Harumi Hisaki

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.

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.

Therapeutic time window in the penumbra during permanent focal ischemia in rats: changes of free fatty acids and glycerophospholipids.

Abstract To better define a therapeutic time window for reducing the extent of damage in ischemic penumbra, the time courses of changes in the glycerophospholipid and free fatty acid (FFA) levels were determined in the rat cerebral cortex following induction of the permanent focal ischemia. Focal ischemia induced a biphasic increase in FFA levels in the cerebral cortex, which had been recognized as the ischemic penumbra during the early stages after permanent occlusion of the middle cerebral artery (MCA). The first increase in FFA levels, in which the polyunsaturated fatty acid (PUFA) contained a large number of arachidonic acid (C20:4) molecules, began at 30 min and reached a peak at 1 h, followed by transient return to each sham level 2-6 h after the onset of MCA occlusion. Thereafter, the delayed increase in FFA levels, showing more increases of docosahexaenoic acid (C22:6) molecules than the C20:4 in PUFA compositions, occurred at 24 h. In contrast, the levels of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) decreased rapidly at 30 min of ischemia and returned transiently to each sham level at 1-6 h. The levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), including polyphosphoinositides (PIPs), began to decrease significantly during the late stages, i.e., 24 h after induction of ischemia. These results suggest that the time-dependent changes in FFA and PIPs levels during the early stages of ischemia (until 6 h after induction) might be an important determinant of the subsequent neuronal death in the ischemic penumbra and that the breakdown of glycerophospholipids in the later stages after the induction of focal ischemia was associated with the development of infarction in the cerebral cortex. [Neurol Res 2000; 22: 393-400]

Mild hypothermia reduces the rate of metabolism of arachidonic acid following postischemic reperfusion

Free fatty acid (FFA) accumulation during cerebral ischemia has been described as an indicator of ischemic damage. Furthermore arachidonic acid (AA) metabolites, liberated from glycerophospholipids, have been confirmed to induce disturbances of membrane functions. Are there differences in AA levels in the hippocampus of normo- and hypothermic gerbils following ischemia-reperfusion? In an attempt to answer this question, we first studied the time course of changes in the amount of AA liberated from glycerophospholipids using gerbils subjected to 5 min of ischemia-reperfusion under normo- and mild hypothermia. FFAs (including AA) were separated from total lipids by Bond Elut (NH2) column chromatography and analyzed by gas-liquid chromatography. Mild intra-ischemic hypothermia (MIH) did not affect the ischemia-induced AA accumulation following of 5 min of forebrain ischemia. The accumulated AA amounts under MIH tend to decrease more slowly to baseline levels from 15 to 30 min of reperfusion than do the levels under normothermia. These results suggested that MIH reduced the rate of metabolism of AA after reperfusion and might suppress the generation of free radical, eicosanoids and other bioactive metabolites.

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.

Effect of detergents on the microsomal cytochrome P-450-dependent monooxygenases from frog and rat liver

Abstract 1. 1. A frog liver microsomal hydroxylase system was more susceptible to inhibition by five detergents (sodium cholate, sodium deoxycholate, Triton X-100, Emulgen 913 and Tween 20) in the assay for laurate hydroxylation than the rat liver microsomal hydroxylase system. This suggests that liver microsomal cytochrome P-450-dependent monooxygenases from different sources may not have the same response to detergents. 2. 2. Emulgen 913 was the most effective detergent for the solubilization of cytochrome P-450 from frog and rat liver microsomes among the five detergents. 3. 3. The average level of cytochrome P-450 as determined in the frog ( 0.401 ± 0.023 nmol/mg protein ) is lower than that in the rat (0.60 or 1.19 ± 0.10 ), rabbit ( 1.45 ± 0.35 ), swine ( 0.59 ± 0.16 ), and approximately the same as in the human ( 0.39 ± 0.14 ) and the cat ( 0.380 ± 0.085 ).

Rewarming eliminates the protective effect of cooling against delayed neuronal death.

Mild intra-ischemic hypothermia provides neuroprotection against delayed neuronal death in the hippocampal CA1. It has recently been reported that reduction in the metabolic rate of arachidonic acid (AA) liberated during ischemia might contribute to this neuroprotection. To examine whether rewarming during the early period of recirculation accelerates AA consumption and eliminates the neuroprotection, we measured the levels of AA in the hippocampus after various recirculation times under normothermia and hypothermia with or without rewarming. The tendency for AA to disappear was significantly different between each pair of groups. Histological examination 7 days after ischemia revealed no protection in the rewarmed group. These results suggest that neuronal injury during rewarming after hypothermia may be attributed to the rate of AA metabolism.

Detergent Induced Changes in Serum Lipid Composition in Rats

The influence ofin vivo administration of detergents on serum lipid composition was studied in rats. Male Wistar rats received 50 mg Emulgen 913 (polyoxyethylene nonylphenylether, a nonionic detergent) or SDS (sodium dodecylsulfate, an anionic detergent) per kg of body weight intraperitoneally for 3 consecutive days. Emulgen 913 and SDS administration increased the level of cholesterol esters and phospholipids, respectively. But Emulgen 913 administration reduced the level of triglycerides in the Serum, and SDS administration reduced also the levels of triglycerides and cholesterol esters. In spite of the changes in serum lipid composition, the administration of these detergents did not affect the amount of total lipids in rat serum. The proportion of palmitic, oleic, and docosahexaenoic acids in phospholipids was decreased by the administration of Emulgen 913 while the level of arachidonic acid was raised. However, the level SDS administration had no effect on the fatty acid composition of the serum phospholipids. On the other hand, both Emulgen 913 and SDS administration showed an effect on the fatty acid composition of triglycerides. It is postulated that liver damage due to administration of detergents is responsible for the changes in serum lipid and fatty acid composition in detergent-treated rats.

Regional distribution of ethanolamine plasmalogen in the hippocampal CA1 and CA3 regions and cerebral cortex of the gerbil

Although ethanolamine plasmalogens (EtnPm) are the predominant phospholipids in neural tissue, their physiological role has not been clarified. The biophysical conformation of EtnPm in the proteoliposome enhances the activity of the sodium-calcium exchanger, which has been proposed to induce intracellular calcium ion accumulation during ischemia and early reperfusion. The levels of EtnPm in the areas of the gerbil brain selectively vulnerable to ischemia, namely the hippocampal CA1 and CA3 regions and the cerebral cortex, were measured by high-performance thin-layer chromatography and gas-liquid chromatography. The concentration of EtnPm in the CA1 region, which is the most vulnerable to ischemic and anoxic stress, was 2.6- and 2.7-fold higher than that in the CA3 region and cerebral cortex, respectively. The significantly higher concentration of EtnPm in the hippocampal CA1 region may enhance sodium-calcium exchanger activity and play an important role in the vulnerability of this region to ischemia.