Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA-depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15-min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.     

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.     

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.     

Regional Studies of Changes in Brain Fatty Acids Following Experimental Ischaemia and Reperfusion in the Gerbil

Regional studies of brain phospholipid metabolism were carried out during a period of ischaemia induced in the gerbil by bilateral carotid occlusion for 60 min. The associated changes in free fatty acids (FFAs) during this period and following recirculation for up to 180 min were noted. Following ischaemia there was a generalised rise in the levels of all FFAs with no selective release of either the unsaturated (arachidonic and docosahexaenoic) or saturated (palmitic and stearic) fatty acids. There were no observed differences between the brain regions studied, which is in contrast to previously reported observations for prostaglandins. There was also no indication of any specific phospholipid fraction being involved in FFA release. This would indicate that the release of FFAs from phospholipids is a nonspecific event, probably due to the action of hydrolytic lipases. Restoration of the circulation resulted in a short, sharp increase (within 5 min) in all FFAs, but in contrast to the observations during ischaemia alone there was a relatively larger rise in the unsaturated FFAs as compared to the saturated FFAs. Following this increase there was a gradual general decline in all FFA levels until 180 min of reperfusion. Since there was no preferential depletion of unsaturated FFAs during reperfusion, when free radical attack is considered to be at its maximum, it is our opinion that free radical peroxidation is unlikely to explain the pathology described in our model.     

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.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Lesions to the Corticostriatal Pathways Ameliorate Hypoglycemia-Induced Arachidonic Acid Release

The concentrations of free fatty acids (FFAs) in the neostriatum of control rats and rats subjected to unilateral cortical ablation were measured during and following severe insulin-induced hypoglycemia. The total FFA concentration in the caudate nucleus contralateral to the lesion increased to approximately 1.5 and 3 times the control level after 5 and 30 min of isoelectricity, respectively, and was similar to the control value following 1 h of recovery. After 5 min of isoelectricity, the total FFA pool was significantly smaller in the decorticated striatum. No difference between hemispheres was noted after 30 min of isoelectricity. After 5 min of isoelectricity the levels of stearic and arachidonic acid were selectively increased whereas palmitic acid and oleic acid remained at control levels. In the decorticated striatum of lesioned animals the arachidonic acid concentration was significantly lower, whereas the level of stearic acid was not significantly different from the control value. After 30 min of isoelectricity the levels of all four FFA species were increased. Apart from a significantly lower level of oleic acid on the decorticated side, there were no interhemispheric differences in the FFA levels. Since the early interhemispheric differences in the FFA levels. Since the early interhemispheric hemispheric differences in the levels of arachidonic and stearic acids coincide with a selective decrease in the levels of glutamate and a decreased energy utilization on the decorticated side, the results suggest that glutamate release during hypoglycemia induces an early receptor-mediated degradation of phospholipids, presumably via the phosphatidylinositol cycle.     

Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

Factors affecting the free fatty acids in rat brain cortex

The mode of free fatty acid (FFA) release from rat brain cortex was examined under various treatments prior to decapitation and at various times after decapitation. Brain FFA are comprised mainly of 16:0, 18:0, 18:1 and 20:4 with smaller amounts of 18:2, 22:4 and 22:6. A biphasic mode of FFA release is observed with respect to post-decapitative ischemic treatment. The initial rapid phase involves a 3-fold increase in 18:0 and 20:4 and this process occurs within the first min of decapitation. The second phase involves a less rapid increase of most fatty acids between 2 and 5 min after decapitation. Besides the ischemia-induced increase in brain FFA, the levels of individual fatty acids are also affected by factors such as handling stress and administration of anesthetic agents. Pentobarbital anesthesia, but not ketamine, caused a partial reduction in 18:0 and 20:4 levels in brain in vivo. Pentobarbital treatment also reduced the rapid phase of FFA increase commencing after decapitation. On the other hand, FFA level was higher in animals subjected to ketamine anesthesia, both during the non-ischemic and ischemic phase. Results obtained from this study indicate that the FFA pool in brain is regulated by a complex mechanism contributed by extrinsic and intrinsic factors.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early 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.     

Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells

Apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. The present study uses an in vitro tissue culture model to investigate whether free fatty acids (FFAs), at concentrations comparable to those found following traumatic brain injury, trigger cell death. Nerve growth factor (NGF)-differentiated PC12 cells exposed to oleic and arachidonic acids (2 : 1 ratio FFA/BSA) showed normal cell survival. However, when cells were exposed to stearic and palmitic acids, there was a dramatic loss of cell viability after 24 h of treatment. The cell death induced by stearic acid and palmitic acid was apoptotic as assessed by morphological analysis, and activation of caspase-8 and caspase-3-like activities. Western blotting showed that differentiated PC12 cells exposed to stearic and palmitic acids exhibited the signature apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP). Interestingly, blockade of caspase activities with the pan-caspase inhibitor z-VAD-fmk failed to prevent the cell death observed induced by palmitic or stearic acid. RT-PCR and RNA blot experiments showed an up-regulation of the Fas receptor and ligand mRNA. These findings are consistent with our hypothesis that FFAs may play a role in the cell death associated with trauma in the CNS and PNS.     

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.     

Accurate Evaluation of 1,2-Diacylglycerol in Gerbil Forebrain Using HPLC and In Situ Freezing Technique

Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.     

Platelet-Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.     

Alterations in lipid and calcium metabolism associated with seizure activity in the postischemic brain

Transient ischemia is known to lead to a long-lasting depression of cerebral metabolic rate and blood flow and to an attenuated metabolic and circulatory response to physiological stimuli. However, the corresponding responses to induced seizures are retained, demonstrating preserved metabolic and circulatory capacity. The objective of the present study was to explore how a preceding period of ischemia (15 min) alters the release of free fatty acids (FFAs) and diacylglycerides (DAGs), the formation of cyclic nucleotides, and the influx/efflux of Ca(2+), following intense neuronal stimulation. For that purpose, seizure activity was induced with bicuculline for 30 s or 5 min at 6 h after the ischemia. Extracellular Ca(2+) concentration (Ca(2+)(e)) was recorded, and the tissue was frozen in situ for measurements of levels of FFAs, DAGs, and cyclic nucleotides. Six hours after ischemia, the FFA concentrations were normalized, but there was a lowering of the content of 20:4 in the DAG fraction. Cyclic AMP levels returned to normal values, but cyclic GMP content was reduced. Seizures induced in postischemic animals showed similar changes in Ca(2+)(e), as well as in levels of FFAs, DAGs, and cyclic nucleotides, as did seizures induced in nonischemic control animals, with the exception of an attenuated rise in 20:4 content in the DAG fraction. We conclude that, at least in the neocortex, seizure-induced phospholipid hydrolysis and cyclic cAMP/cyclic GMP formation are not altered by a preceding period of ischemia, nor is there a change in the influx/efflux of Ca(2+) during seizure discharge or in associated spreading depression.     

Starch-free fatty acid complexation in the presence of whey protein

The effect of whey protein on starch-free fatty acid (FFA) complexation was studied in a model system composed of sorghum starch, whey protein, and different FFAs (palmitic, oleic, linoleic, and lauric acids) in a weight ratio of 20:2:1(w/w/w). Whey protein decreased the enthalpy of the melting of the starch–lipid complex by 20–30% for the FFAs except linoleic acid, and increased the reformation exothermic enthalpy by 150–350% in the DSC cooling cycle. The large difference between enthalpies upon heating and cooling in the starch–FFA sample was diminished by the addition of whey protein. X-ray diffraction data showed more pronounced crystalline order of V-type starch–FFA complexes when whey protein was present. A previously described cooling stage viscosity peak, formed due to starch–FFA–protein complexation, paralleled formation of the better defined V-type crystallite of the starch–FFA complex. Whey protein also significantly decreased the amount of starch–FFA complexation in a dilute system. The effect of protein on starch–FFA complexation was related to the formation of a three-component complex composed of starch, FFA, and protein previously identified in our laboratory.     

Turnover of individual free fatty acids in man.

The use of tracer fatty acids in the study of free fatty acid (FFA) metabolism in man is reviewed in light of the present knowledge of the metabolism of individual FFA. The fractional turnovers of palmitic, stearic and oleic acids are very similar and close to the values reported for the total plasma FFA. The fractional turnover of the polyunsaturated arachidonic acid is higher and there is also a sex difference, women showing 70% higher values than men. Splanchnic fractional uptake differs considerably among the individual FFA, while muscle uptake is virtually independent of the chemical structure of the fatty acid. It is concluded that labeled oleic or palmitic acid can be used as tracers for the total FFA fraction when studying the whole body turnover in the postabsorptive state. In other situations, conclusions from result of tracer experiments should be limited to the particular fatty acid studied. For measurements of splanchnic FFA metabolism, no single fatty acid seems to be adequate as a tracer for the total FFA fraction.     

Cerebral Phosphoinositide, Triacylglycerol, and Energy Metabolism in Reversible Ischemia: Origin and Fate of Free Fatty Acids

Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid, diacylglycerol (DAG), triacylglycerol (TAG), and free fatty acids (FFAs), as well as their fatty acid composition, were determined in rat forebrain during ischemia and postischemic recirculation. Cerebral energy state and electroencephalograms (EEGs) were also studied. Fifteen minutes of ischemia resulted in a decrease in PIP2 and PIP contents but not in PI content, concurrent with an enlargement of the FFA and DAG pools. The latter were enriched in stearate and arachidonate. Prolongation of ischemia did not produce further changes in content of any of the inositol phospholipids, but the increase in levels of FFAs and DAG continued. At the end of 45 min of ischemia, levels of both PIP2 and PIP decreased by 45-50%, and the total phosphoinositide content (PIP2 + PIP + PI) decreased by 21%, whereas levels of FFAs and DAG increased to 14- and 3.6-fold of control levels, respectively. During ischemia, the TAG-palmitate level decreased, but the TAG-arachidonate level increased; the tissue energy state deteriorated severely; and the EEG was suppressed. A 30-min recirculation period after 15 or 45 min of ischemia led to increases in PIP2, PIP, and total phosphoinositide contents, whereas levels of FFAs and DAG promptly decreased toward control values. The TAG-arachidonate level peaked and the TAG-palmitate level returned to a low control value during early recirculation. The ischemic changes in tissue lipids were completely reversed within 3 h of recirculation after both periods of ischemia. Adenylates were fully phosphorylated with as little as 30 min of reflow. The EEG activity partially recovered during reflow after 15 min of ischemia, whereas it remained depressed after prolonged ischemia. Thus, phosphodiesteric cleavage of PIP2 and PIP followed by deacylation of DAG is likely to contribute to the production of FFAs in early ischemia. Deacylation of undetermined lipids plays a role for the increment in levels of FFAs in the later period of ischemia. The rapid postischemic increase in levels of PIP2 and PIP indicates active synthesis not only from existing PI, but probably also by means of accumulated FFAs and DAG. These results indicate that the impaired resynthesis of inositol phospholipids cannot be a cause of the poor EEG activity after prolonged ischemia. Degradation and resynthesis of polyphosphoinositides and formation of TAG-arachidonate may be important for modulation of free arachidonic acid levels in the brain during temporary ischemia.