Utilization of Uniformly Labeled 13C-Polyunsaturated Fatty Acids in the Synthesis of Long-Chain Fatty Acids and Cholesterol Accumulating in the Neonatal Rat Brain

Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18-carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20-and 22-carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo ¹³C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly ¹³C-enriched 16-and 18-carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U-¹³C-polyunsaturated fatty acid mixture, ¹³C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; ¹³C enrichment remained high in the phospholipids and cholesterol for 15 days. ¹³C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in ¹³C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16-and 18-carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long-chain saturated and monounsaturated fatty acids and cholesterol.     

Phospholipid Degradation and Cellular Edema Induced by Free Radicals in Brain Cortical Slices

Abstract: Cellular edema and increased lactate production were induced in rat brain cortical slices by xanthine oxidase and xanthine, in the presence of ferric ions. Lipid peroxidation, as measured by thiobarbituric acid-reactive malon-dialdehyde, was increased 174%. Among the various subcellular fractions of brain cortex, xanthine oxidase-stimulated lipid peroxidation was highest in myelin, mitochondria, and synaptosomes, followed by microsomes and nuclei. Antioxidants, catalase, chlorpromazine, and butylated hydroxytoluene inhibited lipid peroxidation in both homogenates and synaptosomes, indicating H₂O₂ and radicals were involved. Further, several free fatty acids, especially oleic acid (18:1), arachidonic acid (20:4), and docosahexaenoic acid (22:6) were released from the phospholipid pool concomitant with the degradation of membrane phospholipids in xanthine oxidase-treated synaptosomes. These data suggest that Upases are activated by free radicals and lipid peroxides in the pathogenesis of cellular swelling.     

Vanadium and lipid peroxidation: evidence for involvement of vanadyl and hydroxyl radical

The present study was designed to determine which form of vanadium is involved in initiating conjugated diene formation in both purified and partially peroxidized fatty acids, and to determine if active oxygen radicals are involved in this process. We report that vanadyl is the active form of vanadium in initiating conjugated diene formation in micelles prepared from purified fatty acids or partially peroxidized fatty acids. Vanadate did not initiate conjugated diene formation in either case. Hydroxyl radicals were shown to be involved in the initiation of diene conjugation when vanadyl and hydrogen peroxide were added together in a reaction mixture. In this case, there was a rapid burst of conjugated diene formation which quickly leveled off. Using spin trapping techniques, hydroxyl radicals were shown to be generated in the vanadyl-catalyzed break-down of fatty acid hydroperoxides. A comparison was made between the ability of vanadyl or vanadyl chelates to decompose hydrogen peroxide and catalyze the decomposition of fatty acid hydroperoxides. It was found that strongly chelated vanadyl (vanadyl/EDTA) was much less effective in decomposing both hydrogen peroxide and fatty acid hydroperoxides than the weak vanadyl chelates (e.g., vanadyl/ADP). This study suggests a mechanism to explain the effects of vanadium on lipid peroxidation.     

Characterization of a Novel Phospholipase A2 Activity in Human Brain

Abstract: Phospholipases A₂ (PLA₂) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA₂ activity in human CNS. We therefore have characterized some aspects of the PLA₂ activity present in the temporal cortex of human brain. More PLA₂ activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA₂ activity eluted in a high molecular mass fraction (∼180 kDa). PLA₂ activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF₃, γ-linolenoyl amide, and N -methyl γ-linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA₂s, it differs in several key features.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Brain Cortical Fatty Acids and Phospholipids During and Following Complete and Severe Incomplete Ischemia

Abstract: To explore the possibility that peroxtdative degradation of brain tissue lipid constituents is an important mechanism of irreversible ischemic damage, we measured cortical fatty acids and phospholipids during reversible brain ischemia in the rat. Neither complete nor severe incomplete ischemia (5 and 30 min) caused any measurable breakdown of total or individual fatty acids or phospholipids. Except for a small (and reversible) decrease of inositol plus serine phosphoglycerides in the early postischemic period following 30 min of incomplete ischemia, there were no significant losses of fatty acids or phospholipids during recirculation. Since peroxidation, induced in brain cortical tissue in vitro , characteristically involves degradation of polyenoic fatty acids (arachidonic and docosahexaenoic acids) and of ethanolamine phosphoglycerides, the present in vivo results fail to support the hypothesis that peroxidation of membrane lipids is of primary importance for ischemic brain cell damage. Both complete and severe incomplete ischemia caused a similar increase in the tissue content of free fatty acids (FFA). Thus the FFA pool increased by about 10 times during a 30-min ischemic period, to constitute 1 - 2% of the total fatty acid pool. Since there was a relatively larger increase in polyenoic FFA (especially in arachidonic acid) than in saturated FFA, the release of FFA may be the result of activation of a phospholipase A₂ unbalanced by reesterification. Increased levels of FFA persisted during the initial recirculation period, but a gradual normalization occurred and the ischemic changes were essentially reversed at 30 min after restoration of circulation. The pathophysiological implications of the changes in FFA are discussed with respect to mitochondrial dysfunction, formation of cellular edema and prostaglandin-mediated deterioration of postischemic circulation.     

Lipid Hydroperoxides Inhibit Reacylation of Phospholipids in Neuronal Membranes

The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1-14C]arachidonic acid into synaptosomal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration-dependent manner, with 50% inhibition occurring at less than 25 microM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non-competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.     

Postischemic Cerebral Lipid Peroxidation In Vitro: Modification by Dietary Vitamin E

Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE-deficient, -normal, and -supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of alpha-tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE-deficient group, intermediate in the VE-normal group, and smallest in the VE-supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. alpha-Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE-deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation-induced lipid peroxidation suggests free radical mediation.     

Coupling Among Energy Failure, Loss of Ion Homeostasis, and Phospholipase A2 and C Activation During Ischemia

Abstract— The objective of the present experiments was to correlate changes in cellular energy metabolism, dissipative ion fluxes, and lipolysis during the first 90 s of ischemia and, hence, to establish whether phospholipase A₂or phospholipase C is responsible for the early accumulation of phospholipid hydrolysis products. Ischemia was induced for 15–90 s in rats, extracellular K⁺ (K⁺_e) was recorded, and neocortex was frozen in situ for measurements of labile tissue metabolites, free fatty acids, and diacylglycerides. Ischemia of 15-and 30-s duration gave rise to a decrease in phosphocreatine concentration and a decline in the ATP/free ADP ratio. Although these changes were accompanied by an activation of K⁺ conductances, there were no changes in free fatty acids until after 60s, when free arachidonic acid accumulated. An increase in other free fatty acids and in total diacylglyceride content did not occur until after anoxic depolarization. The results demonstrate that the early functional changes, such as activation of K⁺ conductances, are unrelated to changes in lipids or lipid mediators. They furthermore suggest that the initial lipolysis occurs via both phospholipase A₂ and phospholipase C, which are activated when membrane depolarization leads to influx of calcium into cells.     

Carbon isotope analysis of acetaldehyde emitted from leaves following mechanical stress and anoxia

Although the emission of acetaldehyde from plants into the atmosphere following biotic and abiotic stresses may significantly impact air quality and climate, its metabolic origin(s) remains uncertain. We investigated the pathway(s) responsible for the production of acetaldehyde in plants by studying variations in the stable carbon isotope composition of acetaldehyde emitted during leaf anoxia or following mechanical stress. Under an anoxic environment, C3 leaves produced acetaldehyde during ethanolic fermentation with a similar carbon isotopic composition to C3 bulk biomass. In contrast, the initial emission burst following mechanical wounding was 5–12‰ more depleted in ¹³C than emissions under anoxia. Due to a large kinetic isotope effect during pyruvate decarboxylation catalysed by pyruvate dehydrogenase, acetyl-CoA and its biosynthetic products such as fatty acids are also depleted in ¹³C relative to bulk biomass. It is well known that leaf wounding stimulates the release of large quantities of fatty acids from membranes, as well as the accumulation of reactive oxygen species (ROS). We suggest that, following leaf wounding, acetaldehyde depleted in ¹³C is produced from fatty acid peroxidation reactions initiated by the accumulation of ROS. However, a variety of other pathways could also explain our results, including the conversion of acetyl-CoA to acetaldehyde by the esterase activity of aldehyde dehydrogenase.     

Linoleic acid-induced ultra-weak photon emission from Chlamydomonas reinhardtii as a tool for monitoring of lipid peroxidation in the cell membranes

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes.     

Arachidonic Acid and Other Unsaturated Fatty Acids Alter Membrane Potential in PC12 and Bovine Adrenal Chromaffin Cells

Abstract: The action of arachidonic acid and other fatty acids on membrane potential in PC 12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1–40 μ M ) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K⁺-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC 12 and bovine chromaffin cells by modulating K⁺ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca²⁺ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca²⁺ influx and secretion. We propose a model where internally generated fatty acids act as a feed-back to desensitize the stimulated cell via inhibition of receptor-dependent Ca²⁺ influx and induction of membrane hyperpolarization.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

F4 - Isoprostanes as Specific Marker of Docosahexaenoic Acid Peroxidation in Alzheimer's Disease

Abstract : F₂-isoprostanes are prostaglandin-like compounds derived from free radical-catalysed peroxidation of arachidonic acid. Peroxidation of eicosapentaenoic acid produces F₃-isoprostanes, whereas peroxidation of docosahexaenoic acid would give F₄-isoprostanes. This study demonstrates the presence of esterified F₄-isoprostanes in human brain and shows that levels are elevated in certain brain cortex regions in Alzheimer's disease. Our data with Alzheimer's disease suggest that analysis of F₄-isoprostanes will provide new opportunities to study lipid peroxidation in the neurodegenerative diseases.     

Nonesterified fatty acids and lipid peroxidation

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidaton of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.     

Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium-Independent Efflux by Oleic and Arachidonic Acids

Abstract: The release of preloaded [¹⁴C]neuroactive amino acids (glutamic acid, proline, γ-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca²⁺ -independent process. This Ca²⁺-independent efflux is increased by compounds that activate Na⁺ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [¹⁴C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on ²²Na⁺ permeability is observed. Since Na⁺ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na⁺ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na⁺ gradient.     

Membrane fatty acids, lipid peroxidation and adenylate cyclase activity in cultured neural cells

Lipid peroxidation and basal adenylate cyclase activity have been examined in neuroblastoma cultured with a variety of exogenous fatty acids. Formation of cyclic AMP depended upon fatty acid type, with supplementation affecting activities in the order: linoleate greater than cis-vaccenate = linolenate greater than control (132.7, 72.6, 71.9 and 36.0 pmol cAMP formed/mg protein, respectively). Lipid peroxidation, measured by formation of malondialdehyde (MDA), also varied with fatty acid; however, there was little correlation between MDA production and basal cyclase activity. Inclusion of alpha-tocopherol in culture-medium blocked MDA formation without affecting cAMP accumulation. Fe2+-dependent induction of peroxidation was accompanied by a time-dependent inhibition of cyclase activity.     

The nature of diene conjugation in human serum, bile and duodenal juice

Diene-conjugated lipids have been located by HPLC in serum, bile and duodenal juice. Whether esterified or not the same predominant fatty acid is responsible for most of the diene conjugation in all of these biological fluids. Initial attempts to generate this fatty acid in pure lipid by classical lipid peroxidation in vitro were unsuccessful. Ultraviolet irradiation of free fatty acids in the presence of protein produced diene-conjugated lipids similar to those found in vivo. The predominant diene-conjugated fatty acid in vivo is an isomerised C18:2 compound.     

Increased susceptibility to lipid peroxidation in rabbit kidneys: a consequence of warm ischaemia and subsequent reperfusion

Rabbit kidneys were clamped and rendered warm ischaemic (WI) in situ for 60 and 120 min. They were then either removed immediately after the ischaemic insult or after reperfusion with blood for 60 min or 24 hr. Homogenates were assayed for phospholipid-Schiff base fluorescence (Ex. 360 nm, Em. 435 nm) and for diene conjugate formation by u.v. spectrophotometry (240 nm) as indices of lipid peroxidation. No alteration in tissue levels of Schiff base was evident immediately after WI but when the homogenates were incubated at 37 degrees C for 90 min, the rate of peroxidation was significantly elevated compared to controls (P less than 0.02 after WI of 60 min and P less than 0.001 after 120 min of WI). These values were still further elevated after reperfusion with blood for 60 min and 24 hr (P less than 0.001). Diene conjugates were raised after WI alone and further still after reperfusion. Thus an early index of lipid peroxidation (diene conjugation) suggested peroxidative damage during the warm ischaemic period itself, whilst detection of Schiff bases was only possible after in vitro incubation of the tissue. Both indices of oxygen-derived free radical damage were increased after reperfusion in vivo with blood and may relate to the degree of tissue damage sustained during ischaemia and reflow.