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.     

THE INCORPORATION OF [1-14C]ACETATE INTO UNESTERIFIED FATTY ACIDS IN RAT CEREBRAL CORTEX IN VIVO

—The incorporation of [1-¹⁴C]acetate into unesterified fatty acids and into the fatty acids of neutral glycerides and of phospholipids has been measured in rat cerebral cortex in vivo. The most rapid incorporation is seen in the unesterified fatty acids which have a turnover time of 5-6 min. It is suggested that unesterified fatty acids are precursors to neutral glycerides and phospholipids rather than being derived from them by lipase activity.     

Isolation and characterization of lipid in phloem sap of canola

Phloem isolated from canola (Brassica napus L.) stems was found to contain phospholipid, diacylglycerol, triacylglycerol, steryl and wax esters, and comparatively high concentrations of unesterified fatty acids. Indeed, the composition of phloem lipid was markedly different from that of microsomal membranes and cytosol isolated from both leaves and stems. Specifically, phloem lipid consisted predominantly of unesterified fatty acids and was enriched in medium-chain fatty acids, in particular, lauric, myristic and pentadecanoic acids. This unique composition also distinguished phloem lipid from that of well-characterized cytosolic lipid particles such as oil bodies found in plant cells. Moreover, levels of medium-chain fatty acids in the phloem increased when canola plants were stressed by exposure to sublethal doses of ultraviolet irradiation. Phloem levels of lauric acid, for example, increased by 11-fold upon treatment with sublethal ultraviolet irradiation. Spherical lipid particles were discernible in isolated phloem sap by electron microscopy, suggesting that the lipid in phloem is in the form of lipid particles. The presence of lipid in phloem may be reflective of long-distance lipid transport in plants, primarily in the form of free fatty acids.     

Augmentation of unesterified arachidonic acid in the renal inner medulla of dexamethasone-treated rats. Relationship to altered triglyceride metabolism

The present study was designed to investigate the effect of dexamethasone treatment for 2 weeks (2.5 mg/kg/week, subcutaneously) on the level of unesterified fatty acids, particularly arachidonic acid, in the renal medulla of rats, and to relate the observed effect to changes in the tissue concentration and the fatty acid composition of renal medulla phospholipids and triglycerides. Dexamethasone treatment caused an increase in the renal inner medulla level of unesterified fatty acids, including arachidonic acid, that was associated with a reduction of triglycerides and of arachidonic acid esterified into triglycerides, and with an increase in the rate of fatty acids esterification into triglycerides. In contrast, dexamethasone treatment did not affect the renal medulla concentration of phospholipids, the arachidonic acid content of renal medulla phospholipids, or the rate of esterification of fatty acids into renal medulla phospholipids. In the face of increased fatty acid esterification into triglycerides, the finding of reduced triglyceride levels in the renal medulla of dexamethasone-treated rats suggests excessive triglyceride breakdown. If so, fatty acids including arachidonic acid liberated from triglycerides may contribute to elevation of unesterified fatty acid levels in the renal medulla during dexamethasone treatment. The increased level of free arachidonic acid in the renal medulla of dexamethasone-treated rats may explain in part the reported effect of this steroid in increasing urinary prostaglandins.     

Hormonal and Metabolic Studies in Pheochromocytoma

One patient with benign and another with malignant pheochromocytoma have been studied in an attempt to elucidate the effect of increased catecholamines on the response of blood sugar, unesterified fatty acids, insulin and growth hormone to a glucose load. The presence of increased catecholamines in both patients appeared to produce increased fasting plasma unesterified fatty acid levels, carbohydrate intolerance and an unusual plasma growth hormone response to glucose. There was no interference with the normal decrease in plasma unesterified fatty acids after glucose ingestion. The malignant tumour, but not the benign one, was associated with low plasma insulin levels.After removal of the benign tumour the fasting unesterified fatty acid levels became normal. In addition, following glucose ingestion there appeared to be a more normal plasma insulin and growth hormone response and improved glucose tolerance. After removal of the primary malignant tumour it is noteworthy that the distant metastases secreted abnormal amounts of both adrenaline and noradrenaline.     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Astaxanthin from the red crab langostilla (Pleuroncodes planipes): optical R/S isomers and fatty acid moieties of astaxanthin esters

The composition of the fatty acids of astaxanthin esters and the distribution of astaxanthin optical RS isomers in the esterified and unesterified astaxanthin fractions extracted from the meal of the pelagic red crab langostilla (Pleuroncodes planipes; Decapoda, Anomura) were determined. Astaxanthin diesters comprised approximately 70%, monoesterified astaxanthin approximately 12%, and unesterified astaxanthin approximately 10% of total carotenoids, respectively. Unidentified carotenes and minor yellow xanthophylls represented approximately 8% of the total carotenoids. Three astaxanthin diester fractions (ratio 5:4:1) and one monoester fraction were clearly distinguished by thin-layer chromatography, and fatty acid moieties were determined in all of them. Saturated fatty acids accumulated in astaxanthin diesters, but were reduced in the monoester fraction when compared to langostilla crude oil extract (CE). Astaxanthin diesters, but not monoesters were enriched in C16:0 and C18:1n-9, when compared to the CE. Astaxanthin monoesters were rich in polyunsaturated fatty acids (approximately 70% of total fatty acids), in particular C20:5n-3 and C22:6n-3. Acylation of astaxanthin in langostilla seems to be selective rather than specific. The three diesterified astaxanthin fractions of langostilla had a ratio of approximately 3:1:3 between the (3R,3'R)-, (3R,3'S)-, and (3S,3'S)-astaxanthin isomers, whereas in the monoesterified and unesterified fractions the ratio was approximately 4:1:4. The astaxanthin optical RS isomer composition indicates that langostilla is unable to racemize astaxanthin.     

Lipid composition and metabolism in oospores and oospheres ofAchlya americana

Oospores and oospheres ofAchlya americana Humphrey were isolated by sonication and filtration through nylon-mesh cloth of progressively diminishing porosity, and their lipid composition was investigated. The average dry weight of an oospore was 3.2 ng. Approximately 37% of the dry weight was composed of lipid. Triacylglycerols represented 88.7% of the total lipid, unesterified fatty acids made up 9.7%, and sterols, sterol esters, phospholipids, and mono- and diacylglycerols each constituted less than 1% of the total. Palmitic, oleic, and linoleic acids were the predominant fatty acids, along with smaller amounts of myristic, palmitoleic, stearic, arachidonic, and eicosapentaenoic acids. The fatty acid composition of the triacylglycerol fraction was similar to that of the total lipid, while that of the phospholipid fraction was higher in oleic acid. The unesterified fatty acid fraction was higher in saturated components than the total lipid, while the sterol ester fraction was higher in unsaturated fatty acids. In both the total lipid and the various lipid classes, unsaturated fatty acids increased during spore development. The sterol fraction consisted of 72% fucosterol, 22% cholesterol, and 7% 24-methylenecholesterol. In both oospheres and oospores, 1-[¹⁴C] acetate was assimilated most readily into phospholipids, triacylglycerols, and unesterified fatty acids, and was incorporated preferentially into palmitic, palmitoleic, and oleic acids. 1-[¹⁴C]-Arachidonic acid was incorporated by isolated oospheres into eicosapentaenoic acid, indicating that arachidonic acid is the immediate precursor of eicosapentaenoic acid.     

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15–20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.     

Localization of phosphatidylethanolamine in microsomal membranes and regulation of its distribution by the fatty acid composition

Rat liver microsomes were incubated with the monofunctional aminoreagent fluorescamine. Although the probe easily penetrated the membranes, two pools of phosphatidylethanolamine (PE) could be detected. The first pool rapidly reacted with the probe and comprised 80% of the total PE. The second pool exhibited a very slow interaction. The two pools showed differences in fatty acid composition as well as in their sites of attachment. In vivo labeling with ethanolamine, glycerol, and palmitic and stearic acid resulted in a higher specific activity in the first pool after 1 hr; equilibration with the second pool took about 3 hr. No equilibration between the pools could be detected under in vitro conditions. In vivo incorporation of labeled fatty acids showed that palmitic and stearic acids were mainly incorporated into phosphatidylethanolamine by de novo synthesis, while linoleic and arachidonic acids were introduced through deacylation-reacylation processes. Injection of liposomes consisting of labeled synthetic phosphatidylethanolamines into the portal vein was followed by uptake by the hepatocytes and incorporation of the lipids into the microsomal membranes. Depending on the fatty acid composition of the injected lipid, one of either of the two pools became labeled. It is suggested that the fatty acid composition of a given phospholipid molecule exerts a signal function directing the lipid to its final intramembranous location.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

Diet preferences of warblers for specific fatty acids in relation to nutritional requirements and digestive capabilities

During energy-demanding periods of the annual cycle such as migration or during cold days in winter, birds store fat comprised mostly of 16- or 18-carbon unsaturated fatty acids. In such situations, birds may feed selectively on foods with specific fatty acids that enable efficient fat deposition. We offered wild-caught yellow-rumped warblers Dendroica coronata paired choices between semi-synthetic diets that differed only in their fatty acid composition. Warblers strongly preferred diets containing long-chain (18:1; carbon atoms:double bonds) unsaturated, unesterified fatty acids to diets containing long-chain saturated, unesterified fatty acids (18:0) and they preferred diets containing mono-unsaturated fats (18:1) to diets containing poly-unsaturated fats (18:2). The preference for diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids was consistent in birds tested one week after capture at 21°C, one month after capture when cold-acclimated (1°C), and six weeks after capture at 21°C. Birds acclimated to a diet with 50% of the fat comprised of unesterified stearic acid (18:0) lost mass and reduced their food intake when we reduced ambient temperature from 21°C to 11°C over three days. We conclude that especially in energy-demanding situations there are limits to the yellow-rumped warblers' ability to assimilate some long-chain saturated fatty acids and that this digestive constraint can explain in part why yellow-rumped warblers prefer diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Stimulation of mitochondrial reactive oxygen species production by unesterified,unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P < 0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.     

Arachidonic acid and arachidonoyldiglycerols increase in rat cerebrum during bicuculline-induced status epilepticus

Bicuculline-induced status epilepticus was found to be associated with increased amounts offree fatty acids and diacylglycerols in the rat cerebrum. The predominant fatty acid in both lipid pools was arachidonic acid. The accumulation of arachidonoyl-diglycerols decreased at the time of and during behavioral seizures induced by bicuculline, while the amount of free arachidonic acid appeared to increase. We propose a metabolic relationship between these lipids to explain the described changes. The similarities between the composition of the lipid pools and the fatty acid composition of phosphatidylinositol support the hypothesis that these changes may be a result of a convulsion-activated degradation of this phospholipid from excitable membranes.     

Comparative metabolism of free and esterified fatty acids by the perfused rat heart and rat cardiac myocytes

Studies have been conducted on the uptake and metabolism of unesterified oleic acid and lipoprotein triacylglycerol by the perfused rat heart, and of oleic acid, free glycerol and lipoprotein triacylglycerol by rat cardiac myocytes. The perfused heart efficiently extracted and metabolized unesterified fatty acid and the fatty acid released during lipolysis of the recirculating triacylglycerol. The released glyceride glycerol, however, was largely accumulated in the perfusion media. Cardiac myocytes also extracted and rapidly metabolized unesterified fatty acid. As with the intact heart, free glycerol was poorly utilized by cardiac myocytes. Although the cells appeared to extract a small amount of available extracellular triacylglycerol presented as very low density lipoprotein, this was shown to be unmetabolized, suggesting adsorption rather than surface lipolysis and uptake of the released fatty acid. The data suggest that myocytes are unable to metabolize triacylglycerol fatty acids without prior lipolysis by extracellular (capillary endothelial) lipoprotein lipase.     

Accelerated clearance of plasma triglycerides and free fatty acids in omega3-depleted rats.

The present study aims mainly at exploring the effects of a severe depletion in polyunsaturated long-chain omega3 fatty acids upon the fate of circulating lipids. The plasma concentration and fatty acid pattern of triglycerides, diglycerides, free fatty acids, and phospholipids were measured in omega3-depleted and control rats injected intravenously one hour before sacrifice with either saline, a control medium-chain triglyceride:olive oil emulsion or a medium-chain triglyceride:fish oil emulsion recently found to rapidly increase the phospholipid content of C20:5omega3 and C22:6omega3 in different cell types. The estimated fractional removal rate of the injected triglycerides and the clearance of free fatty acids from circulation were both higher in omega3-depleted rats than in control animals. The injection of the lipid emulsions apparently inhibited intracellular lipolysis, this being least pronounced in omega3-depleted rats. The increased clearance of circulating triglycerides and unesterified fatty acids in omega3-depleted rats may favor the cellular accumulation of lipids. In turn, such an accumulation and the lesser regulatory inhibition of tissular lipolysis may match the increased clearance of circulating unesterified fatty acids and, hence, account for the lack of any significant difference in plasma unesterified fatty acid concentration between these and control animals.     

Protein acylation in normoxic and ischemic/reperfused cardiac tissue.

In addition to a prominent role in tissue energy conversion, fatty acids are involved in signal transduction and modulation of cellular protein localization and function. The latter is accomplished by acylation of specific cellular proteins. In the present study the amount of fatty acyl moieties covalently bound to cardiac proteins and the effect of myocardial ischemia and reperfusion on the degree and relative fatty acyl composition of cardiac proteins have been investigated in isolated rat hearts. In the normoxic heart about 0.32% of the cellular fatty acyl pool is covalently bound to proteins. Approximately 90% of these fatty acyl chains are thio-esterified, whereas a relatively minor part is attached to cardiac proteins through amide linkage. Thio-esterified fatty acyl chains are derived from palmitic, stearic, oleic, linoleic, arachidonic and docosahexaenoic acid. In contrast, amide linked protein acylation shows a preference for myristic acyl chains. Acute ischemia and reperfusion inflicted upon the isolated rat heart did enhance significantly the content of (unesterified) fatty acids, but did neither affect the degree of protein acylation nor the relative fatty acyl composition of acylated proteins in cardiac tissue.     

Fatty acid synthesis in aged potato slices

Synthesis of fatty acids has been studied in aged potato slices. Formation of the very long chain fatty acids was inhibited by the presence of fluoride or by high incubation temperatures. Arsenite caused an increase in the percentage incorporation of radioactivity from acetate-[¹⁴C] into palmitic acid, apparently by inhibiting further elongation. The results indicate that the aged potato contains at least three enzymes responsible for saturated fatty acid synthesis, At short incubation times, the newly formed fatty acids were mainly unesterified but later become incorporated into phospholipids. Phosphatidylcholine contained the greatest proportion of radioactive fatty acids. Newly formed polyenoic fatty acids were principally transacylated into phosphatidylcholine and phosphatidylethanolamines. The very long chain fatty acids, on the other hand, were mainly located in the wax ester and unesterified fatty acid fractions, from which they can easily be converted into suberin components.     

METABOLISM OF BRAIN SPHINGOMYELINS: HALF-LIVES OF SPHINGOSINE, FATTY ACIDS AND PHOSPHATE FROM TWO TYPES OF RAT BRAIN SPHINGOMYELIN

Abstract— The metabolism of rat brain sphingomyelins containing short-chain (C₁₆-C₁₈) and long-chain (C₂₀- C₂₄) fatty acids has been studied by determination of the content of radioactivity in the sphingo-sinc. fatty acids and phosphate of the sphingomyelins over a period of 60 days following the intracisternal injection of [¹⁴C]acetate and [³²P]phosphate. From the rate of decrease of the specific radioactivities of the different constituents of short-chain fatty acid sphingomyelins, we have calculated a half-life of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. For the long-chain fatty acid sphingomyelins the half-life of sphingosine was approximately 465 days. The fatty acids and phosphate from these sphingomyelins had fast and slow turnover pools. The half-life for the fast pool was 7 days for the two constituents and the estimated half-lives for the slow pool were 220 days for fatty acids and 480 days for phosphate. These results suggest that one can distinguish at least three metabolic pools of brain sphingomyelins: (a) sphingomyelins with long-chain fatty acids situated in myelin whose half-lives are 465 days for sphingosine, 220 days for fatty acids and 480 days for phosphate; (b) sphingomyelins with long-chain fatty acids located mainly in non-myelin structures having half-lives of 465 days for sphingosine. 7 days for fatty acids and 7 days for phosphate; (c) sphingomyeiins with short-chain fatty acids with half-lives of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. The differences between the half-lives of the three metabolic pools of sphingomyelin, together with the subcellular localizations of the two molecular species of these compounds, suggest that the metabolism of the different molecular species of sphingomyelin are independent and that in various subcellular fractions the long-chain fatty acid and short-chain fatty acid sphingomyelins have different turnover rates.