In Vitro Induction of Starfish Oocyte Maturation by Cysteine Alkylesters: (oocyte maturation/starfish/1-MeAde/SH-group).

The effect of various disulfide-reducing agents including cysteine and its alkylesters on the induction of germinal vesicle breakdown (GVBD) in starfish (Asterina pectinifera) oocytes was investigated in vitro. Although cysteine did not induce GVBD, its alkylesters were effective. Cysteine alkylesters significantly mimicked the effect of 1-methyladenine (1-MeAde), the naturally occurring maturation-inducing hormone of starfish, on oocyte maturation. However, the effective concentrations and pH optimum for stimulation of oocyte maturation varied between 1-MeAde and the cysteine alkylesters. By comparing pKa values of the disulfide-reducing agents to pH of the medium, it is suggested that the redox potential of a disulfide-reducing agent is an important indicator its ability to induce oocyte maturation. With the use of fluorescent probes for thiol groups, it was shown that the fluorescence in oocyte cortices increased within 5 min after administration of 1-MeAde. The fluorescence intensity in the cortices also increased after treatment with cysteine and its alkylesters, although the intensity was much stronger with the latter. Furthermore, both 1-MeAde and the disulfide-reducing agents were suggested to cause reduction of thiol groups within the plasma membrane as opposed to those on the external and internal surfaces. Thus, it is suggested that disulfide-reducing agents and 1-MeAde induce starfish oocyte maturation by changing the redox state of the thiol groups located within the oocyte plasma membrane.

In vivo influence of ceramide accumulation induced by treatment with a glucosylceramide synthase inhibitor on ischemic neuronal cell death.

It has been shown that exogenous ceramide induces delayed neuronal death (DND) of cultured hippocampal neurons. To evaluate the role of endogenous ceramide in ischemic DND, the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), was used to generate ceramide in gerbil hippocampi in vivo. The trimethylsilylated derivatives of ceramide were analyzed directly by gas chromatography mass spectrometry, after separation with high-performance thin-layer chromatography. The ceramide compositions in vehicle hippocampus consisted mainly of C18:0 fatty acyl sphingosine (87.9%), with C16:0 and C20:0 ceramides being minor components (7.1% and 5.1%, respectively). Ceramide level in the hippocampi from gerbils subjected to D-PDMP treatment was 1.5-fold higher than those from vehicle-treated gerbils. In spite of the accumulation of ceramide observed in the D-PDMP group, the histological studies did not reveal any ischemic neuronal death in hippocampal CA1 neurons with the gerbils that had been subjected to a sham operation (2-min sublethal ischemia). These results suggest that the ceramide accumulation induced by blocking the de novo synthesis of glucosylceramide with D-PDMP may be independent of the metabolic pathway underlying ischemic DND.

Ethanolamine plasmalogens protect cholesterol-rich liposomal membranes from oxidation caused by free radicals.

The aim of the present study is to investigate the effect of ethanolamine plasmalogens on the oxidative stability of cholesterol-rich membranes by comparing it with that of diacyl glycerophosphoethanolamine, using bovine brain ethanolamine plasmalogen (BBEP) or egg yolk phosphatidylethanolamine (EYPE)-containing large unilamellar vesicles (LUVs) and the water-soluble radical initiator AAPH. Electron microscopic observation and particle size measurement visually demonstrated that ethanolamine plasmalogens protect cholesterol-rich phospholipid bilayers from oxidative collapse. Lipid analyses suggested that the effect of ethanolamine plasmalogens in stabilizing membranes against oxidation is partly due to the antioxidative action of plasmalogens involved in scavenging radicals at vinyl ether linkage.

Comparison of the oxidizability of various glycerophospholipids in bilayers by the oxygen uptake method.

The aims of this study were twofold: develop a convenient and rapid procedure for assessing the oxidizability of small quantities of glycerophospholipids in bilayers by the oxygen uptake method, and determine and compare the oxidizability of various glycerophospholipids in bilayers. Our purpose was to elucidate phospholipid oxidation characteristics in membranes. The quantitative autoxidation kinetics of dilinoleoyl phosphatidylcholine (DLPC) (18:2/18:2) was studied in large unilamellar vesicles (LUV) in aqueous dispersions with water-soluble initiator--2,2'-azobis(2-amidinopropane) dihydrochloride--and inhibitor 2-carboxy-2,5,7,8-tetramethyl-6-chromanol. The kinetic data indicated a high efficiency of free radical production, resulting in shortening of measuring time; the very low kinetic chain length, particularly in the induction period, suggested the possibility of including large errors in the kinetics data. Nevertheless, the autoxidation of DLPC obeyed the classic rate law: Rp = kp[LH]Ri(1/2)/(2kt)(1/2) (where Rp = rate of oxygen consumption, kp = rate constant for chain propagation, [LH] = substrate concentration; Ri(1/2) = square root of rate of chain initiation, and 2kt = rate constant for chain termination) in a mixed bilayer system with saturated dimyristoyl PC (14:0/14:0), which provided precise and reproducible data. Therefore, the system was used to assess the relative oxidizability of each glycerophospholipid DLPC (18:2/18:2), dilinolenoyl PC (18:3/18:3),1-palmitoyl-2-linoleoyl PC (16:0/18:2), 1-palmitoyl-2-arachidonoyl PC (16:0/20:4), 1-palmitoyl-2-docosahexaenoyl PC (16:0/22:6), and dilinoleoyl PE (18:2/18:2) in bilayers. The results suggested that the oxidizability of glycerophospholipid in bilayers is substantially influenced by the number of intramolecular oxidizable acyl chains and the content of bis-allylic hydrogen in a structured environment, and showed deviation of the rate law for autoxidation in PC and PE mixed LUV, which possibly was due to non-homogeneous phospholipid distribution in vesicles.

Action of 1-(11-selenadodecyl)-glycerol and 1-(11-selenadodecyl)-3-trolox-glycerol against lipid peroxidation.

The antioxidant action on lipid peroxidation of the synthesized selenium compounds 1-(11-selenadodecyl)-glycerol (SeG) and 1-(11-selenadodecyl)-3-Trolox-glycerol (SeTrG, where Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was investigated. We compared the reactivity of the selenium compounds toward peroxyl radicals and their inhibitory effect on lipid peroxidation, induced by several kinds of initiating species such as azo compounds, metal ions, and superoxide/nitric oxide in solution, micelles, membranes, and rat plasma. SeTrG, but not SeG, scavenged peroxyl radicals. SeG reduced methyl linoleate hydroperoxides in organic solution and in methyl linoleate micelles oxidized by ferrous ion (Fe2+)/ascorbic acid. In rat plasma SeG and SeTrG decreased the formation of lipid hydroperoxides generated by hydrophilic azo compounds. SeG and SeTrG spared alpha-tocopherol (alpha-TOH) consumption in multilamellar vesicle membranes oxidized by hydrophilic or lipophilic initiators, and only SeTrG spared alpha-TOH in superoxide/nitric oxide oxidized membranes. In rat plasma oxidized by radical initiators (either hydrophilic or lipophilic) or superoxide/nitric oxide, SeTrG suppressed alpha-TOH consumption, but SeG had no effect. The two selenium-containing compounds showed inhibitory effects on lipid peroxidation that depended on their structure, the medium where they acted, and the oxidant used.

Ceroid accumulation in rat kidneys caused by uptake of ferric nitrilotriacetate.

Ceroid accumulation in kidneys caused by lipid peroxidation in vivo was studied during intraperitoneal (i.p.) injections of ferric nitrilotriacetate (Fe3+-NTA) solution, which is a weak iron chelate compound and generates free radicals in the presence of oxygen. Fe3+-NTA solution (0.5-0.75 mg/100 g body weight) was injected into rats (n=6) for 4 days a week. After 2 weeks of Fe3+-NTA loading, the rats were not treated for 1 month as a rest interval, and then the iron loading was repeated for 35 weeks including the rest intervals. Two rats in each group, Fe3+-NTA loading and control, were sacrificed at 19 and 40 weeks of age, and portions of a kidney and the liver of each rat were fixed in 10% formalin buffered to pH 7.0 for the histological studies. We found ceroid accumulates in greater amounts and more quickly within macrophages and epithelial cells of proximal convoluted tubules of the kidney and Kupffer cells of the liver in iron-loaded rats.

Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion (125I3-).

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.

Ethanolamine plasmalogen and cholesterol reduce the total membrane oxidizability measured by the oxygen uptake method.

To investigate the effects of ethanolamine plasmalogen, phosphatidylethanolamine, cholesterol, and alpha-tocopherol on the oxidizability of membranes, various large unilamellar vesicles (LUVs) including these lipids and antioxidant were examined for their total membrane oxidizabilities, evaluated as R(p)/R(i)(1/2) value (where R(p) is rate of oxygen consumption and R(i)(1/2) is the square root of rate of chain initiation) by the oxygen uptake method with water-soluble radical initiator and inhibitor. Incorporation of bovine brain ethanolamine plasmalogen (BBEP) into vesicles as well as cholesterol led to lower the total membrane oxidizability dose-dependently. The effect of BBEP was more efficient in the presence of cholesterol in vesicles. On the other hand, diacyl counterpart, egg yolk phosphatidylethanolamine, and a typical radical scavenger, alpha-tocopherol, had no effect on the membrane oxidizability. Alpha-tocopherol only prolonged an induction period dose-dependently in the present oxidizing system, suggesting a novel antioxidant mechanism of ethanolamine plasmalogens besides the action of scavenging radicals.

Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers.

The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (CH) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant alpha-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k (p)/(2k (t))(1/2)=R (p)/[LH]R(i)(1/2) by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability. A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals.