Brain Arachidonic Acid Incorporation and Precursor Pool Specific Activity During Intravenous Infusion of Unesterified [3H]Arachidonate in the Anesthetized Rat

Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [³H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly ( t _1/2 < 1 min) to steady-state values that averaged <5% of plasma specific activity. Incorporation was rapid, as >85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.     

Phospholipid supplementation reverses behavioral and biochemical alterations induced by n-3 polyunsaturated fatty acid deficiency in mice

This study investigated the effects of a diet deficient in alpha-linolenic acid followed or not by supplementation with phospholipids rich in n-3 polyunsaturated fatty acids (PUFA) on behavior and phospholipid fatty acid composition in selected brain regions. Three weeks before mating, two groups of mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or a diet deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group was supplemented with n-3 PUFA from either egg yolk or pig brain phospholipids for 2 months. In the open field, rearing activity was significantly reduced in the deficient group. In the elevated plus maze (anxiety protocol), the time spent on open arms was significantly smaller in deficient mice than in controls. Using the learning protocol with the same task, the alpha-linolenic acid deficiency induced a learning deficit. Rearing activity and learning deficits were completely restored by supplementation with egg yolk or cerebral phospholipids, though the level of anxiety remained significantly higher than that of controls. There were no differences among the 4 diet groups for either the Morris water maze or passive avoidance. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all other regions analysed. The frontal cortex and the striatum were the most markedly affected by the deficiency. Supplementation with phospholipids restored normal fatty acid composition in brain regions except for frontal cortex. Egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for reversing behavioral changes and altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.     

Characterization and metabolism of ovine foetal lipids

1. Total phospholipid concentrations in liver, kidney and brain of the 140-day ovine foetus were only half of those in comparable maternal tissues. 2. Phosphatidylcholine was the predominant phospholipid in all foetal tissues examined. The most striking difference between foetal and maternal tissues in individual phospholipids was in the heart; foetal heart contained more ethanolamine plasmalogen than choline plasmalogen, whereas in adult tissue the concentration of these was reversed. Sphingomyelin content of foetal brain was only one-sixth of that of maternal brain tissue. 3. Oleic acid (18:1) was the predominant acid in the phospholipid extracted from foetal tissues, except in brain where palmitic acid (16:0) was slightly higher. In phospholipids from adult tissues there was a higher proportion of unsaturated fatty acids (linoleic acid, 18:2, and linolenic acid, 18:3) and a correspondingly lower proportion of oleic acid (18:1). The distribution of fatty acids in the neutral lipid fraction of foetal and maternal tissues was very similar; oleic acid (18:1) was generally the principal component. 4. (14)C derived from [U-(14)C]-glucose and [U-(14)C]fructose infused into the foetal circulation in utero was incorporated into the neutral lipids and phospholipids of heart, liver, kidney, brain and adipose tissue. 5. Phospholipid analysis revealed that the specific activity of phosphatidic acid was higher in liver than in other tissues. The specific activity of phosphatidylethanolamine was less than that of phosphatidylcholine in heart, but in other tissues they were about the same. The specific activities of phosphatidylinositol and phosphatidic acid in brain were very similar and were higher than the other components. The specific activity of phosphatidylserine was highest in liver and brown fat. 6. The pattern of incorporation of (14)C derived from [(14)C]glucose and [(14)C]fructose into foetal neutral lipids was similar. Diglyceride accounted for most of the radioactivity in brain, whereas triglyceride had more label in heart, liver, kidney and fat.     

Quantitative properties and biosynthesis of phospholipids in rat embryonic tissues in the process of development

The content of lipid phosphorus and the rate of [1-14C]palmitate incorporation into individual phospholipids of rat embryonic liver, kidney, spleen, brain, and placenta at different stages of prenatal development were studied. It was shown that the level of neutral phospholipids at all stages is much higher than that in acid fractions; however, the rate of the acid fraction exchange is 10 times higher depending on the age of the embryos. The specific radioactivity of individual fractions of embryonic rat tissue largely exceeds that in adult animals.     

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.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Specific phospholipid fatty acid composition of brain regions in mice. Effects of n-3 polyunsaturated fatty acid deficiency and phospholipid supplementation

This study examined the effects of dietary alpha-linolenic acid deficiency followed or not by supplementation with phospholipids rich in n;-3 polyunsaturated fatty acid (PUFA) on the fatty acid composition of total phospholipids in 11 brain regions. Three weeks before mating, mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group were supplemented with n;-3 polyunsaturated fatty acids (PUFA) from either egg yolk or pig brain phospholipids for 2 months. Saturated and monounsaturated fatty acid levels varied among brain regions and were not significantly affected by the diet. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all regions. alpha-Linolenic acid deficiency decreased the level of 22:6 n-3 and was compensated by an increase in 22:5 n-6 in all regions. However, the brain regions were affected differently. After the pituitary gland, the frontal cortex, and the striatum were the most markedly affected with 40% reduction of 22:6 n-3. Supplementation with egg yolk or cerebral phospholipids in deficient mice restored a normal fatty acid composition in brain regions except for the frontal cortex. There was a regional distribution of the fatty acids in the brain and the impact of deficiency in alpha-linolenic acid was region-specific. Dietary egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for the recovery of altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Origin of the arachidonic acid released post-mortem in rat forebrain.

To determine the origins of the arachidonic acid released post-mortem in brain tissue, [3H]arachidonic acid was injected by the intracerebro-ventricular route and radioactivity monitored in complex lipids and free arachidonic acid at various times after decapitation. The specific activity of the released arachidonic acid was close to that in the total phospholipid fraction and much lower than that of the neutral lipids. The phospholipid with the closest specific activity to the free arachidonic acid recovered at the end of the post-mortem period was phosphatidylinositol. Phosphatidylcholine showed a small but significant decrease in radioactivity post-mortem and could contribute 37% of the arachidonic acid released to the free fatty acid fraction. Arachidonic acid released in rat forebrain after decapitation thus comes from a mixture of phospholipids with phosphatidylinositol and phosphatidylcholine being the major source. Phosphatidylserine and phosphatidic acid did not make important contributions to the free arachidonic acid. In the microsomal fraction, the specific activity of the free arachidonic acid was very close to that in phosphatidylinositol.     

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.     

Phosphoinositides of the brain and liver of rats in ethanol poisoning]

Alcohol intoxication induced in the rat brain significant decrease in the monophosphoinositides level, but the di- and triphosphoinositides quantity did not change; the content of all the three studied fractions of the inositol-containing phospholipids in the liver decreased. The tendency to retain the normal level of di- and triphosphoinositides in the brain following alcohol intoxication may be considered as an important factor providing physiological condition of the brain metabolism.     

Dietary tryptophan does not alter the function of brain serotonin neurons

The hypothesis that alterations in dietary tryptophan modify the functional activity of brain serotonin-containing neurons was tested by recording the electrophysiological activity of single serotonergic cells in awake, behaving cats after meal ingestion of diets containing varying proportions of tryptophan and the neutral amino acids that compete with tryptophan for uptake into the brain. The data revealed that while the various diets produced significant changes in brain serotonin and its major metabolite, 5-hydroxyindoleacetic acid, there was no change in the activity of serotonin-containing dorsal raphe cells following meal ingestion. Furthermore, a pulse injection of tritiated labeled tryptophan following the various diets produced no significant change in the release of tritiated serotonin into the lateral ventricles, while tritiated 5-hydroxyindoleacetic acid was significantly increased. These data suggest that dietary tryptophan does not alter the functional activity of central serotonergic neurons, in contrast with current popular beliefs that such dietary manipulations alter brain function.     

Normal Myelin Composition and Phospholipid Synthesis in Adrenalectomized Rats with Reduced Brain Myelin and Glycerol 3-Phosphate Dehydrogenase Activity

The composition of CNS myelin was investigated in rats adrenalectomized at day 14 and killed 7 days later, previously shown to result in a 25% reduction in the amount of bulk-isolated myelin and a 40% decrease in brain glycerol 3-phosphate dehydrogenase activity. The proportions of the major myelin proteins, as well as the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase, were the same in the myelin from both adrenalectomized and control animals. The amount of total phospholipid and the proportions of individual phospholipids were also normal in myelin from the adrenalectomized animals. The amount of nonmyelin phospholipid in whole brain was unchanged by adrenalectomy. Labeling studies carried out 4 days after adrenalectomy of 14-day-old animals showed no change in the synthesis rates of the major myelin phospholipids as compared with the synthesis rate of nonmyelin phospholipids. Furthermore, incorporation of [1,(3)-14C]glycerol into the glycerol moiety of ethanolamine plasmalogen, which requires glycerol 3-phosphate dehydrogenase, was also normal, showing that the reduced oligodendroglial glycerol 3-phosphate dehydrogenase activity following adrenalectomy was not rate-limiting for myelin phospholipid synthesis.     

Changes in lipid peroxidation, superoxide dismutase activity, ascorbic acid and phospholipid content in liver of freshwater catfish Heteropneustes fossilis exposed to elevated temperature

1. 1. Lipid peroxidation, superoxide dismutase (SOD) activity, ascorbic acid (AsA) and individual phospholipid contents in liver of fresh water cat fish Heteropneustes fossilis were measured after exposure to different temperatures (25, 27, 32, 37°C) at various times (1–4 h).

2. 2. Lipid peroxidation and superoxide dismutase activity were significantly increased with increases in temperature at various times.

3. 3. Ascorbic acid content was depleted when temperature was increased.

4. 4. After temperature exposure, phosphatidyl inositol was increased while phosphatidyl choline, phosphatidyl serine and phosphatidyl ethanolamine were depleted. Phosphatidic acid level did not change.

5. 5. The findings indicated an increased oxidative stress in liver following increases in temperature at various times. Concurrent with the increase in lipid peroxidation, superoxide dismutase activity and ascorbic acid from the liver of fish varied. It is suggested that depletion of major individual phospholipids following temperature exposure could be due to superoxide created oxidative stress in the liver.

     

Phospholipid and Ca++ dependency of phorbol ester receptors

The phospholipid and Ca++ dependency of a partially purified phorbol ester apo-receptor from the soluble fraction of mouse brain homogenates was studied. This apo-receptor is believed to be identical with the Ca++ and phospholipid-dependent protein kinase C. Binding of phorbol esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences (greater than 100-fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100-fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo-receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]phorbol 12,13-dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with dioleoyl-phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by phorbol esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to phorbol esters is discussed.     

Neutral amino acids in the brain: changes in response to food ingestion

Abstract— The brain levels of each of the aromatic and branched-chain amino acids change 2 h after fasting rats begin to consume either a carbohydrate-fat diet or a similar diet containing 18% or 40% protein. Carbohydrate-fat ingestion elevates the concentrations of each of the aromatic amino acids in brain, while substantially depressing those of the branched-chain amino acids. The inclusion of protein in this diet suppresses the increases in brain aromatic amino acids and attenuates the decreases in the branched-chain amino acids. The changes in the brain level of each neutral amino acid following the ingestion of any of these diets correlate extremely well with the effects of the diet on the serum neutral amino acid pattern, specifically on the serum concentration ratio of each neutral amino acid to the sum of the other neutral amino acids. The diet-induced changes in the brain level of each of the amino acids also correlate surprisingly well with the calculated rate of brain influx for each amino acid.     

Dynamics of Docosahexaenoic Acid Metabolism in the Central Nervous System: Lack of Effect of Chronic Lithium Treatment

Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h^–1, respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 mol.g^–1), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A₂ activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithium's action is an arachidonic acid-selective phospholipase A₂.     

Altered lipid composition of adipose tissue in human muscular dystrophy

Lipids of subcutaneous adipose tissue from normal and dystrophic human samples were investigated with particular reference to the fatty acid composition of neutral lipids and phospholipids. The free fatty acid pattern did not show much change. Triglyceride, which together with cholesterol composed most of the neutral lipid fraction, showed significant changes in fatty acid composition by gas-liquid chromatography (GLC). There was a decrease in the total phospholipids. Analysis of the levels of individual phospholipids, however, showed an increase in sphingomyelin and ethanolamine phosphoglyceride contents. Fatty acid composition of the different phospholipid classes analyzed by GLC showed significant changes.