Hiroyuki Shimasaki

Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C

The oxidation of human low density lipoprotein (LDL) initiated by free radical initiator and its inhibition by vitamin E and water-soluble antioxidants have been studied. It was found that the kinetic chain length was considerably larger than 1, suggesting that LDL was oxidized by a free radical chain mechanism. Vitamin E acted as a lipophilic chain-breaking antioxidant. Water-soluble chain-breaking antioxidants such as ascorbic acid and uric acid suppressed the oxidation of LDL initiated by aqueous radicals but they could not scavenge lipophilic radicals within LDL to break the chain propagation. Ascorbic acid acted as a synergistic antioxidant in conjunction with vitamin E.

Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen

The oxidation of human and rat erythrocyte ghost membranes by molecular oxygen has been performed in an aqueous suspension at 37 degrees C. A constant rate of oxygen uptake was observed in the presence of radical initiator. alpha-Tocopherol in the membrane suppressed the oxidation and the induction period was clearly observed. alpha-Tocopherol decreased linearly during the induction period and when it was depleted the induction period was over and a rapid oxidation started. The rate of oxidation was proportional to the square root of the rate of initial radical generation. The kinetic chain length, the ratio of the rate of propagation to that of initiation, was long, ranging from 7 to 100. These results indicate that the erythrocyte ghost membranes are oxidized by a free radical chain mechanism by molecular oxygen. Among the fatty acids of membrane lipids, polyunsaturated fatty acids were oxidized exclusively. Proteins as well as polyunsaturated fatty acids were oxidized and the formation of the high- and low-molecular-weight proteins and the decrease of protein bands were observed on gel electrophoresis.

Double-Blind Controlled Study on the Effects of Dietary Diacylglycerol on Postprandial Serum and Chylomicron Triacylglycerol Responses in Healthy Humans

Objective: The effects of dietary diacylglycerol (DG) on postprandial lipemia in healthy humans were investigated. Methods: Forty normolipidemic male volunteers ingested fat emulsions containing either DG oil or triacylglycerol (TG) oil, at different doses: 10 g (n = 13), 20 g (n = 10) and 44 g (n = 17). Two test emulsions were given at seven-days intervals in random order. Fatty acid compositions of the test oils had been adjusted to be equal. Fasting and postprandial serum lipid concentrations in each group and plasma lipoprotein lipids in the 20 g-fat ingestion group were measured during the postprandial intervals. Results: When DG emulsion was ingested, serum TG concentrations were significantly lower (p < 0.05) in the late postprandial phase, i.e., 4 hours, 6 hours as compared to the TG emulsion. The magnitude of postprandial lipemia (the area bounded by the curve above the fasting concentration) after ingestion of 44 g-DG emulsion was significantly less than that of 44 g-TG emulsion (6.54 ± 5.12 and 8.45 ± 7.54 mmol · h/L, mean ± SD, respectively). Chylomicron TG, cholesterol, and phospholipid concentrations at 4 hours after ingestion of DG emulsion were significantly lower (p < 0.05) than those after the ingestion of TG emulsion at the same time point. No marked differences were observed for VLDL, LDL and HDL lipids between the test emulsions. Conclusion: In the usual range of fat intake (10–44 g), postprandial response after ingestion of DG emulsion was significantly less than that after ingestion of TG emulsion in healthy human subjects.

Brain Free Fatty Acids, Edema, and Mortality in Gerbils Subjected to Transient, Bilateral Ischemia, and Effect of Barbiturate Anesthesia

Abstract: Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono‐ and polyunsaturated FFAs increased approximately 12‐fold in total at the end of a 30‐min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic acid.

Inhibition of Oxidation of Low Density Lipoprotein by Vitamin E and Related Compounds

The oxidation of low density lipoprotein (LDL) was carried out aiming specifically at elucidating the anti-oxidant action of alpha-tocopherol. Lipophilic and hydrophilic azo compounds and copper induced the oxidation of LDL similarly to give cholesterol ester and phosphatidylcholine hydroperoxides as major products. The antioxidant potency of alpha-tocopherol in LDL was much poorer than in homogeneous solution. Doxyl stearic acids were used as spin probe and incorporated in LDL. The rate of reduction of doxyl nitroxide in LDL by ascorbate decreased with increasing distance from the LDL surface. From the competition between the spin probe and alpha-tocopherol in scavenging radical, it was found that the efficacy of radical scavenging by alpha-tocopherol became smaller as the radical went deeper into the interior of LDL. On the other hand, 2,2,5,7,8-pentamethyl-6-chromal spared the spin label regardless of the position of nitroxide. The antioxidant activity of chromanols against LDL oxidation increased with decreasing length of isoprenoid side chain at the 2-position. All these results were interpreted by location and low mobility of alpha-tocopherol in LDL. The tocopherol mediated propagation was observed notably at low rate of radical flux, but this was suppressed by reductant such as ascorbic acid and ubiquinol.

Lethal forebrain ischemia stimulates sphingomyelin hydrolysis and ceramide generation in the gerbil hippocampus.

Ceramide, a hydrolyzed product of sphingomyelin, is reported to play an important role in apoptosis. In this study, we measured the sphingomyelin and ceramide levels in the hippocampus of the gerbil after transient forebrain ischemia for 5 min (lethal) or 2 min (sublethal). The aim was to examine alterations in the sphingomyelin cycle during delayed neuronal death, which we considered could be due to apoptosis. Sphingolipids were separated on high-performance thin-layer chromatography plates and analyzed by gas-liquid chromatography. At 30 min and 24 h after lethal ischemia, sphingomyelin levels were decreased and ceramide levels were increased compared with control levels. No significant changes were observed after sublethal ischemia. These results suggest that the sphingomyelin cycle may have a role in neuronal death.

Fatty acid composition of the chronic subdural hematoma: with reference to its recurrence

The fatty acid composition of aspirated chronic subdural hematoma (CSDH) was measured by gas liquid chromatography and the relationship between fatty acid and recurrence of the hematoma was assessed. Thirty patients with CSDH were operated on through a single burr-hole; 4 patients developed recurrent hematoma (13%). The lipid composition of CSDH was mainly phosphatidylcholine, sphingomyelin, cholesterol, free fatty acid, triacylglycerol and cholesterol ester. The fatty acid constituents were palmitic, stearic, oleic, linoleic, arachidonic and docosahexanoeic acids. Analysis of the polyunsaturated fatty acids demonstrated that hematoma taken from patients with recurrent CSDH contained more linoleic acid (n-6) than those with non-recurrent CSDH. Linoleic and arachidonic acids are known to induce angiogenesis in cultured aortic endothelial cells. Change in fatty acid composition of recurrent hematoma might be associated with rebleeding from the hematoma capsule.

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation.

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.

Comparison of the fatty acid composition of total lipids and phospholipids in breast milk from Japanese women.

Abstract Fatty acid (FA) composition of total lipids (TL) and phospholipids (PL) in breast milk obtained from 20 normal delivery healthy women in Tokyo, Japan was analyzed. Total lipids were extracted from the samples and then PL, consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (Sph) and phosphatidylinositol (PI), were separated by two‐dimensional thin‐layer chromatography. The FA composition of TL and PL was analyzed by gas liquid‐chromatography. Compared with previous reports, the contents of eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3) in TL from Japanese women were higher than those from Chinese and Canadian women, which may be caused by different dietary habits and food types consumed by those populations. The contents of arachidonic acid (AA; 20:4n‐6), EPA and DHA in PE and PI were much higher than those in PC. In addition, no significant correlation of EPA or DHA content was found between TL and PL. The findings indicate that PL especially PE and PI in human milk may be a source of EPA and DHA for infants in the rapid developmental stage. These results should be considered in infant formula production.

Oxidized low-density lipoprotein induces the production of superoxide by neutrophils

Exposure of guinea pig peritoneal neutrophils to ox‐LDL led to the production of superoxide, which was measured by the formation of superoxide‐dependent chemiluminescence. The cells exposed to unoxidized LDL, e.g. native LDL, acetyl‐LDL, and self‐aggregates of LDL showed no production of superoxide. The superoxide production was correlated with the levels of oxidative modification of LDL and reached a maximum between 10 and 30 min during incubation, but preincubating the cells with cytochalasin B decreased the superoxide production. These findings indicate that neutrophils rapidly take up ox‐LDL by phagocytosis and generate superoxide which may cause superoxide‐mediated lipid peroxidation in vivo.