((80HdA) in urine is developed. The used mass spectrometer - a PE Sciex API 365 triple

quadrupole mass spectrometer is equipped with a turbo ionspray source and is used in the multiple reaction monitoring (MRM) mode. Except for a centrifugation and addition of internal standards labelled with stable isotopes no sample preparation is required. The detection limit for 80HdG is around 25 fmol (corresponding to a concentration of approx. 1nM) and for 8OHdA the detection limit is around 75 fmol (corresponding to a concentration of approx. 3nM).

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Daniela Hwsch and Wolfgang Avgustm. Depnrtmentof Medud Faculty, Otto-van-Guencke-University Mngdeburg.

Lipid peroxidation is an important consequence of free radical-mediated damage to cells and tissues. The aim of our investigations was to establish specific markers of lipid peroxidation in general and in particular in brain. We introduced two sensitive and specific GC-MS methods for the determination of monohydroxyeicosatetaenoic acids (HETEs) and isoprostanes in comparison with common markers of oxidative stress (TBARS, LOOH, I-HNE). Q uantitative measurements were achieved using GC-MS in the negative ion chemical ionisation mode allowing detection in the femtomol range. The monohydroxy fatty acids determined in freshly isolated functionally intact rat brain mitochondria were 2-, 3., 5., 8+9-, 11+12- and 15-HETEs. After induction of an oxidative stress with iron/ascorbate especially 5; S-12- and 15-HETEs increased significantly, with a TBARS like kinetics, whereas 2-HETE was characteristically enhanced during the induction phase of peroxidation, when mitochondrial functions and antioxidants decline. Most part of the HETEs remain esterified in membrane bound phospholipids. Chiral phase analysis proves that 12- and 15-HETE constitute a racemic mixture, formed nonenzymatically. F2-isoprostanes identified in mitochondrial samples (E-epi-PGF2a and 9a,llfl-PGFZa) are stable products, which exhibit remarkable increases following oxidative stress. Furthermore we introduced the analysis of F4-isoprostanes (l), derived from docosahexaenoic acid, an essential constituent of nervous tissue. We could demonstrate the presence and oxidant-induced increase of two FCisoprostane isomers. The methods are being applied to detect lipid peroxidation in body fluids from patients with different diseases. 1) J. Nourooz-Zadeh et al., 1999, J. Neurochem. 72,

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