Synthetic phospholipids with high purity fatty acids

Phospholipids derived from fatty acids are increasingly used by the pharmaceutical industry as excipients and also as active pharmaceutical ingredients (API) themselves. Newly developed HPLC‐CAD (charged aerosol detection) methods allow the resolution of phospholipids with the same headgroup but different fatty acid chains. A new manufacturing process has been established on multi‐Kg scale, which enables the manufacture of phospholipids meeting the quality requirements for use as excipients or APIs. Such products can now be obtained in significantly higher chemical purities than previously possible, with no unqualified impurity exceeding the International Conference on Harmonisation (ICH) limit of 0.15%.     

Volatile fatty acids of frass of certain omnivorous insects

The frass of the following omnivorous insects reared on natural and artificial diets was analyzed for volatile fatty acids:Blattella germanica, Acheta domesticus, Blaberus discoidalis. Acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids were identified in all frass samples. The possible significance of volatile fatty acids in frass is discussed.     

Brain mitochondrial incorporation of elongated fatty acids into phospholipids

The characteristics patterns and asymmetric distribution of phospholipid fatty acids suggest precise control mechanisms. Our investigations were designed to assess mitochondrial fatty acid elongation and their pattern of incorporation into complex lipids. Fatty acid chain elongation in the total lipid fraction occurred primarily with the more abundant fatty acids present. Elongation patterns in free fatty acids were similar to the total lipid fraction except C_20:4 and C_22:4 were formed to slightly greater extent. Choline glycerophosphatide and ethanolamine glycerophosphatide displayed different patterns of elongation. Choline glycerophosphatide contained more elongated longer chain polyunsaturated fatty acids, while ethanolamine glycerophosphatide contained greater amounts of elongated shorter chain saturated and monounsaturated fatty acids. These results suggest that fatty acid elongation may play a specific role in fulfilling mitochondrial phospholipid fatty acid requirements.     

Abnormal metabolism of polyunsaturated fatty acids and phospholipids in diabetic glomeruli

Studies were done on changes in phospholipid content and fatty acid composition of phospholipids and on the role of the acylation pathway in synthesis of phospholipids in the development of abnormal fatty acid composition in the glomeruli of rats 2 and 10 mo after induction of diabetes with streptozotocin. The proportions of individual phospholipids in the glomeruli of rats were not changed 2 mo after induction of diabetes, but the proportion of phosphatidylethanolamine (PE) decreased and that of sphingomyelin increased 10 mo after induction of diabetes. In contrast, in liver the proportion of PE was increased and that of phosphatidylcholine was decreased. These results showed that changes of individual phospholipids in glomeruli were time-dependent and tissue-specific. Two mo after induction of diabetes, the main change in the phospholipid fatty acid composition of diabetic glomeruli was a decrease in arachidonic acid (AA); the main change in serum free fatty acids (FFA) was an increase in linoleic acid (LA) and a decrease in AA. Ten mo after induction of diabetes, the main changes in the phospholipid fatty acid composition of glomeruli were an increase in LA and a decrease in AA; the main change of the serum FFA composition was a decrease in AA. Thus, the fatty acid composition of glomerular phospholipids was not directly correlated to that of the serum in diabetic rats. Acyl-CoA synthetase and acyltransferase activities increased in diabetic glomeruli with either AA or LA as substrate, but activity toward LA increased more at 2 mo after induction of diabetes. Acyl-CoA synthetase activity increased in diabetic glomeruli with LA as substrate, but that did not change with AA as substrate at 10 mo after induction of diabetes. Furthermore, acyltransferase activity decreased in diabetic glomeruli with AA as substrate, although that did not change with LA as substrate at 10 mo after induction of diabetes.     

Essential fatty acids in maternal diet and in rat milk phospholipids

The administrations of semisynthetic diets supplemented with oils and fats containing different levels of linoleic acids to lactating rats result in corresponding changes in the polyunsaturated fatty acids of triglycerides in the collected milk. Milk phospholipids show a quite different trend, polyunsaturated acids of the linoleic acid family being highest with low dietary linoleic acid supply, and vice versa, suggesting a control in the secretion of polyunsaturated fatty acids in milk.     

Component fatty acids of some cruciferae oils

Summary The oils from yellow mustard seed (Brassica alba), black mustard seed (Brassica nigra) of Indian origin, and rapeseed (Brassica Compestris) of unknown origin have been analyzed for their fatty acid composition without preliminary resolution of fatty acids by lead-salt-alcohol or fractional crystallization methods. The results compare very favorably with those determined by other recently developed methods. It may be concluded therefore that this method can be favorably employed for the determination of fatty acid composition of fats containing higher unsaturated acids. Confirmatory evidence has been obtained for the presence of eicosenoic acid in rapeseed oil. The nature and amount of fatty acids of yellow mustard seed oil of Indian origin do not differ in any significant manner from those of other cruciferous seed oils. The present analysis of black mustard seed oil reveals a higher amount of linolenic acid, and the presence of a C₂₀ monoethenoid acid, not heretofore reported. Contribution No. 708 from the Department of Chemistry, University of Pittsburgh. Presented in part at the Spring meeting of the American Oil Chemists’ Society, held in New Orleans, La., May, 1948. Baliga and Hilditch’s paper. “The Component Acids of Rapeseed Oil” (J. Soc. Chem. Ind.67, 258–262 (1948).     

Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids

Previous studies have shown that exogenous free n-3 polyunsaturated fatty acids (PUFA) can prevent tachyarrhythmias caused by specific agents in isolated cardiac myocytes. However, the question as to whether incorporation of the n-3 PUFA into membrane phospholipids has the same immediate protective effects remained to be answered. To answer this question, we increased the content of n-3 PUFA in the phospholipids of cultured neonatal rat myocytes by growing them 2–3 d in a culture to which eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in 15 μM concentration was added. Analysis of the fatty acid composition of membrane phospholipids revealed a significantly higher level of EPA and DHA (from 0.2 to 7.6% and from 1.2 to 6.5%) in cells supplemented with EPA or DHA, respectively. The responses of the myocytes grown in normal media or in media enriched with the PUFA to arrhythmogenic agents were examined after free fatty acids were removed from the medium and the cells. The arrhythmogenic agents used were the β-adrenergic agonist isoproterenol or an elevated extracellular concentration of calcium. The results showed that there was no significant difference in the induction of tachyarrhythmias by isoproterenol or by elevated [Ca²⁺]_o in cells grown in media enriched with PUFA, as compared with cells grown in normal media in the absence of the free PUFA. Under the conditions of this study, only the unesterified PUFA were able to protect the cardiomyocytes against induced arrhythmias. There was no antiarrhythmic effect due to an increased fraction of EPA or DHA in membrane phospholipids.     

Synthesis of phospholipids and phospholipid fatty acids by isolated perfused rat lung

Synthesis of phospholipids and phospholipid fatty acids in isolated perfused rat lung was studied. The perfusion fluid was a Krebs-Ringer bicarbonate buffer containing a¹⁴C labeled substrate. It was found that 1-¹⁴C-acetate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, or U-¹⁴C-D-glucose was incorporated into tissue lipids in the isolated perfused lung at a rate geeater than that in incubated minced tissue. However, the patterns of the newly synthesized lipids from these two systems were similar. In 1 hr of perfusion, 6.8, 3, 14.5, 7.5, 7, and 2% of the initial¹⁴C-radioactivity of 1-¹⁴C-acetate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, and U-¹⁴C-D-glucose, respectively, were incorporated into phospholipids. Phospholipid fatty acids accounted for 95–96% total phospholipids-¹⁴C when¹⁴C-substrates, other than glucose, were used. For glucose, only 20% phospholipids-¹⁴C was in phospholipid fatty acids. More than 80% phospholipid fatty acids-¹⁴C was in palmitic acid when 1-¹⁴C-acetate and U-¹⁴C-D-glucose were used, while 37, 61, 80, and 94% phospholipid fatty acid-¹⁴C from 1-¹⁴C-laurate, 1-¹⁴C-sterate, 1-¹⁴C-oleate, and 1-¹⁴C-palmitate, respectively were recovered in the original form of the fatty acid used. The newly synthesized phospholipid fatty acid (13–24%) from 1-¹⁴C-laurate, 1-¹⁴C-stearate, and 1-¹⁴C-oleate was palmitic, and 10% of phospholipid fatty acid from 1-¹⁴C-stearate was in oleic acid. Hydrolysis by phospholipase A showed that¹⁴C from perfused substrates was esterified to both α and β positions of phospholipids. It was found that positional selectivity of phospholipid fatty acids was determined by chain length, degree of unsaturation, and source of fatty acid.     

Positional analysis of triglycerides and phospholipids rich in long-chain polyunsaturated fatty acids

Four sources of long-chain polyunsaturated fatty acids (LCP) differing in their chemical structure (triglycerides or phospholipids) and in their origin (tuna triglycerides, fungal triglycerides, egg phospholipids, and pig brain phospholipids) were analyzed to determine the distribution of the component fatty acids within the molecule. Lipase and phospholipase A₂ hydrolysis was performed to obtain 2-monoacylglycerols and lysophospholipids, respectively, which allowed us to determine the distribution of fatty acids between the sn-2 and sn-1,3 positions of triglycerides or between the sn-1 and sn-2 position of phospholipids. Fatty acids in the LCP sources analyzed were not randomly distributed. In tuna triglycerides, half of the total amount of 22∶6n−3 was located at the sn-2 position (49.52%). In fungal triglycerides, 16∶0 and 18∶0 were esterified to the sn-1,3 (92.22% and 91.91%, respectively) 18∶1 and 18∶2 to the sn-2 position (59.77% and 62.62%, respectively), and 45% of 20∶3n−6 and only 21.64% of 20∶4n−6 were found at the sn-2 position. In the lipid sources containing phospholipids, LCP were mainly esterified to the phosphatidylethanolamine fraction. In egg phospholipids, most of 20∶4n−6 (5.50%, sn-2 vs. 0.91%, sn-1) and 22∶6n−3 (2.89 vs. 0.28%) were located at the sn-2 position. In pig brain phospholipids, 22∶6n−3 was also esterified to the sn-2 (13.20 vs. 0.27%), whereas 20∶4n−6 was distributed between the two positions (12.35 vs. 5.86%). These results show a different fatty acid composition and distribution of dietary LCP sources, which may affect the absorption, distribution, and tissue uptake of LCP, and should be taken into account when supplementing infant formulas.     

Composition of the phospholipids and their fatty acids in the ROC-1 oligodendroglial cell line

ROC-1 cells are a hybrid of C−6 rat glioma and rat oligodendroglia cells. Biochemically these cells resemble the oligodendroglia parent, but their lipid composition is unknown. The phospholipid composition in mole % was: cardiolipin, 1.0; phosphatidylglycerol, 1.2; ethanolamine glycerophospholipids, 27.6; phosphatidylinositol, 5.8; lysophosphatidylethanolamine, 0.8; phosphatidylserine, 5.6; choline glycerophospholipids, 43.7; sphingomyelin, 13.7; phosphatidylinositol-4-monophosphate, 0.8; and lysophosphatidylcholine, 0.6. The choline and ethanolamine plasmalogens made up 7.2 and 18.4% of the total phospholipids, respectively. The phospholipid composition reflects that of both parental cells. The cells had moderate to high levels of 20∶3n−9 indicating n−6 series fatty acid deficiency. The phosphatidylinositol had very high 20∶3n−9 levels with a 20∶3n−9/20∶4n−6 ratio of 2.1 compared to 0.44 and 0.58 for ethanolamine glycerophospholipids (EtnGpl) and choline glycerophospholipids (ChoGpl) respectively. The saturated/polyenoic fatty acid ratios were 0.40 for EtnGpl, 3.38 for ChoGpl and 1.48 for phosphatidylinositol.     

Sphingophosphonolipids, phospholipids, and fatty acids from aegean jellyfish Aurelia aurita

The goal of this study is to elucidate and identify several sphingophosphonolipids from Aurelia aurita, an abundant but harmless Aegean jellyfish, in which they have not previously been described. Total lipids of A. aurita were 0.031-0.036% of fresh tissue, and the lipid phosphorus content was 1.3-1.7% of total lipids. Phosphonolipids were 21.7% of phospholipids and consisted of a major ceramide aminoethylphosphonate (CAEP-I; 18.3%), as well as three minor CAEP (II, III, IV) methyl analogs at 1.3, 1.1, and 1.0%, respectively. The remaining phospholipid composition was: phosphatidylcholine, 44.5%, including 36.2% glycerylethers; phosphatidylethanolamine, 18.6%, including 4.5% glycerylethers; cardiolipin, 5.6%; phosphatidylinositol, 2.6%; and lysophosphatidylcholine, 5.0%. In CAEP-I, saturated fatty acids of 14-18 carbon chain length were 70.8% and were combined with 57.3% dihydroxy bases and 23.4% trihydroxy bases. The suite of the three minor CAEP methyl analogs were of the same lipid class based on the head group, but they separated into three different components because of their polarity as follows: CAEP-II and CAEP-III differentiation from the major CAEP-I was mainly due to the increased fatty acid unsaturation and not to a different long-chain base, but the CAEP-IV differentiation from CAEP-I, apart from fatty acid unsaturation, was due to the increased content of hydroxyl groups originated from both hydroxy fatty acids and trihydroxy long-chain bases. Saturated fatty acids were predominant in total (76.7%), polar (83.0%), and neutral lipids (67.6%) of A. aurita. The major phospholipid components of A. aurita were comparable to those previously found in a related organism (Pelagia noctiluca), which can injure humans.     

Isolation and characterization of novel 2-hydroxy fatty acids from the phospholipids of the spongeSmenospongia aurea

The Caribbean spongeSmenospongia aurea revealed the presence of six novel branched α-hydroxy fatty acids: 2-hydroxy-17-methyloctadecanoic acid, 2-hydroxy-21-methyldocosanoic acid, 2-hydroxy-22-methyltricosanoic acid, 2-hydroxy-22-methyltetracosanoic acid, 2-hydroxy-24-methylpentacosanoic acid, and 2-hydroxy-23-methylpentacosanoic acid. These novel α-hydroxy fatty acids were associated with phosphatidylethanolamine. The spongesAplysina lacunosa andAplysina fistularis also contained considerable amounts of α-hydroxy fatty acids, the very long-chain 5,9,23-tricontatrienoic acid (30∶3), and phytanic acid. The sterol composition of the three sponges was also studied. It indicated thatA. lacunosa andA. fistularis contained large amounts of aplysterol and verongulasterol, whileS. aurea did not show any of these sterols. The results are discussed in terms of the taxonomy of the species.     

Polarized infrared spectra of some crystalline fatty acids

Summary Polarized infrared spectra of thin films of representative samples of form C′, B′, and A′ n-alkyl monocarboxylic acids have been observed. The data obtained on C′ heptadecanoic acid confirm that the type C and type C′ structures are very similar and that the major difference is in the packing of the CH₃ end-groups. The results on type B′ and A′ acids indicate that meaningful polarization data can be obtained on triclinic crystals of n-alkyl carboxylic acids, provided that careful consideration is given to the sample orientation and to the symmetry characteristics of certain portions of the molecules. The P₁ space structure of these crystals and the orthorhombic substructure of the B′ configuration as well as the triclinic substructure of the A′ configuration were confirmed. Some strong infrared bands of the highly irregular triclinic A′ structure did not seem to be appreciably polarized. The data on group-frequency bands which did show clear-cut polarization were in agreement with prediction. The spectra of form A′, B′, and C′ crystals did not coineide with data obtained on KBr pellets. It is concluded that in these pellets fatty acids exist in a form which is not easy to duplicate under more conventional conditions. Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

Water activity-adjusted enzymatic partial hydrolysis of phospholipids to concentrate polyunsaturated fatty acids

Selective partial hydrolyses of egg yolk phospholipid and squid skin phospholipid were carried out. By keeping the water activity (a _w) of Lipozyme IM at an intermediate level, it was easy to concentrate docosahexaenoic acid (DHA). It was also possible to concentrate both DHA and arachidonic acid (AA) simultaneously to a certain level under this a _w range. However, it was impossible to concentrate AA alone when DHA was present. Though there is a limitation in concentrating AA exclusively, the proposed a _w-adjusted hydrolytic reaction is a promising way for preparing phospholipids rich in DHA.     

Incorporation of fatty acids into phospholipids in L cells stimulated by antibody

Binding antibodies to surface membranes stimulated incorporation of fatty acids (FA) into phospholipids of L cells. Antibodies stimulated at least a 3.4-fold greater incorporation of arachidonic acid into phosphatidylinositol than into any other class of phospholipid when compared on a molar basis (p<0.003). This enhanced incorpoation was selective, depending on the character of the FA, because antibodies stimulated the incorporation of arachidonic acid at least 2.4-fold more than oleic acid, palmitic acid or stearic acid (p<0.001). Surprisingly, an antibody-stimulated incorporation of palmitic acid into sphingomyelin (SM) was at least 2.2-fold greater than that into any other class of phospholipid (p<0.001) and the antibody-stimulated incorporation of palmitic acid into SM was at least 60-fold greater than that of arachidonic acid, stearic or oleic acid (p<0.001). Nontoxic doses of ethylenediamine tetraacetic acid (EDTA), dexamethasone, 4-bromophenacylbromide and indomethacin inhibited the antibody-stimulated incorporation of arachidonic acid into cellular phospholipids, principally phosphatidylinositol (PI), and similarly inhibited the antibody stimulation of DNA synthesis. We conclude that when antibody binds to surface antigens on L cells, a rapid and selective incorporation of fatty acids into certain cellular phospholipids occurs, possibly mediated by calcium-dependent phospholipases. Degradation products of arachidonic acid, i.e., prostaglandins, may be important in these antibody stimulation events, as well. These early changes in phospholipid metabolism may serve as an important signal or mechanism for the subsequent stimulation of DNA synthesis in L cells.     

Component fatty acids and composition of some oils and fats

Nineteen different samples of oils and fats have been examined for their component acids and composition by gas-liquid chromatography. Under programmed-temperature operations, the temperatures at which different components start to elute bear a straight-line relationship with their respective carbon numbers. Chromatograms, under programmed-temperature conditions, of methyl esters from such oils as coconut, groundnut, mustard, etc., are used for identifying the components of an unknown oil by comparing its chromatogram taken under nearly identical conditions. For confirmatory identifications, such plots as logarithm of retention times versus carbon numbers for saturated acids (14:0 to 24:0), monoenoic acids (14:1 to 24:1), and dienoic acids (18:2 to 24:2), under isothermal conditions, have also been used. Some new fatty acids, noted for the first time in traditional oils, are 15:0 in cottonseed oil, 20:1 in sesame oil, 22:0 in soybean oil, and 24:2 in mustard oil. Odd-carbon chain acids from 11∶0 to 23:0 have been observed in such vegetable oils as peanut germ, rice bran, andMesua ferrea. Fatty acid composition by GLC for new samples like peanut lecithin, peanut germ oil,Myristica attenuata, Myristica kanarica, Myristica magnifica, Mesua ferrea, Vateria indica, Schleichera trijuga, and shark-liver stearine are presented. Industrial utilization of these new oils and fats is discussed.     

Substituted fatty acids in the leaves of some higher plants

Substituted (hydroxy-, epoxy- and dicarboxy) fatty acids have been analyzed in the needles of two gymnosperms (Pinus sylvestris andJuniperus communis) and the leaves of four angiosperms (Betula verrucosa, Populus nigra, Quercus petraea andCorylus avellana) growing in the Lake Léman basin. The differences in the distributions of the acids that have been analyzed are greater between the various species than between the spring and autumn samples of a given species. Moreover, the isomeric composition of the 8,16-, 9,16- and 10,16-dihy-droxypalmitic acid mixture is specific for each species.     

Isolation of brominated long-chain fatty acids from the phospholipids of the tropical marine spongeAmphimedon terpenensis

Preliminary investigation of the phospholipid fatty acid composition of the tropical marine spongeAmphimedon terpenensis by gas chromatography/mass spectrometry revealed the presence of some novel brominated fatty acids. Two new brominated fatty acids, (5E, 9Z)-6-bromo-5,9-tetracosadienoic acid (2a) and (5E, 9Z)-6-bromo-5,9-pentacosadienoic acid (3a) were subsequently isolated from a chloroform/methanol (3∶1, vol/vol) extract of the sponge and characterized as their methyl esters 2b and 3b. The known brominated fatty acid (5E, 9Z)-6-bromo-5,9-hexacosadienoic acid (4a) was also isolated. The new fatty acid methyl esters were confirmed as brominated δ5,9 acid derivatives by chemical ionization mass spectrometry. The position of the bromine substituent was determined to be C-6 by nuclear magnetic resonance techniques while the stereochemistry of the two double bonds was deduced by nuclear Overhauser enhancement difference spectroscopy. The biosynthetic implications of the co-occurrence of the three brominated acids are discussed.     

Composition and metabolism of fatty acids in phospholipids of density-separated red cells of rats

Fatty acid compositions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and the rates of fatty acid esterification to these phospholipids (PL) were measured in intact rat red cell populations of different ages separated by density gradient centrifugation in order to clarify changes in membrane lipids of red blood cells during in vivo aging. Fatty acid compositions of PC and PE altered progressively as red cells became denser. Changes in unsaturated fatty acids occurred predominantly at the 2-position of PC and PE and those in saturated fatty acids at both positions. The esterification rates of 5 major fatty acids decreased as red cells became denser and those of oleic acid, linoleic acid and arachidonic acid to both PC and PE of fraction I cells (oldest cells) were 37–51% those of fraction IV cells (youngest cells). Reduction in the rates of fatty acid esterification appeared to occur in the course of red cell maturation because reticulocyte-enriched cell fractions showed 4.5–14.5 times higher rates of linoleic acid and arachidonic acid esterifications to PC and PE.