Mechanism of Arachidonic Acid Liberation During Ischemia in Gerbil Cerebral Cortex

Once brain ischemia was induced in the gerbil cerebral fronto-parietal cortex, serial changes occurred in energy metabolites and various lipids. The amounts of inositol-containing phospholipids began to decrease immediately after energy failure, followed by an increase in the amount of 1,2-diacylglycerol with a subsequent liberation of arachidonic acid and other free fatty acids. The fatty acid compositions of inositol-containing phospholipids, of 1,2-diacylglycerols produced by ischemia, and of free fatty acids liberated during ischemia were quite similar. The amount of stearic acid liberated was much larger than that of arachidonic acid between 30 s and 1 min of ischemia. On the other hand, there was no significant decrease in the amount of the other phospholipids except for phosphatidic acid. Furthermore, there was also no change in the fatty acid composition of phosphatidylcholine or phosphatidylethanolamine throughout 15 min of ischemia. The amount of cytidine-monophosphate reached a peak (36.7 nmol/g wet wt) at 2 min of ischemia. These results indicated that arachidonic acid was predominantly liberated from inositol-containing phospholipids by phospholipase C, and by the diglyceride lipase and monoglyceride lipase system rather than from phosphatidylcholine or phosphatidylethanolamine by phospholipase A2 or plasmalogenase or choline phosphotransferase during the early period of ischemia.     

Effect of the conditions of analysis and obtaining of the sample on the level of free fatty acids in the brain of the rat

Free fatty acids (FFA) have been determined in the rat brain by gas-liquid chromatography after isolation by thin-layer chromatography on silica gel. The brains were removed under three experimental conditions, 1) after freezing in situ with liquid nitrogen, 2) after immersion of the animal in liquid nitrogen, 3) after decapitation, the brain being frozen 3 minutes later. The total FFA level was found to be equal respectively to 20.1, 33.1 and 168 micrograms/g. In any case, the main fatty acids were palmitic, stearic and oleic acid but there were marked increases in arachidonic and docosahexaenoic acids following decapitation. A cause of error in the FFA determination originated in the use of commercial silica gel which contained significant amounts of fatty acids.     

Biosynthesis of diacylglycerols by rat intestinal mucosa in vivo.

The biosynthesis of diacylglycerols was studied in rat intestinal mucosa during in vivo absorption of a low molecular weight fraction fraction of butter oil and of the corresponding medium and long chain fatty acids. The experimental fat solutions were given by stomach tube to the animals after a 24-h fast and mucosal scraping were collected 3 h later. The lipids were isolated and the acylclycerols determined by combined thin-layer chromatography gas-liquid chromatography techniques and stereospecific analyses. Free fatty acid feeding led mainly to sn-1,2-diacyl-glycerols, which contained exogenous and endogenous fatty acids. During triacylglycerol feeding, both sn-1,2-and sn-2,3-diacylglycerols were recovered in significant amounts from the intestinal mucosa. The composition of the sn-2,3-diacylglycerols corresponded to that with exogenous fatty acids but the sn-1,2-diacylglycerols clearly contained both exogenous and endogenous fatty acids. In all cases it was possible to isolate endogenous sn-1,2-diacylglycerols made up largely of species with linoleic and arachidonic acids in the 2 position and palmitic and stearic acids in the 1 position, which apparently were not converted to triacylglycerols. The in vivo reacylation of 2-monoacylglycerols via both sn-1,2- and sn-2,3-diacylglycerols is in agreement with similar findings in vitro with everted sacs of rat intestinal mucosa.     

Norepinephrine-induced 1,2-diacylglycerol accumulation and change in its fatty acid composition in the isolated perfused rat heart

Phosphoinositide hydrolysis is elicited by -adrenoceptor stimulation in the myocardium, resulting in the generation of 1,2-diacylglycerol by the direct activation of phospholipase C. However, the physiological role of 1,2-diacylglycerol accumulation in the heart has been largely unexplored. Therefore, we studied the effects of norepinephrine on the accumulation of 1,2-diacylglycerol and its fatty acid composition, as well as its function in isolated perfused rat hearts. A 30 min perfusion with norepinephrine following a stabilization period of 25 min caused increases of 68% and 57% in 1,2-diacylglycerol levels in the heart at 10^–6 M and 5 × 10^–6 M, respectively, compared to controls. Analysis of its fatty acid composition showed a significant elevation in the percentages of 18:2 and 20:4 although the absolute amounts of these increases in fatty acids were relatively low when compared to the elevation in the total amount of 1,2-diacylglycerol. The change in contractility was not consistently related to an increase in 1,2-diacylglycerol. These results indicate that increase in 1,2-diacylglycerol level in response to norepinephrine perfusion was accompanied by a change in fatty acid composition of 1,2-diacylglycerol.     

Versatile methods for the synthesis of mixed-acid 1,2-diacylglycerols

Two methods for synthesizing mixed-acid 1,2-diacylglycerols starting from 2,3-epoxy-1-propanol (glycidol) or 3-chloro-1,2-propanediol have been described. This first method involves fatty acid addition to a protected glycidol derivative and solid-state isomerization. The second approach exploits the specificity of the trityl group for primary alcohols and the nucleophilic replacement of chlorine by a carboxylate ion in an aprotic solvent. The second method proves to be more general: with 3-chloro-1-O-trityl-1,2-propanediol as an intermediate compound apparently all types of mixed-acid, saturated and unsaturated, chiral and racemic 1,2-diacylglycerols can be prepared in good yields. In the first method tritylglycidol is a good starting compound. The use of this method, however, is restricted because only the 2-position of glycerol can be occupied with an unsaturated fatty acid. For de-blocking protected 1,2-diacylglycerols, the trityl group and other protecting groups were exchanged for the trifluoroacetyl group, which group could then be removed without any detectable acyl migration (< 1%). To this end, the 1,2-diacyl-3-trifluoroacetyl-glycerols were dissolved at room temperature in methanol containing pyridine, whereby the trifluoroacetyl group was split off, giving the 1,2-diacylglycerol.     

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.     

Relation Between Free Fatty Acid and Acyl-CoA Concentrations in Rat Brain Following Decapitation

To ascertain effects of total ischemia on brain phospholipid metabolism, anesthetized rats were decapitated and unesterified fatty acids and long chain acyl-CoA concentrations were analyzed in brain after 3 or 15 min. Control brain was taken from rats that were microwaved. Fatty acids were quantitated by extraction, thin layer chromatography and gas chromatography. Long-chain acyl-CoAs were quantitated by solubilization, solid phase extraction with an oligonucleotide purification cartridge and HPLC. Unesterified fatty acid concentrations increased significantly after decapitation, most dramatically for arachidonic acid (76 fold at 15 min) followed by docosahexaenoic acid. Of the acyl-CoA molecular species only the concentration of arachidonoyl-CoA was increased at 3 min and 15 min after decapitation, by 3–4 fold compared with microwaved brain. The concentration of docosahexaenoyl-CoA fell whereas concentrations of the other acyl-CoAs were unchanged. The increase in arachidonoyl-CoA after decapitation indicates that reincorporation of arachidonic acid into membrane phospholipids is possible during ischemia, likely at the expense of docosahexaenoic acid.     

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.     

Cholesterol Esters of the Human Brain During Fetal and Early Postnatal Development: Content and Fatty Acid Composition

Abstract: The content and fatty acid composition of cholesterol esters of the human brain during development from 13 weeks' gestation up to 26 months of age was studied. The three major brain areas, the forebrain, cerebellum, and the brain stem, were studied separately. The concentration of the esters in each brain region was the highest at the earliest fetal age of 13 weeks and fell during growth. However, transient rises in the concentration were observed, at about birth in the forebrain and at 4–5 months after birth in the cerebellum The peak concentration during the transient period (125–150 μg/g fresh tissue of forebrain and 100–125 μg/g of cerebellum) was similar to the concentrations observed in the two parts respectively during early fetal ages. The brain stem also showed similar transient peak at about a few weeks before birth, but only when the esters were expressed as amount per cell. In absolute terms, a clear transient period was evident in the forebrain between birth and 9 months, while in the cerebellum or the brain stem, the total amount of the esters increased up to about 1 year of age and then remained almost unchanged. The major fatty acids of the esters were palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acid. Most of these fatty acids showed certain changes in relative proportions during development. Thus, in the forebrain, palmitic and oleic acid decreased from about 32% and 40% (weight percentages) at 13–15 weeks of gestation to about 20% and 25% respectively at 26 months of age. During this period, linoleic and arachidonic acid increased from about 3% and S% to about 10% and 24%, respectively. Most of these changes occurred after birth. The cerebellum and the brain stem differed only slightly from the forebrain in either the fatty acid composition or the pattern of the developmental changes in the composition.     

The fatty acid composition of 1,2-diacylglycerol and polyphosphoinositides from human erythrocyte membranes.

The fatty acid compositions of 1,2-diacylglycerol and polyphosphoinositides have been determined in human erythrocyte membranes that have been incubated in the presence or in the absence of Ca2+. The results show that the diacylglycerol that is formed in Ca2+-treated membranes has a fatty acid composition closely similar to that of the inositides, consistent with previous indications that Ca2+ stimulates the activity of a polyphosphoinositide phosphodiesterase in the membranes. In contrast with some previous results, it appears that these plasma-membrane inositides and their derived diacylglycerols are rich in stearic acid and arachidonic acid.     

Measurements of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.     

Age features of glycerolipid metabolism in rat liver under short term exposure to thyroxine

The age specificity of the regulation by thyroid hormones of 1,2-diacylglycerol production in rat liver has been studied. It was found that L-thyroxine-stimulation of the 3-month old rats liver cells resulted in a rapid rise in 1,2-diacylglycerol concentration in hepatocytes and simultaneous degradation of phospholipids. The endogenous phosphatidylcholine and phosphatidylethanolamine are the sources of 1,2-diacylglycerol in a liver. Under the action of hormone liver cells of young rats may product 1,2-diacylglycerol from exogenous 1-acyl, 2-[14C]arachidonyl-phosphatidylethanolamine. Thyroxine had no effect on de novo 1,2-diacylglycerols formation and their release from triacylglycerol. In liver cells of elder rats, 1,2-diacylglycerol and individual phospholipids content are unaffected by hormones.     

Biphasic Liberation of Arachidonic and Stearic Acids During Cerebral Ischemia

The mode of free fatty acid (FFA) liberation from the mouse brain during ischemia was investigated at various times after decapitation and under nizofenone treatment. Normal nonischemic brain FFAs consist mainly of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) with smaller amounts of arachidonic acid (20:4), docosahexaenoic acid (22:6), and others. Postdecapitative ischemia induced a rapid, biphasic release of 20:4 after a short lag of less than 30 s. The first phase showed a rapid 6.4-fold increase within 1 min of decapitation, followed by the second phase involving a slow release at less than one-fifth the rate of the first phase and lasting for at least 10 min. A similar, but not so marked, biphasic liberation was observed with 18:0. However, all of the other fatty acids (16:0, 18:1, 22:6, and others) were released only in a single phase at a slow rate. The time course for the rapid and specific liberation of 20:4 coincided with the time course for the decrease in brain ATP concentration during ischemia. Pretreatment of the animals with nizofenone resulted in a marked suppression of both FFA liberation and ATP depletion during ischemia. This suppression was particularly noteworthy with 20:4 and 18:0. The present study indicates that there is a specific and rapid liberation of 20:4 and 18:0 in a very early stage of ischemia and that this liberation seems to depend on availability of ATP in the brain. The physiological role of this transient 20:4 liberation during ischemia is discussed.     

Biosynthesis of triacylglycerols by rat intestinal mucosa in vivo.

The structure of mucosal triacylglycerols was studied in rat intestinal mucosa in vivo during the absorption of a low molecular weight fraction of butter oil and of the corresponding free fatty acids of medium and long chain length. The mucosal lipids were isolated by solvent extraction and the acylglycerol structures were determined by combined AgNO3- thin-layer chromatography and gas-liquid chromatography techniques and stereospecific analysis. Evidence was obtained for a rapid biosynthesis of triacylglycerols from diacylglycerols arising from the operation of both the monoacylglycerol and the phosphatidic acid biosynthetic pathways. Both sn-1,2- and sn-2,3-diacylglycerols appeared to be converted to triacylglycerols at significant rates, but a preferential utilization of sn-1,2-diacylglycerols could not be excluded. Endogenous dilution varied from a miniumum of 5% during triacylglycerol biosynthesis from monoacylglycerols to 15% during their synthesis from free fatty acids, and was characterized by a preferential placement of the endogenous acids in the sn-3 and 2 positions of the triacylglycerol molecules. Exogenous myristic acid was preferentially associated with the sn-3 position, and stearic acid became preferentially bound to the sn-1 position. The complexity of the triacylglycerol end products prevented an exact estimate of the contribution of the phosphatidic acid pathway, but the acylglycerol structures were compatible with a minimum of 20% of total triacylglycerol yield at all times.     

Polyphosphoinositides as a Probable Source of Brain Free Fatty Acids Accumulated at the Onset of Ischemia

The quantitative relationship between phosphoinositides and free fatty acids (FFAs) in brain ischemia was studied by measuring contents of individual fatty acids in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DAG), and the FFA pool. Various periods of complete ischemia (1, 3, 10, and 30 min) were produced by decapitation. Ischemia of 1-3 min caused rapid decreases in PIP2 and PIP content together with preferential production of stearic and arachidonic acids in the DAG and FFA pools. The decrement in levels of these fatty acid residues in polyphosphoinositides was sufficient to account for their increment in levels in the enlarged DAG and FFA pools. After 10 min of ischemia, levels of PIP2, PIP, and DAG approached plateau values, but levels of all FFAs continued to increase. The increases in content of DAG and FFAs at later ischemic periods could not be accounted for by the decreases in content of PIP2 and PIP, PI and PA levels showed only transient and subtle changes. These results indicate that, at the onset of ischemia, phosphodiesteric cleavage of PIP2 and PIP and subsequent deacylation by lipases are primarily responsible for the preferential increase in levels of free stearic and arachidonic acids and that, later, hydrolysis of other phospholipids plays a major role in the continuous accumulation of FFAs.     

Biosynthesis of triacylglycerols containing short-chain fatty acids in lactating cow mammary gland. Activity of diacylglycerol acyltransferase towards short-chain acyl-CoA esters

1. Microsomal 1,2-diacylglycerol acyltransferase from lactating cow mammary gland incorporated equal molar amounts of microsomal-bound 1,2-dipalmitoyl [2-3H]glycerol and [1-14C]-butyrate, [1-14C]hexanoate or [1-14C]palmitate from their CoA esters into triacylglycerol. The enzyme could also utilize exogenous 1,2-diacylglycerols in the presence of ethanol. 2. The pH optimum of the enzyme was 6.1 and 6.4 with butyryl-CoA and hexanoyl-CoA respectively. Values of V were approximately the same (2.7 and 2.4 nmol-min-1-mg-1, respectively), but values of Km were different (34 and 10 muM, respectively) with these two substrates. Mg2+ was not required as cofactor. 3. The presence ofa Mg2+-dependent phosphatidate phosphatase in the microsomal fraction was demonstrated. 4. It is proposed that triacylglycerols containing butyric and hexanoic acid are biosynthesized in cow mammary gland by the glycerolphosphate pathway, in which long-chain 1,2-diacylglycerols derived from phosphatidic acid are acylated at the sn-3 position by short-chain acyl-CoA esters.     

PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.     

Hydrolysis of chylomicron arachidonate and linoleate ester bonds by lipoprotein lipase and hepatic lipase

Chylomicrons labeled with [3H]arachidonic and [14C]linoleic acid were incubated with bovine milk lipoprotein lipase or rat postheparin plasma, containing both lipoprotein lipase and hepatic lipase. During incubation with bovine lipoprotein lipase, [3H]arachidonic acid was released from chylomicron triacylglycerols at a slower rate than [14C]linoleic acid. Only small amounts of [14C]linoleic acid were found as 1,2(2,3)-diacylglycerols, whereas a transient accumulation as [14C]monoacylglycerols was observed. In contrast, significantly more [3H]arachidonic acid was found as 1,2(2,3)-diacylglycerols than as monoacylglycerols at all time intervals investigated. The initial pattern of triacylglycerol hydrolysis by postheparin plasma was similar to that of bovine lipoprotein lipase. However, in contrast to the results obtained with bovine lipoprotein lipase, little [3H]1,2(2,3)-diacylglycerol accumulated. The addition of antiserum to hepatic lipase increased the amount of 3H found in 1,2(2,3)-diacylglycerols and inhibited the formation of free [3H]arachidonic acid. The antiserum also caused a significant inhibition of the hydrolysis of [3H]-but not of [14C]triacylglycerol. With regard to chylomicron phospholipids, the rate of hydrolysis of [14C]linoleoyl phosphatidylcholine with milk lipoprotein lipase was twofold higher than that of the [3H]arachidonyl phosphatidylcholine. However, the hepatic lipase of postheparin plasma had similar activity towards the two phosphatidylcholine species. Postheparin plasma rapidly hydrolyzed chylomicron 3H-labeled and 14C-labeled phosphatidylethanolamine to the same degree, and lipoprotein lipase similarly hydrolyzed 3H-labeled and 14C-labeled phosphatidylethanolamine at approximately equal rates. Antiserum to hepatic lipase inhibited the postheparin plasma hydrolysis of phosphatidylethanolamine and 3H-labeled phosphatidylcholine by about 60%, but the 14C-labeled phosphatidylcholine by only 27%.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,2-Diacylglycerols do not potentiate the action of phospholipases A2 and C in human platelets

1,2-Diacylglycerol has recently been reported to potentiate the ability of phospholipases A and C to hydrolyze phospholipids in a cell-free system. The present study has been undertaken to investigate whether 1,2-diacylglycerol can also perform this function in intact cells using the platelet as a test system. Exogenous 1-oleoyl-2-acetyl-glycerol ( OAG ) and 1,2- didecanoylglycerol , at concentrations sufficient to produce maximal phosphorylation of a 40,000 dalton protein, caused no significant formation of [3H]inositol phosphates and [32P]phosphatidic acid (products of phospholipase C activation) or [14C]arachidonic acid metabolites and lysophosphatidyl[3H]inositol (products of phospholipase A2 activation). These data therefore imply that 1,2-diacylglycerols do not potentiate the actions of phospholipases A2 and C in intact platelets at concentrations that are physiologically relevant.     

Modification of GABA and calcium uptake by lipids in synaptosomes from normoxic and ischemic brain

The effects of membrane lipid disturbances induced by ischemia and exogenously added lipids on the uptake of GABA and Ca²⁺ were investigated in gerbil brain synaptosomes. Ischemia was produced by bilateral ligation of common carotid arteries in Mongolian gerbil for 10 min. The level of the free fatty acids (FFA) increased significantly in ischemic synaptosomes. Incorporation of [1-¹⁴C]arachidonate into membrane phosphatidylinositol and phosphatidylcholine was decreased by about 20–35%. Furthermore ischemia exerted an inhibitory effect on GABA uptake but remained without effect on calcium accumulation. Thiopental application in dose of 100 mg per kg body weight 30 min before ischemia caused a protective effect on membrane lipid disturbances induced by ischemia and enhanced GABA uptake. Unsaturated fatty acids (arachidonate and docosahexanoate) in concentration of 10⁻⁵−10⁻⁴ mol/l and lysocompounds (lysophosphatidylcholine and lysophosphatidylethanolamine) in concentrations higher than 10⁻⁴ mol/l decreased GABA and Ca²⁺ uptake in synaptosomes from normoxic brains. No effect was seen with saturated stearic acid. These results suggest that the inhibition of GABA uptake into ischemic synaptosomes resulted from an action of unsaturated fatty acids, arachidonic and docosahexanoic acids which were liberated during ischemia. Moreover the transient higher local concentration of lysophospholipids close to GABA carrier system may also have contributed to the inhibition observed during ischemia.