Calcium-translocating and cardiotonic properties of oxidation products of linoleic acid

Calcium-translocating activity of linoleic acid and its lipoxygenase (linoleate: oxygen oxidoreductase; EC 1.13.11.12) metabolites or autoxidation products was determined in vitro by estimation of 45Ca transport from a bulk aqueous to a bulk organic phase. Fresh commercial linoleic acid, tested immediately after removal from a sealed vial, stimulated calcium translocation only at concentrations greater than 1 mM. In contrast, 45Ca translocation by linoleic acid exposed to air was detectable at 10 microM. Oxidation products of linoleic acid obtained either by incubation with lipoxygenase or by autoxidation were much less potent than the calcium ionophore A23187. The products obtained by enzymic oxidation of linoleic acid enhanced contractility in the Langendorff-perfused guinea pig heart up to 45% over control (at 3 X 10(-8) M). The inotropic response was transient with rapid onset and not affected by the beta-adrenergic antagonist, propranolol. The autoxidation products of linoleic acid increased cardiac contractility up to 43% at 10(-6) M. In contrast, fresh linoleic acid caused only a negative inotropic effect at 10(-8) to 3 X 10(-7) M, progressing to contracture at 10(-6) M. These findings suggest that conflicting reports on the cardiostimulant effect of linoleic acid may be due to varying levels of the autoxidation products. Linoleic acid metabolites in vivo may have a physiological role in myocardial function related to their Ca2+-ionophoric activity.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis

High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Liberation and oxygenation of polyenoic acids in stimulated platelets

Radiolabeled polyenoic acids were incorporated into human platelet lipids using albumin as vector. Platelets were then triggered with 0.1 or 1 U/ml thrombin, and 0.5 or 2 x 10(-6) M calcium ionophore A23187. Lipid extracts were analyzed for neutral lipids, free fatty acids, monohydroxylated acids, prostanoids and glycocerophospholipid subclasses. During platelet activation induced by thrombin or by ionophore, arachidonic and eicosapentaenoic acids were liberated from phospholipids in large amounts and were subsequently oxygenated via platelet oxygenases. Substantial amounts of lipoxygenase products and thromboxanes were produced from these acids. Liberation and oxygenation of linoleic, alpha-linolenic, and docosahexaenoic acids were much less pronounced. Polyenoic acid liberation from phospholipid subclasses also behaved quite differently. Apart from alpha-linolenic and adrenic acids, which were poorly liberated, all the others were freed from phosphatidylinositol. In addition, arachidonic, eicosapentaenoic, and 5, 8, 11-eicosatrienoic acids were liberated from phosphatidylcholine at high concentrations of agonists and partially reincorporated into phosphatidylethanolamine. Finally, linoleic acid was deacylated from phosphatidylinositol and phosphatidylserine and almost entirely reacylated into phosphatidylcholine, whereas docosahexaenoic acid was deacylated from phosphatidylcholine and phosphatidylinositol reacylated into phosphatidylethanolamine, respectively. It is concluded that these polyenoic acids, all for which modulate platelet functions, exhibit very different metabolisms. They may act via their oxygenated derivatives and/or at the membrane phospholipid level.     

Association of calmodulin inhibition, erythrocyte membrane stabilization and pharmacological effects of drugs

Isolated liver cells from rats fed a diet deficient in essential fatty acids were used to study the oxidation, esterification and, especially, the desaturation and chain elongation of [1-14C]linoleic acid. 14C-labelled arachidonic acid (20:4) and smaller amounts of eicosatrienoic acid (20:3) were recovered mainly in the phospholipids, while gamma-linolenic acid (18:3) was found in both the phospholipids and the triacylglycerol fraction. Lactate strongly increased the formation of arachidonic acid, which was found mainly in the phosphatidylcholine and the phosphatidylinositol fractions. Lactate reduced the amounts of gamma-linolenic acid. Glucagon and (+)-decanoylcarnitine reduced the formation of arachidonic acid, and (+)-decanoylcarnitine increased the incorporation of gamma-linolenic acid especially, in the triacylglycerol fraction. Increasing concentrations of the [1-14C]linoleic acid substrate increased the formation of arachidonic acid and of the other chain-elongated or desaturated fatty acids. Lactate also stimulated the formation of arachidonic acid in liver cells from animals fed adequate amounts of essential fatty acids. It is suggested that dietary and hormonal factors which can change the intracellular levels of malonyl-CoA may influence both the ratio of arachidonic acid/gamma-linolenic acid formed and the total amounts of desaturated and chain-elongated fatty acids formed from linoleic acid.     

Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Oxidation of Linoleyl Alcohol by Potato Tuber Lipoxygenase: Possible Mechanism and the Role of Carboxylic Group in Substrate Binding

We have studied the aerobic oxidation of linoleyl alcohol (LAL) by potato tuber lipoxygenase in the presence of 0.02% (w/v) non-ionic detergent Lubrol PX (and its analog C₁₂E₁₀) and 0.1 mM sodium dodecyl sulfate to investigate the role of carboxylic group in substrate binding. While the enzyme displayed a comparable affinity toward LA and LAL, the rate of LAL oxidation was approximately one-fourth of that of linoleic acid. The pH-profile of the reaction suggests that the rate of LAL oxidation is controlled by two ionizable groups with pK_avalues of 5.3 and 7.5, with optimal pH being 6.4±0.1. Since LAL is not ionizable at this pH, we conclude that the rate of the reaction is controlled by two ionogenic groups of the enzyme. The primary dioxygenation product(s) of LAL had a maximal absorbance at 233±1 nm. The products have been isolated, catalytically hydrogenated with H₂over Pd on carbon, and analyzed by GC-MS. Two major equimolar products were found to be 9- and 13-hydroxystearyl alcohols, indicating that 9- and 13-hydroperoxylinoleyl alcohols are the primary dioxygenation products. Based on these results we propose that the carboxyl group of polyunsaturated fatty acid may not be involved in substrate binding of potato tuber lipoxygenase.     

The lipoxygenase product, 5-hydroperoxy-arachidonic acid,augments chemotactic peptide-stimulated arachidonic acid release from HL60 granulocytes

The chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys stimulates the release of arachidonic acid from endogenous phospholipids of the human promyelocytic leukemia cell line HL60. This release of unsaturated fatty acid is augmented by the presence of exogenously added lipoxygenase product, 5-hydroperoxy-arachidonic acid (5-HPETE). Other hydroperoxy- and hydroxy-arachidonic acid derivatives are less potent. In addition, saturated fatty acids and hydrogen peroxide do not possess this ability to augment arachidonic acid release. None of the arachidonic acid derivatives are capable of stimulating the release of arachidonic acid from endogenous phospholipids in the absence of the chemotactic peptide.     

Effect of alcohols on arachidonic acid metabolism in murine mastocytoma cells and human polymorphonuclear leukocytes

The effects of alcohols on the formation of leukotrienes, 5-HETE and prostaglandin D2 in mastocytoma cells and human neutrophils were studied. In murine mastocytoma cells, alcohols appear to have at least two different effects on the production of these arachidonic acid metabolites. At low levels of cellular arachidonic acid achieved after stimulation with calcium ionophore A23187 or addition of low levels of exogenous arachidonic acid, alcohols appear to have a general inhibitory effect on the production of lipoxygenase metabolites. In the presence of higher concentrations of cellular arachidonic acid, ethanol and methanol stimulated the production of lipoxygenase metabolites, but had no large stimulatory effect on the cyclo-oxygenase metabolite, prostaglandin D2. Under these conditions, n-propanol and t-butanol have inhibitory effects on leukotriene production. Human neutrophils are less sensitive to ethanol than mastocytoma cells, but stimulatory effects were still found at high ethanol concentrations (220-430 mM).     

Characteristics of a membrane-associated lipoxygenase in tomato fruit

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2^* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30°C, the formation of fatty acid oxidation products from 16:0/18:2^* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22°C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2^* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2^* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K_m of 0.52 millimolar and an apparent V_max of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.     

The transfer of fatty acids across the sheep placenta

Maternal and fetal plasma concentrations of free fatty acids, triacylglycerols and phospholipids and the profile of their fatty acids were measured in three catheterized and unanaesthetized sheep. Fetal concentrations of all three lipid fractions were low and did not correlate with maternal concentrations. There were no measurable umbilical venous-arterial differences. Linoleic acid concentrations were low in both mother and fetus. The fatty acid composition of fetal adipose tissue, liver, lung and cerebellum of five animals was analysed. Again linoleic acid levels were very low, but phospholipids contained 2-8% arachidonic acid. [14C] linoleic acid and [3H] palmitic acid were infused intravenously into three ewes. Only trace amounts of labelled fatty acids were found in fetal plasma and these were confined to the free fatty acids. 14C-label was incorporated into fetal tissue lipids, but most of this probably was due to fetal lipid synthesis from [14C] acetate or other water-soluble products of maternal [14C] linoleic acid catabolism. It is concluded that only trace amounts of fatty acids cross the sheep placenta. They are derived mainly from the maternal plasma free fatty acids and might just be sufficient to be the source of the small amounts of essential fatty acids found in the lamb fetus, but are insignificant in terms of energy supply or lipid storage.     

Adrenaline stimulation of phospholipid metabolism in adipocyte ghosts

The incorporation of long-chain fatty acids into phospholipids has been detected in adipocyte ghosts that were incubated with [1-14 C] stearic, [1-14 C] linoleic or [1-14 C] arachidonic acid. Adrenaline and adenosine activated this incorporation within 15 s of exposure of the ghosts to the hormones and the response was dose dependent. Maximum incorporation of labelled linoleic acid occurred at 10(-5) M adrenaline and 10(-7) M adenosine. The alpha-agonist phenylephrine and the beta-agonist isoproterenol were also shown to stimulate the incorporation of fatty acid in a dose dependent manner. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were each labelled preferentially with linoleic or arachidonic acid. p-Bromophenacylbromide, quinacrine and centrophenoxine inhibited the adrenaline-stimulated incorporation of fatty acids into ghost membrane phospholipids, and p-bromophenacylbromide also reduced the activation of adenylate cyclase by adrenaline. NaF, an activator of adenylate cyclase, like adrenaline, stimulated the incorporation of linoleic acid into ghost membrane phospholipids.     

Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.     

A unique cardiac cytosolic acyltransferase with preferential selectivity for fatty acids that form cyclooxygenase/lipoxygenase metabolites and reverse essential fatty acid deficiency

The rabbit heart contains a cytosolic enzyme which selectively incorporates polyunsaturated fatty acids into phosphatidylcholine. This unique acyltransferase is selective for fatty acids, thus far tested, that are substrates for cyclooxygenase or lipoxygenase (i.e., arachidonic, eicosapentaenoic, linoleic and dihomo-gamma-linoleic acids) or which reverse the symptoms of essential fatty acid deficiency (columbinic acid). On the other hand, palmitic, oleic, 5,8,11-eicosatrienoic (n-9, Mead acid), and docosatetraenoic acid (n-6, adrenic acid) were not incorporated in phospholipids by the cytosolic acyltransferase. No such fatty acid selectivity was exhibited by the cytosolic acyl-CoA synthetase or by the acyltransferase activities present in cardiac microsomes and mitochondria.     

Plasma fatty acids and [13C]linoleic acid metabolism in preterm infants fed a formula with medium-chain triglycerides

Most preterm infant formulas contain medium-chain triacylglycerols (MCT), but the effects of MCT on polyunsaturated fatty acid status and metabolism are controversial. Thus, we studied the effects of MCT on linoleic acid metabolism using stable isotopes. Enterally fed preterm infants were randomized to receive for 7 days 40% of fat as MCT (n = 10) or a formula without MCT (n = 9). At study day 5, infants received orally 2 mg/kg body weight of (13)C-labeled linoleic acid. Fatty acids in plasma lipid classes and (13)C enrichment of phospholipid fatty acids were measured and tracer oxidation was monitored. Compared with the control group, the MCT group showed lower breath (13)CO(2) and higher plasma triacylglycerol contents of octanoic acid, of decanoic acid, and of total long-chain polyunsaturated fatty acids (57.1 +/- 4.4 micro mol/l vs. 37.9 +/- 4.8 micro mol/l, P < 0.01). Concentrations of several polyunsaturated fatty acids in plasma phospholipids and non esterified fatty acids were higher in the MCT group. (13)C concentrations in phospholipid n-6 fatty acids indicated no difference in the relative conversion of linoleic to arachidonic acid. We conclude that oral MCT effectively reduce polyunsaturated fatty acid and long chain polyunsaturated fatty acid oxidation in preterm infants without compromising endogenous n-6 long chain polyunsaturated fatty acid synthesis.     

Differential effect of external calcium on the oxygenated metabolism of endogenous and exogenous arachidonic acid in platelets

The oxygenation of arachidonic acid into thromboxane B2 (TXB2), 12-hydroxy-heptadecatrienoic (HHT) and 12-hydroxy-eicosatetraenoic (12-HETE) acids has been examined in human platelets in the absence or presence of 1mM calcium. From endogenous arachidonic acid, external calcium did not affect the formation of cyclo-oxygenase products (TXB2 and HHT) but enhanced that of 12-HETE when thrombin at high concentrations was the agonist. Dose-response curves performed with thrombin and collagen revealed that increased stimulation resulted in higher ratios of 12-HETE/HHT. On the other hand external calcium did not alter significantly the synthesis of either products from exogenous arachidonic acid and the total conversion of the substrate was unchanged. We conclude that extracellular calcium may facilitate the liberation of arachidonic acid from platelet phospholipids when induced by high thrombin concentrations. The excess of arachidonic acid liberated would then be diverted towards the lipoxygenase pathway.     

Biotransformation of linoleic acid by porcine leukocytes

Linoleic acid is an important essential fatty acids of leukocyte cell membrane phospholipids from some animals, e.g. from pigs and rabbits, and is a known substrate for lipoxygenase(s), especially in plant systems. Lipoxygenase activity has also been well documented in leukocytes using arachidonic acid as a substrate. These findings and our own interest in the fate of linoleic acid have prompted us to investigate the biotransformation of this essential fatty acids in leukocytes.Porcine leukocytes were isolated from whole blood by dextrane precipitation of the erythrocytes and by centrifugation. Broken cells were incubated with exogenous linoleic acid and four major biotransformation products, X₁, X₂, X₃ and X₄, were formed. Following isolation by silicagel column chromatography and thin layer chromatography, the products were derivatized and characterized by GC/MS. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation, and various types of derivatives were made in order to facilitate the structure elucidation. The major product X₁, which represented 60.5% of the total metabolites formed, was identified as 13-hydroxy-9,11-octadecadienoic acid. Product X₂ (16.2%) was shown to be 11-hydroxy-12,13-epoxy-9-octadecenoic acid. Products X₃ and X₄ (respectively 5.2 and 7.5%) resulted in identical thermore, each of the products X₃ and X₄ was shown to be a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic acid (70%) and 9,10,13-trihydroxy-12-octadecenoic acid (30%). With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butylboronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol position.The metabolism of linoleic acid in porcine leukocytes is analogous to that by cereal lipoxygenases. A major difference however is that porcine leukocyte lipoxygenase predominantly yields products, which arise through 13-lipoxygenation, whereas, in cereals, transformation products of 9-hydroperoxy-10,12-octadecadienoic acid are formed to the same extent as metabolites of 13-hydroperoxy-9,11-octadecadienoic acid.