Influence of oxidative stress on fusion of pre-synaptic plasma membranes of the rat brain with phosphatidyl choline liposomes, and protective effect of vitamin E.

Influence of oxidative stress on fusion of pre-synaptic plasma membranes with phosphatidylcholine (PC) liposomes as a model of synaptic vesicle was investigated. The inhibitory effect of vitamin E on the decline in the fusion caused by oxidative stress was also assessed. Rats subjected to hyperoxia as oxidative stress showed significant increases in the levels of lipid hydroperoxides and protein carbonyl moieties in pre-synaptic plasma membranes in the brain. The zeta potential of pre-synaptic membrane surface was decreased markedly. When synaptosomes were incubated with PC liposomes labeled by either rhodamine B or calcein as a fluorescence probe, or 12-doxyl stearic acid as an ESR spin trapping agent, translocation of each probe into oxidatively damaged pre-synaptic membranes was decreased significantly. Fatty acid composition analysis in pre-synaptic membranes obtained from normal rats revealed a marked increase in linoleic acid and a moderate decrease in docosahexaenoic content after the incubation with liposomes. However, rats subjected to hyperoxia did not show marked changes in these fatty acid contents in their pre-synaptic membranes after the incubation. Such changes caused by hyperoxia were inhibited by vitamin E treatment of rats. These results suggest that oxidative damage of pre-synaptic membranes caused by oxidative stress lowers the lipid-mixing for the membrane fusion. The results of this study imply that vitamin E prevents the deficit in neurotransmission at nerve terminals due to the decline in fusion between pre-synaptic membrane and synaptic vesicles caused by oxidative membrane damage.

Down-modulation of toll-like receptor 2 expression on granulocytes and suppression of interleukin-8 production due to in vitro treatment with cellulose acetate beads.

The Adacolumn, which is filled with cellulose acetate beads (CA beads), has been used as a medical device for inflammatory diseases. The CA beads selectively adsorb granulocytes and monocytes and remove them from the peripheral blood. The anti-inflammatory effects of the Adacolumn are possibly caused by removal of these cells but also due to the functional changes in the processed cells. In this study, we investigated the effects of CA beads treatment on modulation of the expression of innate immunity receptors such as the Toll-like receptor (TLR) family and production of an inflammatory cytokine, interleukin-8 (IL-8). Changes in the expressions of TLR1, 2, 4 and 6 in peripheral leukocytes exposed to CA beads were examined by flow cytometry. TLR2 expression on the surface of granulocytes exposed to CA beads was decreased, but the amount of intracellular TLR2 was increased, possibly by internalization. These changes were not observed in monocytes or lymphocytes. Peptidoglycan (PGN) treatment produced similar changes in TLR2 on granulocytes. We also measured the amounts of IL-8 in cultured blood treated with lipopolysaccharide (LPS) and PGN, which are known TLR agonists. PGN-induced IL-8 production was lower in CA beads-treated leukocytes than that in non-treated leukocytes, but LPS did not induce these changes. Based on these findings, we conclude that the down-modulation of TLR2 and suppression of IL-8 production on granulocytes by CA beads, may play an important role in the anti-inflammatory effects of the Adacolumn.

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, ß-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO·) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H(2)O=7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k(s)) for α-TocH in MeOH/H(2)O solution with EYPC vesicles were apparently 3.45×10(5)M(-1)s(-1), which was about 8 times higher than that (4.50×10(4)M(-1)s(-1)) in MeOH/H(2)O solution without EYPC vesicles. The corrected k(s) of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10mM EYPC vesicles than in the bulk MeOH/H(2)O solution, was 2.60×10(3)M(-1)s(-1), which was one-seventeenth that in MeOH/H(2)O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k(s) of vitamin E in membranes increased in the order of tocol<δ-TocH<γ-TocH∼ß-TocH<α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H(2)O solution among vitamin E analogues [k(s)(vesicle)/k(s) (MeOH/H(2)O)=7.7, 10.0, 9.5, 7.4, and 5.1 for α-, ß-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k(s)(micelle)/k(s)(EtOH)=100, 47, 41, 15, and 6.3 for α-, ß-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol<δ-TocH<γ-TocH∼ß-TocH<α-TocH.

Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay.

Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe(3+)-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe(3+) because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M(-1) cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 610), which were 79,100 and 115,700 M(-1) cm(-1) in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.