Effects of Maturation on the Phospholipid and Phospholipid Fatty Acid Compositions in Primary Rat Cortical Astrocyte Cell Cultures

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Barbiturate Attenuation of Brain Free Fatty Acid Liberation During Global Ischemia

Abstract: To find a biochemical basis for the increased tolerance of the brain to anoxia during barbiturate anesthesia, we studied whole-brain free fatty acids (FFA) at various times after decapitation of awake and pentobarbital-anesthetized rats. Post-decapitation, the brains were kept at 37°C for 1 to 60 min before freezing in liquid N₂. Nonischemic brains were frozen in liquid N₂, using a rapid sampling technique. Whole-brain arachidonic, stearic, oleic, linoleic, and palmitic acids were quantitated by gas-liquid chromatography. In unanesthetized, nonischemic brain, total FFA was 1226 ± 121 nmol/g brain ( n = 12) and was unaffected by pentobarbital anesthesia (1126 ± 86 nmol/g brain, n = 11), except for a reduction in arachidonic acid. Total FFA in unanesthetized and pentobarbital-anesthetized rats transiently declined between 0 and 1 min of ischemia, and then rose linearly for up to 60 min, with consistently lower values in pentobarbital-treated rats, the greatest attenuation being that of arachidonic and stearic acid liberation. Brain FFA liberation during global ischemia is the first known biochemical variable directly correlated with the duration (i.e., severity) of global ischemia. The attenuation of brain FFA liberation and especially of arachidonic and stearic acids may be the biochemical basis of barbiturate attenuation of ischemic brain injury.     

Free Fatty Acids in the Rat Brain in Moderate and Severe Hypoxia

Abstract: The effects of mild, moderate, and severe hypoxia on cerebral cortical concentrations of free fatty acids (FFAs) were investigated in artificially ventilated rats under nitrous oxide anaesthesia. No change occurred during either mild (arterial Po₂ 35–40 mm Hg) or moderate (Po₂ 25–30 mm Hg) hypoxia. The effects of severe hypoxia (Po₂ about 20 mm Hg) combined with hypotension (mean arterial blood pressure 80–85 mm Hg) varied with the EEG pattern and the tissue energy state. Thus, a major increase in total as well as in individual FFAs occurred first when EEG was severely depressed (almost isoelectric) and energy homeostasis disrupted. On a relative basis the greatest change occurred in free arachidonic acid. It is concluded that hypoxia is associated with an increase in the concentrations of FFAs in brain tissue, provided that tissue oxygen deficiency is severe enough to cause tissue energy failure. However, an increase in FFAs does not invariably accompany minor reductions in the adenylate energy charge (EC) of the tissue.     

Free Fatty Acid Composition of Human and Rat Peripheral Nerve

Abstract: The free fatty acid (FFA) composition of peripheral nerve resembles that of erythrocytes but the composition of both is different from that of brain and other tissues. Approximately 75% of FFAs of nerve and erythrocytes are saturated and <5% are polyunsaturated whereas in brain and other tissues, 30-45% of FFAs are saturated and 25-50% are polyunsaturated. Approximately 10-15% of the total FFA of nerve have very long chain lengths [C₂₄, C₂₆, C₂₈, and C₃₀]. The presence of these very long-chain FFAs in endoneurium cannot be accounted for by the retention of erythrocytes or by lipid degradation. During Wallerian degeneration a significant increase of 18:1, associated with a decrease of saturated FFAs, was found in rat sciatic endoneurium, but normal values were approached when fiber regeneration was well under way. The FFA composition with chain length ≥C₂₆ were not, however, significantly altered with degeneration or repair of nerves. The metabolic significance of this striking difference between nerve and brain FFA composition is unknown but may reflect different functional properties.     

Free Fatty Acid Content and Release Kinetics as Manifestations of Cerebral Lateralization in Mouse Brain

Free fatty acid (FFA) content was analyzed in mouse cerebral hemispheres and cerebellum under basal and postdecapitative ischemic conditions. Total FFA content immediately after decapitation (2 s) was about two-fold higher in the left hemisphere than in the right. Marked dissimilarities between hemispheres were also apparent when FFA levels were measured during short periods of ischemia. Whereas in the right side a significant FFA release took place as early as 10 s, no accumulation was detected in the left in the 2-20 s interval. The highest rates of total fatty acid release occurred in the 20-30 s interval in both hemispheres and decreased afterwards (3 min). Individual FFA, especially stearate and arachidonate, differed in their rates of production, the right cerebral hemisphere being more active in releasing arachidonic acid. In cerebellum, FFA levels were lower and accumulation was slower than in cerebrum in both intervals. When subjected to 3 min ischemia, the same difference in FFA levels between right and left hemispheres (50%) was observed in heads kept at 20 or 30 degrees C. The differences between hemispheres are interpreted as manifestations of an inherent lateralization in the regulation of acylation-deacylation reactions of complex lipids.     

Fatty Acid Transport and Utilization for the Developing Brain

Abstract: To determine the transport and utilization of dietary saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography-mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n-6 and n-3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.     

Changes in Cerebral Acyl-CoA Concentrations Following Ischemia-Reperfusion in Awake Gerbils

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl-CoAs via acyl-CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia-reperfusion on brain fatty acid and acyl-CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia-reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4-fold in the ischemic-reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl-CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl- and arachidonoyl-CoA and a marked decrease in palmitoyl- and docosahexaenoyl-CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl-CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.     

Metabolic Engineering of Plants to Produce Very Long-chain Polyunsaturated Fatty Acids

Very long-chain polyunsaturated fatty acids (VLCPUFAs) are essential for human health and well-being. However, the current sources of these valuable compounds are limited and may not be sustainable in the long term. Recently, considerable progress has been made in identifying genes involved in the biosynthesis of VLCPUFAs. The co-expression of these genes in model systems such as plant embryos or yeast provided many valuable insights into the mechanisms of VLCPUFA synthesis. The recent successful reconstitution of pathways leading to the synthesis of arachidonic acid, eicosapentaenoic acid and finally docosahexaenoic acid in oil-seed plants indicates the feasibility of using transgenic crops as alternative sources of VLCPUFAs. The various approaches used to attain these results and the specific constraints associated with each approach are discussed.     

Differential Effects of Polyunsaturated Fatty Acids on Membrane Capacitance and Exocytosis in Rat Pheochromocytoma-12 Cells

The fusion of synaptic vesicles with the plasma membrane during exocytosis can be recorded by membrane capacitance measurements under voltage-clamp conditions. These measurements enable high time-resolution quantitation of exocytosis. The present study was carried out using the above technique to elucidate the effects of various polyunsaturated fatty acids on exocytosis in a neuroendocrine cell, the rat pheochromocytoma-12 (PC12) cell. External application of eicosapentaenoic acid and arachidonic acid resulted in an increase in capacitance of PC12 cells, indicating fusion of secretory vesicles with cell membranes and exocytosis. In contrast, docosahexaenoic acid, linoleic acid, oleic acid, and vehicle control had no significant effect on capacitance. The above findings show differential effects of polyunsaturated fatty acids on exocytosis in PC12 cells. It is postulated that besides arachidonic acid, eicosapentaenoic acid could also play an important role in exocytosis and neurotransmitter release, in neurons and hormone-secreting cells. Wee-Liat Ong and Bin Jiang - These authors contributed equally to the work.     

Effect of Randomly Interesterified Triacylglycerol Containing Medium- and Long-Chain Fatty Acids on Hepatic Fatty Acid Oxidation after a Single Administration to Rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

The Influence of Bicuculline-Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum

Abstract: Using ventilated rats maintained on N₂O-O₂ (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15-20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10-fold and stearic acid 2- to 3-fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5- to 10-fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20-min values were only about 20% of those observed in the cerebral cortex and the hippocampus.     

Changes of Uric Acid Level in Rat Brain After Focal Ischemia

Changes of uric acid level in rat cerebral hemisphere after left middle cerebral artery (MCA) occlusion were studied by reversed-phase HPLC with electrochemical detection. Uric acid level in the normal group was 2.98 nmol/g tissue. Uric acid concentration of the left hemisphere in the left MCA-occluded group progressively increased after occlusion, and reached a maximum value of 67.26 nmol/g tissue 24 h after ischemia. Uric acid levels in the right hemisphere remained unchanged. Uric acid concentration of the left hemisphere in sham-operated group was 9.29 nmol/g tissue 24 h after the operation.     

Lipid and Fatty Acid Composition of Brain Tissue from Adrenoleukodystrophy Patients

Abstract: White matter and active plaque tissue from adrenoleukodystrophy (ALD) patients were analysed for lipid class and fatty acid compositions and the results compared with white matter from normal brain. ALD white matter was characterized by increased levels of cholesteryl esters and decreased levels of phosphatidylethanola- mine, including phosphatidylethanolamine plasmalogen, in comparison with normal brain white matter. In addition to even higher levels of cholesteryl esters, ALD plaque tissue had reduced levels of cerebrosides as well as phosphati-dylethanolamines. The loss of phosphatidylethanolamine plasmalogen is indicative of early demyelination. Total lipid from ALD white matter and ALD plaque tissue contained nearly five times and seven times, respectively, more 26:0 than total lipid from normal brain white matter. The 26:0 in ALD white matter was elevated in all lipid classes except phosphatidylinositol, but was located mainly in cerebrosides, phosphatidylcholine, sphingomyelin, and sulfatides. Most of the 26:0 in ALD plaque tissue was present in cholesteryl esters, followed by phosphatidylcholine and sphingomyelin, with reduced amounts in cerebrosides as compared with ALD white matter. The results are consistent with an initial accumulation of very-long-chain fatty acids in ALD white matter, primarily in sphingolipids and phosphatidylcholine, and subsequent accumulation of very-long- chain fatty acids in cholesteryl esters during demyelination. In addition, it was notable that the sphingolipids, especially sphingomyelin in ALD brain, had decreased levels of 24:1 and increased levels of 18:0, as well as increased levels of very-long-chain fatty acids. The extent to which the data shed light on mechanisms of demyelination in ALD is discussed.     

硒、谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠脑亚细胞中的分布

采用梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分。分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这些成分在鼠脑亚细胞中的分布呈现明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三种不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘肽过氧化物酶（ＥＣ１．１１．１．９）．还就机体的自我保护机制和硒在脑组织中的重要作用进行了讨论。     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Increased Uric Acid in the Developing Brain and Spinal Cord Following Cytomegalovirus Infection

Abstract: Tissue concentrations of uric acid were determined in the spinal cord, cerebellum, caudate-putamen, and cerebral cortex of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. Transient signs of neurological impairment were observed in MCMV-infected animals beginning on days 13–16 and continuing until days 19–21. At the onset of neurological impairment, uric acid concentrations in tissues from infected animals were 17–60-fold greater than in control animals. On postnatal day 70, 60 days after inoculation and 40 days after resolution of neurological signs, uric acid levels were still two- to threefold greater in infected animals. Histological examination revealed signs of focal ischemia in the cerebral and cerebellar cortices of MCMV-infected mice only at the onset of neurological impairment, with ischemic cell changes in some pyramidal neurons of the cerebral cortex. These results indicate that uric acid may be a sensitive marker of persistent vascular pathology resulting from cytomegalovirus infection of the developing nervous system     

Ascorbic Acid in Fetal Rat Brain

Ascorbic acid in fetal rat brain increases from 374 mg/g on the 15th day of gestation to 710 mg/g by the 20th day and remains at that level until birth. There is an 18% drop from this plateau after birth.     

Role of Acetyl-l-Carnitine in Rat Brain Lipogenesis: Implications for Polyunsaturated Fatty Acid Biosynthesis

Abstract: This study was undertaken to explore the metabolic fate of acetyl- l -carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1-¹⁴C]acetyl- l -carnitine into the lateral brain ventricle of conscious rats. After injection of [1-¹⁴C]acetyl- l -carnitine, the majority of radioactivity was recovered as ¹⁴CO₂ expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl- l -carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl-CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl- l -carnitine following intracerebroventricular injection. In summary, these data implicate acetyl- l -carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.