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.     

Cytotoxic effects of ether lipids and derivatives in human nonneoplastic bone marrow cells and leukemic cells in vitro

The effects of 2-lysophosphatidylcholine (2-LPC), the alkyl lysophospholipid derivatives (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and 1-O-hexadecyl-sn-glycero-3-phospho-trimethyl-ammonio-hexanol, the 2-acetamide analog of platelet-activating factor (PAF) 1-O-octadecyl-2-acetamide-sn-glycero-3-phosphocholine, the thioether lysophospholipid derivative (TLP) BM 41.440 and the ether-linked lipoidal amine CP-46,665 on tritiated thymidine uptake and trypan blue dye exclusion were tested in vitro in various freshly explanted cell samples from human nonneoplastic bone marrow and human leukemias. In both assay systems, a dose range of 1–20 μg/ml of the compounds was tested after 24, 48 and 72 hr of coincubation with the cells. The trypan blue dye exclusion revealed statistically significant preferential cytotoxicity in leukemic cells for three compounds with the order of quantitative selectiveness: ET-18-OCH₃>BM41.440>2-acetamide analog of PAF. CP-46,665 was the most toxic compound, but did not reveal significant differences between nonneoplastic bone marrow and leukemic cells when added in concentrations greater than 1 μg/ml. The trimethyl-ammoniohexanol compound showed only minor activity in the majority of tests, when added at concentrations <20 μg/ml. 2-LPC was rather ineffective. The tritiated thymidine uptake showed only preferential antiproliferative effects towards leukemic cells of ET-18-OCH₃ and, sometimes, within the dose time frame tested of BM 41.440. All compounds tested except 2-LPC and the trimethyl-ammonio-hexanol compound were active also in this assay (inhibition of uptake>50% of the controls). Based on these results, ET-18-OCH₃ and BM 41.440 are recommended for experimental bone marrow purging.     

TMAH-Catalyzed Transesterification of EPA and DHA in Encapsulated Fish Oil Products

Tetramethylammonium hydroxide (TMAH)-catalyzed transesterification was developed as a rapid and reliable method using gas chromatography (GC) to determine the total fatty acid profile and to quantify the ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in 20 brands of encapsulated fish oil products. The AOAC method with boron trifluoride (BF₃) as a catalyst was used as a reference. After the respective transesterifications of BF₃ and TMAH, seven brands of encapsulated fish oil showed a single peak of EPA or DHA in the chromatograms, while 13 brands showed a single peak in the chromatograms after BF₃ esterification, but doublet peaks of EPA or DHA after TMAH esterification. By comparing with the GC/MS NIST library and authentic standard fatty acids of ethyl esters, the two pairs of doublet peaks were confirmed the ethyl and methyl esters of EPA and DHA, while the sum of the peak areas of the doublet represented the content of EPA or DHA. The reaction time course concluded that optimal TMAH transesterification was obtained at 25 °C for 10 min and using GC columns of low to medium polarity including Rtx-wax and Rtx-2330 were able to differentiate and quantify the ethyl- and methyl-esterified EPA and DHA, while RT-2560 column with higher polarity than the two other columns was unable to resolve the ethyl ester from the methyl ester of EPA or DHA. An EPA/DHA ratio of ≥1.10 may serve as an indicator of fish oil fortified with the ethyl ester of EPA.     

Lipase-catalyzed incorporation of n−3 polyunsaturated fatty acids into vegetable oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

Enzymatic and chemical synthesis of phosphatidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid

Regioselective incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into phosphatidylcholine (PC) was carried out using enzymatic and chemical synthesis. Incorporation at the sn‐1 position was successfully achieved by lipase‐catalysed esterification of 2‐palmitoyl‐lysophosphatidylcholine (LPC), although in most cases, the enzymes incorporated EPA and DHA at lower rates than other fatty acids. For the incorporation of DHA, Candida antarctica lipase B was the only useful enzyme, while incorporation of EPA was efficiently carried out using either this enzyme or Rhizopus arrhizus lipase. The highest yields in the lipase‐catalysed reactions were obtained at the lowest water activity (close to 0). However, by carrying out the reactions at a higher water activity of 0.22, more EPA and DHA were incorporated. Esterification of 2‐palmitoyl‐LPC with pure EPA at this water activity converted 66 mol‐% of LPC to PC using Rhizopus arrhizus lipase as catalyst. When the fatty acid was DHA and the catalyst Candida antarctica lipase B, 45 mol‐% of PC was obtained. For incorporation of EPA and DHA at the sn‐2 position, phospholipase A₂ was used, but the reaction was very slow. Chemical coupling of 1‐palmitoyl‐LPC and EPA or DHA was more efficient, resulting in complete conversion of LPC.     

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.     

Incorporation of polyunsaturated fatty acids into CT-26, a transplantable murine colonic adenocarcinoma

Previous studies in our laboratory have shown that marine oils, with high levels of eicosapentaenoic (EPA, 20∶5n−3) and docosahexaenoic acids (DHA, 22∶6n−3), inhibit the growth of CT-26, a murine colon carcinoma cell line, when implanted into the colons of male BALB/c mice. Anin vitro model was developed to study the incorporation of polyunsaturated fatty acids (PUFA) into CT-26 cells in culture. PUFA-induced changes in the phospholipid fatty acid composition and the affinity with which different fatty acids enter the various phospholipid species and subspecies were examined. We found that supplementation of cultured CT-26 cells with either 50 μM linoleic acid (LIN, 18∶2n−6), arachidonic acid (AA, 20∶4n−6), EPA, or DHA significantly alters the fatty acid composition of CT-26 cells. Incorporation of these fatty acids resulted in decreased levels of monounsaturated fatty acids, while EPA and DHA also resulted in lower levels of AA. While significant elongation of both AA and EPA occurred, LIN remained relatively unmodified. Incorporation of radiolabeled fatty acids into different phospholipid species varied significantly. LIN was incorporated predominantly into phosphatidylcholine and had a much lower affinity for the ethanolamine phospholipids. DHA had a higher affinity for plasmenylethanolamine (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) than the other fatty acids, while EPA had the highest affinity for phosphatidylethanol-amine (1,2-diacyl-sn-glycero-3-phosphoethanolamine). These results demonstrate that,in vitro, significant differences are seen between the various PUFA in CT-26 cells with respect to metabolism and distribution, and these may help to explain differences observed with respect to their effects on tumor growth and metastasis in the transplantable model.     

Asymmetric <Emphasis Type="Italic">in vitro</Emphasis> synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30°C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30–40% 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) and 60–70% 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3′ position of the glycerol molecule.     

Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase D-catalyzed transphosphatidylation

A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested. The reaction proceeded well, giving almost no hydrolysis of PtdCho to PtdOH in a biphasic system consisting of diethyl ether and acetate buffer at 30°C. The isopropylidene protective group was removed by heating the diastereomeric isopropylidene PtdGro at 100°C in trimethyl borate in the presence of boric acid to obtain the desired PtdGro diastereomers. The purities of the products, which were determined by chiral-phase HPLC, were exclusively dependent on the optical purities of the original isopropylideneglycerols used. The present method is simple and can be utilized for the synthesis of pure PtdGro diastereomers having saturated and unsaturated acyl chains.     

Synthesis of 6-phosphatidyl-L-ascorbic acid by phospholipase D

Phospholipase D (EC 3.1.4.4) ofStreptomyces species was found to catalyze transphosphatidylation to L-ascorbic acid from phosphatidylcholine (PC) in a biphasic reaction system. The product was identified as 1,2-diacyl-sn-glycero-3-phospho-6′-L-ascorbic acid (PA-AsA) by mass spectrometry and nuclear magnetic resonance spectroscopy. The optimal pH of transphosphatidylation was 4.5 and the rate of PA-AsA formation increased as concentrations of L-ascorbic acid increased. The conversion of PC to PA-AsA was greater than 80%. PA-AsA was found to be more resistant to hydrolysis by phospholipase D than was PC.     

Fractionation of menhaden oil ethyl esters using supercritical fluid CO2

Supercritical fluid CO₂ was used to fractionate menhaden oil fatty acid ethyl esters to obtain concentrates of the esters of allcis-5,8,11,14,17-eicosapentaenoic acid (EPA) and allcis-4,7,10,13,16,19-docosahexaenoic acid (DHA). Separation of the ethyl esters was found to occur primarily by carbon number, thus limiting the degree to which the ethyl esters of EPA and DHA could be concentrated. Urea fractionation of whole esters in order to remove saturates, monoenes and dienes prior to fractionation with supercritical fluid CO₂ resulted in concentrates of EPA and DHA in purities exceeding 90%. Several criteria are given for the selection of crude oils in order to maximize both purity and yield of concentrates.     

An enzymatic method for the synthesis of mixed-acid phosphatidylcholine

The enzymatic synthesis of PC with decanoic acid in the sn-1 and hexanoic acid in the sn-2 position is described. The procedure comprises the following enzymatic steps: (i) treatment of egg yolk with phospholipase A₂ (PLA₂) to hydrolyze egg yolk PC to 1-acyl lysophosphatidylcholine (LPC); (ii) esterification of 1-acyl LPC with hexanoic acid catalyzed by PLA₂ to yield PC with hexanoic acid in the sn-2 position; (iii) removal of the FA in the sn-1 position by lipase-catalyzed ethanolysis to yield 2-hexanoyl LPC; and finally (iv) introduction of decanoic acid in this position by lipase-catalyzed esterification of 2-hexanoyl LPC to yield 1-decanoyl-2-hexanoyl-PC. Two egg yolks with a weight of 16 g were required to obtain 160 mg of the desired product. The chemical purity of the PC product and the positional purity of the FA were around 99%. The method is applicable for the synthesis of other mixed-acid PC species as well.     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Regioisomers of Phosphatidylcholine Containing DHA and Their Potential to Deliver DHA to the Brain: Role of Phospholipase Specificities

Because neurons cannot synthesize docosahexaenoic acid (DHA), a dietary supplement of DHA in the form of phospholipids is recommended for maintaining proper brain functions. A model for delivering dietary sn-2-DHA phosphatidylcholine (PtdCho) to the brain involves phospholipase A₂ based deacylation/reacylation cycle followed by delivery of DHA through high-density lipoproteins that bind to the brain capillary endothelial cells in the blood–brain barrier (BBB). Our previous study demonstrated preference of endothelial lipase (EL) for PtdCho species that contain sn-2-DHA, resulting in production of sn-2-DHA lysoPtdCho that is preferentially taken up by the brain. However, since CoA-dependent reacylation of lysoPtdCho with DHA at the sn-2 position is not favored in vivo, we proposed that sn-1-DHA PtdCho in the diet may be a superior source of DHA for the brain. To test this hypothesis, DHA PtdCho regioisomers were prepared, and their hydrolysis by physiologically relevant phospholipases was determined. The data presented here show that: (1) group X secretory PLA₂ (sPLA₂) is about threefold more active than group V sPLA₂ in releasing sn-2 fatty acids from DHA regioisomers, and (2) EL shows its specificity for DHA PtdCho species in a concentration independent manner, suggesting that the enzyme could play a major role in generating free sn-1-DHA or/and sn-2-DHA lysoPtdCho from the regioisomers in the BBB. We propose that PtdCho species containing sn-1-DHA may have the advantages of both “preserving” DHA in deacylation/reacylation cycle and releasing free DHA in the BBB for uptake by the brain.     

Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase

The objective of this study was to investigate the use of lipases as catalysts for separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil by kinetic resolution. Transesterification of various fish oil triglycerides with a stoichiometric amount of ethanol by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation. When free fatty acids from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. By this modification, complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into the various positions of the triglycerides were avoided. As an example, when tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained after 24 h. The residual glyceride mixture contained 49% DHA and 6% EPA (8:1), with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were directly esterified with ethanol, 68% conversion was obtained after only 8h. The residual free fatty acids comprised 74% DHA and only 3% EPA (25:1). The recovery of both DHA into the residual free fatty acid fraction and EPA into the ethyl ester product remained very high, 83 and 87%, respectively.     

The Identification and Quantification of Phospholipids from Thermus and Meiothermus Bacteria

Structural identities of the major phospholipid (PL-2), minor phospholipid (PL-1) and trace phospholipid (PL-0) from representative strains of the genera Thermus and Meiothermus were established. Phospholipids were quantified using phosphorus-31 nuclear magnetic resonance (³¹P-NMR). The structures of the major phospholipid (PL-2) from Thermus filiformis MOK14.7 and Meiothermus ruber WRG6.9 were identified as 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine (GlcNAc-PGAA) and 2′-O-(2-acylalkyldiol-1-O-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine (GlcNAc-diolPGAA). Interestingly, M. ruber contained only a diacyl form of GlcNAc-PGAA (87 %), while T. filiformis contained both GlcNAc-PGAA (59 %) and GlcNAc-diolPGAA (18 %). The structures of the minor phospholipid (PL-1) were established as 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-glucosaminyl)-N-glyceroyl alkylamine (GlcN-PGAA, 13 %) in T. filiformis and 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-galactosaminyl)-N-glyceroyl alkylamine (GalN-PGAA, 19 %) in M. ruber. This is the first reliable discovery of phosphatidylglyceroylalkylamines modified by glucosamine or galactosamine with a free amino group. No signs of diol-based phosphatidylglyceroylalkylamines were found in PL-1 phospholipids. Similar to PL-2, trace phospholipid (PL-0) from T. filiformis contained both unsubstituted diol-based phosphatidylglyceroylalkylamine (diolPGAA) and PGAA, while M. ruber contained only free PGAA. Unlike analysis using TLC, the diol form of phosphatidylglyceroylalkylamines is clearly resolved from the diacyl form via ³¹P-NMR.     

Wax Ester Rich Oil From The Marine Crustacean,Calanus finmarchicus,is a Bioavailable Source of EPA and DHA for Human Consumption

Oil from the marine copepod, Calanus finmarchicus, which contains >86 % of fatty acids present as wax esters, is a novel source of n‐3 fatty acids for human consumption. In a randomized, two‐period crossover study, 18 healthy adults consumed 8 capsules providing 4 g of Calanus^® Oil supplying a total of 260 mg EPA and 156 mg DHA primarily as wax esters, or 1 capsule of Lovaza^® providing 465 mg EPA and 375 mg DHA as ethyl esters, each with an EPA‐ and DHA‐free breakfast. Plasma EPA and DHA were measured over a 72 h period (t = 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h). The positive incremental area under the curve over the 72 h test period (iAUC_{0‐72 h}) for both EPA and DHA was significantly different from zero (p < 0.0001) in both test conditions, with similar findings for the iAUC_{0–24 h} and iAUC_{0–48 h}, indicating the fatty acids were absorbed. There was no difference in the plasma iAUC_{0–72 h} for EPA + DHA, or DHA individually, in response to Calanus Oil vs the ethyl ester condition; however, the iAUC_{0–48 h} and iAUC_{0–72 h} for plasma EPA in response to Calanus Oil were both significantly increased relative to the ethyl ester condition (iAUC_{0–48 h}: 381 ± 31 vs 259 ± 39 μg*h/mL, p = 0.026; iAUC_{0‐72 h}: 514 ± 47 vs 313 ± 49 μg*h/mL, p = 0.009). These data demonstrate a novel wax ester rich marine oil is a suitable alternative source of EPA and DHA for human consumption.     

Fractionation of urea-pretreated squid visceral oil ethyl esters

Ethyl esters of squid (Illex argentinus) visceral oil contained 11.8% eicosapentaenoic acid (EPA) and 14.9% docosahexaenoic acid (DHA). The esters were treated with urea to increase the contents of EPA and DHA. The non-urea complexing ethyl esters of squid visceral oil contained 28.2% EPA and 35.6% DHA. This mixture was fractionated by molecular distillation to further increase the EPA or DHA content. The fraction collected in the 110°C distillate had an EPA content of 39.0% with 0.26 g/100 g of cholesterol, while the 130°C distillate contained 65.6% DHA and 0.42 g/100 g of cholesterol. Ethyl esters prepared from visceral oil of squid Ommastrephes bartrami had 4.5% EPA and 12.7% DHA. After urea pretreatment, the EPA and DHA contents were raised to 10.1 and 30.0%, respectively. When this mixture was further fractionated by molecular distillation, 16.9% EPA with 0.35 g/100 g cholesterol was found in the 110°C distillate and 52.6% DHA with 0.70 g/100 g cholesterol was found in the 130°C distillate. Cholesterol in the squid visceral oil ethyl esters was concentrated in the final residue of molecular distillation when the polyunsaturated ethyl esters were enriched by the urea complexation method prior to molecular distillation. For example, the cholesterol content in the ethyl esters from O. bartrami squid visceral oil was 2.28 g/100 g originally. It was enriched to 64.15 g/100 g in the final residue from the molecular distillation.     

Positional distribution of FA in TAG of enzymatically modified borage and evening primrose oils

Stereospecific analysis was carried out to establish positional distribution of FA in the TAG of DHA, EPA, and (EPA+DHA)-enriched oils. In this study, TAG of enzymatically modified oils were purified using a silicic acid column. The TAG were then subjected to positional distribution analysis using a modified procedure involving reductive cleavage with Grignard reagent. The results showed that in DHA-enriched borage oil (BO), DHA was randomly distributed over the three positions of TAG, whereas γ-linolenic acid (GLA) was mainly esterified at the sn-2 and-3 positions. In DHA-enriched evening primrose oil (EPO), however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this FA was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In (EPA+DHA)-enriched BO, EPA and DHA were mainly esterified at the sn-1 and -3 positions of TAG, whereas GLA was mainly located at the sn-2 position. In (EPA+DHA)-enriched EPO, GLA was mainly located at the sn-2 and-3 positions; EPA was preferentially esterified at the sn-1 and-3 positions, and DHA was found mainly at the sn-3 position.