Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase

The immobilized 1,3-regiospecific Rhizomucor miehei lipase (Lipozyme™) was employed to catalyze the transesterification reaction (acidolysis) of 1,2-diacyl-sn-glycero-3-phosphatidylcholine with n-3 polyunsaturated fatty acids under nonaqueous solvent-free conditions. With a concentrate of 55% eicosapentaenoic acid (EPA) and 30% docosahexaenoic acid (DHA) and pure phosphatidylcholine from egg yolk, phospholipids of 32% EPA and 16% DHA content were obtained, presumably as a mixture of phosphatidylcholine and lysophosphatidylcholine. ³¹P nuclear magnetic resonance (NMR) analysis turned out to be a valuable technique to study the details of the reactions involved. It revealed that when 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine was transesterified with 98% pure EPA, a substantial amount of hydrolysis side reaction took place (39%), leading to a product mixture of 39% phosphatidylcholine, 44% lysophosphatidylcholine, and 17% sn-glycerol-3-phosphatidylcholine. The lysophosphatidylcholine constituent comprised 70% EPA, whereas the phosphatidylcholine component contained 58% EPA. The ³¹P NMR technique provided valid information about the mechanism of the reaction. It became evident that a high dosage of lipase containing 5% water afforded optimal conditions for the optimal extent of EPA incorporation into the phospholipids, under which the extent of hydrolysis side reaction remained relatively high.     

An extended method for separating and quantitating molecular species of phospholipids

AN improved and extended method for separating and quantitating molecular species of four phospholipid classes is presented. Crude lipid extract is first separated into phospholipid classes on a silica column. Each phospholipid class is then separated into molecular species without derivatization using high-performance liquid chromatography on columns packed with octadecyl silica. Quantitation of individual species is achieved by measuring absorbance at 205 nm. Factors for converting absorbancies to mol fractions have been determined. Quantitation by absorbance at 205 nm agrees well with quantitation by gas chromatography which is preferred to quantitation by phosphate analysis. One hundred phospholipid species have been identified. A table of relative retention times of molecular species is provided. Examples of quantitative analyses of species composition are presented.     

Preparation of polyunsaturated phospholipids by lipase-catalyzed transesterification

Transesterifications were investigated to determine a means for preparing polyunsaturated phospholipids simply from soy phospholipid, sardine oil, and two kinds of microbial lipases originating fromCandida cylindracea andRhizopus delemar. The optimum reaction conditions forCandida cylindracea lipase were: 4 g of sardine oil, 10 mL of water, 0.7 g of lipase, 10 mL of hexane, 48 hr of reaction time at 37°C for 3 g of soy phospholipid, for which the transesterification ratio reached approximately 45%. Recovery of phospholipid was low, because hydrolysis also occurred under these reaction conditions. However, hydrolysis could be suppressed by using glycerine instead of water, and the recovery of phospholipid increased to 47%, although the transesterification ratio was reduced to 32%. Rhizopus delemar lipase has 1,3-specificity for triglycerides, and the transesterification ratio was approximately 37% in the 1-position of phospholipid. The resulting phospholipid was rich in polyunsaturated fatty acids and linoleic acid, while the total percentage of polyunsaturated fatty acids incorporated was 18.4%. Therefore, polyunsaturated phospholipids can be prepared easily by transesterification of soy phospholipid with fish oil by means of commercial lipases.     

Trans-monoenoic and polyunsaturated fatty acids in phospholipids of aVibrio species of bacterium in relation to growth conditions

AVibrio species of bacterium known to contain the polyunsaturated fatty acid 20∶5n−3 was grown in both freshwater and seawater media at 5 and 20°C and examined for adaptive changes in lipid composition. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), together with a smaller proportion of nonesterified fatty acids (NEFA), comprised almost all the lipid under all growth conditions examined. Temperature had a more pronounced effect than the salinity of the medium on lipid composition. The proportion of PE in total lipid was always higher at 5 than at 20°C. Conversely, the proportion of NEFA was lower at 5 than 20°C whereas that of PG was not altered. The levels of saturated fatty acids in total lipid, PE and PG were all decreased by growth at 5°C. No differences were observed with respect to growth temperature in the levels ofcis 16∶1n−7, the principal monoenoic fatty acid in both PE and PG.Trans 16∶1n−7 was found to comprise 12.8–15.2% of fatty acids in PE and PG of bacteria grown at 5°C but only 4.4–8.5% of phospholipid fatty acids in bacteria cultured at 20°C. Regardless of medium composition, a reduction in growth temperature from 20 to 5°C also caused the proportions of 20∶5n−3 to increase from around 0.8 to 4.4% in PE and from around 4 to 20% in PG. The simultaneous occurrence oftrans 16∶1n−7 and 20∶5n−3 is unique to thisVibrio species of bacterium. The increased proportions of both these fatty acids with decreasing temperature suggest that they have a role in retailoring biomembrane phospholipids during temperature acclimation of the bacterium.     

Dietary eritadenine-induced alteration of molecular species composition of phospholipids in rats

The effect of dietary eritadenine, a hypocholesterolemic compound found in the mushroomLentinus edodes, on the fatty acid and molecular species profiles of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver microsomes was investigated in relation to the hypocholesterolemic action of the compound in rats. Animals were fed the control or eritadenine-supplemented (50 mg/kg diet) diet for 14 d. Eritadenine supplementation significantly decreased the plasma concentrations of cholesterol and phospholipids, but not triglycerides. The PC/PE ratio of liver microsomes, but not plasma, was also markedly decreased by eritadenine. Eritadenine supplementation was found to increase the proportion of 18:2n-6 and, inversely, to decrease the proportion of 20:4n-6 and 22:5n-6 in plasma PC and liver microsomal PC and PE, indicating that eritadenine depressed the metabolism of linoleic acid. The effect of eritadenine on the profile of n-3 fatty acids was dissimilar in PC and PE. These changes in fatty acid composition were selectively reflected in the molecular species composition of both PC and PE; the extent of increase in 16:0–18:2 molecular species or decrease in 18:0–20:4 molecular species was apparently greater than that of other molecular species containing 18:2 or 20:4 in the sn-2 position. These results suggest that, in addition to the decrease in liver microsomal PC/PE ratio, the alteration of plasma PC molecular species composition might also participate in the hypocholesterolemic action of eritadenine.     

Distribution of the molecular species of phospholipids in human umbilical cord blood

The composition of phosphatidylcholine (PC) and sphingomyelin (SM) was studied in cord blood lipoproteins to determine whether equilibration of the molecular species of phospholipids among lipoproteins was comparable with that reported for adults. The molecular species distributions of PC in low density lipoprotein (LDL) differed from that of high density lipoprotein (HDL). Whereas LDL PC was richer in combinations of fatty acids with 16 and 18 carbon atoms than HDL, the HDL was markedly enriched in combinations of fatty acids with 18 and 20 carbon atoms. Sphingomyelins in LDL were richer in palmitic acid than HDL while HDL had a greater proportion of long chain sphingomyelin than LDL. The molecular species of PC and SM do not equilibrate in cord blood. The results for the SM distributions were similar to other reports for adult human lipoprotein. However, the marked differential distribution of PC among lipoproteins appears unique to cord blood. The mechanisms responsible for equilibrating PC among lipoproteins are less well developed in the neonate when compared with the adult.     

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.     

Effect of diet on the fatty acid and molecular species composition of dog retina phospholipids

Dogs were born to mothers fed commercial diets low or enriched in n-3 fatty acids and raised on those diets until they were about 50 d old. Retinas were removed, lipids were extracted, and total phospholipids were anlyzed for fatty acid and molecular species composition. Animals from the low n-3 group had significantly lower retinal levels of 22∶6n-3 and higher levels of n-6 fatty acids, especially 20∶4n-6 and 22∶5n-6. There was no difference in the retinal levels of 18∶2n-6, and only small differences were found in saturated and monounsaturated fatty acids. The most dramatic differences in molecular species occurred in 22∶6n-3-22∶6n-3 (4.7 vs. 0.8%) and 18∶0-22∶6n-3 (27.6 vs. 14.4%); total molecular species containing 22∶6n-3 were significantly lower in the low n-3 group (45.5 vs. 24.0%). Molecular species containing 20∶4n-6 and 22∶5n-6 were greater in the low n-3 animals (13.0 vs. 25.7%), as were molecular species containing only saturated and monounsaturated fatty acids (40.8 vs. 35.4%). These results show that modest differences in the amount of n-3 fatty acids in the diets of dogs can have profound effects on the fatty acid and molecular species composition of their retinas.     

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.     

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.     

Competitive inhibitions in the metabolism of polyunsaturated fatty acids studied via the composition of phospholipids,triglycerides and cholesteryl esters of rat tissues

Male rats which had been kept on fat-free diet and which were deficient in essential fatty acids were divided into ten groups. All ten groups received 0.8% of calories of linolenate, and nine received one of three levels of either linoleate, γ-linolenate or arachidonate for a period of six days. The rats were sacrificed, the livers, kidneys and testes were extracted, and the phospholipids, triglycerides and cholesteryl esters were separated by thin-layer chromatography. The fatty acid composition of each was determined by gas-liquid chromatography. The inhibition of the metabolism of linolenic acid by linoleate, γ-linolenate and arachidonate was evidenced in all three lipid classes and in all tissues. The activities in suppressing linolenate metabolism were in the order 20:4 >18:3 >18:2. Presented before the AOCS, Houston, April, 1965.     

Marine phospholipids as dietary carriers of long‐chain polyunsaturated fatty acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids (PUFA) of the n‐3 series. Fish oil is a classical source of n‐3 PUFA, where they occur in the form of triacylglycerols (TAG). However, new sources of n‐3 PUFA esterified in phospholipids (PL) are emerging. We prepared liposomes from a natural marine lipid extract and examined their behaviour under conditions mimicking that of the gastrointestinal tract. This physicochemical approach proved that liposomes could be used as an effective oral PUFA delivery system. In vivo studies in rats were performed to examine the metabolic fate of EPA (20:5 n‐3) and DHA (22:6 n‐3) delivered either in PL from liposomes or in TAG from oil. Liposome ingestion increased PUFA bioavailability in lymph compared with fish oil. The proportion of n‐3 PUFA esterified in the sn‐2 position of chylomicron TAG depended on the dietary lipid source. Complex time‐course profiles were observed for plasma lipids with liposome supplementation over a 2‐week period, suggesting time‐dependent regulations. Taken together, the type of PUFA, EPA or DHA, as well as its intramolecular distribution in chylomicron TAG seemed to influence the metabolic fate of the fatty acids and their physiological activities.     

Surfactant properties of low molecular weight phospholipids

Surface tensions, critical micelle concentrations (CMCs), contact angles on hydrophobic polyethylene, and foaming characteristics of phosphatidic acids, phosphatidylcholines, phosphatidylethanolamines, and phosphatidylglycerols were measured to determine their suitability as substitutes for traditional surfactants. These phospholipids have fatty acid chains of 5 to 12 carbon atoms, a range over which they are soluble at room temperature. Their surface tensions decrease with increasing concentrations until their CMCs are reached, above which their plateau surface tensions are as low as 21 mN/m, indicating excellent surface activities. In general, plateau surface tensions decrease with increasing chain length within each phospholipid type. The classical relationship for In CMC vs. chain length is followed with slopes typical of anionic surfactants for phosphatidic acids and phosphatidylglycerols and resembling zwitterionic surfactants for phosphatidylcholines and phosphatidylethanolamines, consistent with the charge on the hydrophilic group. The wetting capabilities of aqueous solutions on polyethylene are good and foam heights and stabilities are high, the latter two properties being comparable to traditional anionic (sodium dodecylsulfate) and nonionic (octylphenol polyethoxylate) surfactants. Some anomalies are observed regarding the effect of chain length on wetting and foaming, probably due to the depletion effect. Many phospholipids slowly degrade in aqueous solution. We conclude that short-chain phospholipids exhibit excellent surfactant properties and may be useful in many applications.     

Nuclear magnetic resonance investigation of hydrocarbon chain packing in bilayers of polyunsaturated phospholipids

²H nuclear magnetic resonance (NMR) on chaindeuterated phospholipids has been used to study the influence of the degree of unsaturation on lipid chain packing and on area per molecule at the lipid water interface. Order and motions of deuterated stearic acid in position sn-1 of phosphatidylcholines (PC) containing 18∶0, 18∶1n-9, 18∶2n-6, 18∶3n-3, 20∶4n-6, 20∶5n-3, or 22∶6n-3 in position sn-2 were investigated in pure PC and in mixtures of PC in a phosphatidylethanolamine (PE) matrix. Results reveal that lipid packing in bilayers is mainly controlled by packing requirements at the lipid water interface. Increasing degrees of unsaturation lower chain order and increase area per PC molecule, whereas inclusion of PE in model membranes has the opposite effect. Chain order and motions in highly unsaturated lipid membranes are less sensitive to changes in temperature. Temperature sensitivity decreases further upon incorporation of PC into a PE matrix. Unsaturation induces chain disordering, which may be interpreted as an increase in area per molecule of lipids toward the center of the bilayer. This may result in a lower packing density of unsaturated lipids at the lipid water interface. We hypothesize that these differences in lipid packing and dynamics may influence activity of membrane proteins.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. IV. Peroxidation and interconversion of polyunsaturated fatty acids in muscle phospholipids

It has been suggested that the net changes which take place in the composition of the muscle phospholipid fatty acids of the antioxidant-deficient rat represent the balance of two opposing processes. To compensate for (A) the preferential peroxidative destruction of the most highly polyunsaturated fatty acids in the tissue there occurs (B) an increase in the conversion of available precursors to the higher polyunsaturated fatty acids. Analysis of the data in terms of peroxidation kinetics indicated that the onset of creatinuria in one group after 3 weeks and in a second group after 7 weeks on an antioxidant-deficient diet occurred in both cases concomitant with the peroxidative “disappearance” of approximately 125 μg of phospholipid polyunsaturated fatty acid per gram wet weight of tissue or 2% of the total muscle phospholipid fatty acids.     

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes

The biosynthesis of [¹⁴C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [¹⁴C] phosphatidic acid by preincubation of unlabeled microsomes withsn-[¹⁴C]glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgC1₂. The newly formed [¹⁴C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [¹⁴C] phosphatidate and [¹⁴C]CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP:phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation ofsn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol. Presented in part at the Annual Meeting of the Canadian Federation of Biological Societies, Winnipeg, June 1975.     

Composition of phospholipids of white muscle of six tuna species

A comparative study of the phospholipids of white muscle of six of the comercially utilized tuna species, including quantitative analyses of phospholipid classes and studies of the acyl composition of the major components. Plasmalogen compounds also were identified and quantified. Choline and ethanolamine glycerophospholipids were the most abundant classes in all the samples, as well as the only molecules containing plasmalogens (16∶0, 18∶0, and 18∶1 alkenyl ether chains). The patterns of fatty acid distribution within each of the phospholipid classes showed general similarities in the species analyzed. However, ratios between certain saturated and polyunsaturated fatty acids in different phospholipid classes showed remarkable diffences. The high content of n−3 polyunsaturated fatty acids in the principal phospholipids, such as the plasmalogens, and taking into account the fatty acids possible importance in human nutrition, indicates that the white muscle of tuna species may be a potentially important dietary item.     

Fatty acid and molecular species composition of rat brain phosphatidylcholine and-ethanolamine from birth to weaning

The fatty acid composition of diacyl phosphatidylcholine and phosphatidylethanolamine from the brain of rats 3, 6, 9, 12, 15, 18, and 21 days old were determined. In phosphatidylcholine, the relative amounts of stearic and oleic acid increased from 25% to 33% while the relative amounts of myristic, palmitic, and palmitoleic decreased from 65% to 50% during this time period. The same pattern was seen in phosphatidylethanolamine with stearic and oleic increasing from 38% to 49% and the shorter chain acids decreasing from 17% to 13%. The polyunsaturated fatty content of phosphatidylcholine was approximately 10% and increased slightly during the first 3 weeks, while the polyunsaturated content of phosphatidylethanolamine decreased from 44% to 37%. The molecular species composition of phosphatidylcholine and phosphatidylethanolamine was determined in brains of rats 3, 6, and 9 days old. The relative amounts of the molecular species remained nearly constant during this time period with phosphatidylcholine containing 35% saturated, 40% monoenoic, 6% dienoic, 11% tetraenoic, 2% pentaenoic, and 5% hexaenoic. Phosphatidylethanolamine contained 1% saturated, 8% monoenoic, 3% dienoic, 40% tetraenoic, 9% pentaenoic, and 37% hexaenoic species. Analysis of the fatty acid composition of the molecular species reveals that in phosphatidylcholine the polyunsaturated fatty acids 20∶4 and 22∶6 are predominately paired with 16∶0, while in the phosphatidylethanolamine these two unsaturated fatty acids are paired with 18∶0. Furthermore, dipalmitoyl phosphatidylcholine accounts for approximately 25% of the total molecular species of that lipid.