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%.     

Lipid peroxidation of isolated chylomicrons and oxidative status in plasma after intake of highly purified eicosapentaenoic or docosahexaenoic acids

Fourteen healthy male volunteers were given two separate high-saturated-fat meals with and without the addition of 4 g highly purified ethyl esters of either eicosapentaenoic acid (EPA) (95% pure, n=7) or docosahexaenoic acid (DHA) (90% pure, n=7) supplied as 1-g capsules each containing 3.4 mg vitamin F. The chylomicrons were isolated 6 h after the meals, at peak concentrations of n−3 fatty acids (FA). Addition of n−3 FA with the meal caused a 10.4-fold increase in the concentration of n−3 FA in chylomicrons compared to the saturated fat meal without addition of n−3 FA. After the saturated-fat meal, the concentration of thiobarbituric acid-reactive substances (TBARS) was 327.6±34.6 nmol/mmol triacylglycerol (TAG), which increased to 1015.8±212.0 nmol/mmol TAG (P<0.0001, n=14) after EPA and DHA were added to the meal. There was no significant correlation between the concentrations of TBARS and vitamin E in the chylomicrons collected 6 h after the test meal. The present findings demonstrate an immediate increase in chylomicron peroxidation ex vivo provided by intake of highly purified n−3 FA. The capsular content of vitamin E was absorbed into chylomicrons, but the amount of vitamin E was apparently not sufficient to protect chylomicrons against lipid peroxidation ex vivo. Daily intake of 4 g n−3 FA either as EPA or DHA for 5 wk did not change the plasma concentration of TBARS. Although not significantly different between groups, DHA supplementation decreased total glutathione in plasma (P<0.05) and EPA supplementation increased plasma concentration of vitamin E (P<0.05). The other lipid-soluble and polar antioxidants in plasma remained unchanged during 5 wk of intervention with highly purified n−3 FA.     

Chemoenzymatic synthesis of structured triacylglycerols containing eicosapentaenoic and docosahexaenoic acids

There are indications in the recent literature that the location of polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in triacylglycerols (TAG) may influence their oxidative stability. To address that question, two types of structured lipids were designed and synthesized: firstly, a TAG molecule possessing pure EPA or DHA at the mid-position with stearic acid at the outer positions; and secondly, a TAG molecule possessing pure EPA or DHA located at one of the outer positions with stearic acid at the mid-position and the remaining end position. The former adduct was synthesized in two steps by a chemoenzymatic approach. In the first step 1,3-distearolyglycerol was afforded in good yield (74%) by esterifying glycerol with two equivalents of stearic acid in ether in the presence of silica gel using Lipozyme^TM as a biocatalyst. This was followed by a subsequent chemical esterification with pure EPA or DHA using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a coupling agent in the presence of 4-dimethylaminopyridine in dichloromethane in excellent yields (94 and 91, respectively). The latter adduct was synthesized in two enzymatic steps. In the first step tristearoylglycerol was prepared in very high yield (88%) by esterifying glycerol with a stoichiometric amount of stearic acid under vacuum at 70–75°C using an immobilized Candida antarctica lipase without a solvent. That adduct was subsequently treated in an acidolysis reaction with two equivalents of EPA or DHA without solvent at 70–75°C or in toluene at 40°C in the presence of Lipozyme to afford the desired product in moderate yields (44 and 29%, respectively). This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists’ Society, San Diego, CA.     

Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on fatty acid absorption, incorporation into serum phospholipids and postprandial triglyceridemia

Fourteen healthy volunteers were randomly allocated to receive 4 g highly purified ethyl esters of eicosapentaenoic acid (EPA) (95% pure, n=7) or docosahexaenoic acid (DHA) (90% pure, n=7) daily for 5 wk in supplement to their ordinary diet. The n−3 fatty acids were given with a standard high-fat meal at the beginning and the end of the supplementation period. EPA and DHA induced a similar incorporation into chylomicrons which peaked 6 h after the meal. The relative uptake of EPA and DHA from the meal was >90% compared with the uptake of oleic acid. During absorption, there was no significant elongation or retroconversion of EPA or DHA in total chylomicron fatty acids. The concentration of EPA decreased by 13% and DHA by 62% (P<0.001) between 6 and 8 h after the meal. During the 5-wk supplementation period, EPA showed a more rapid and comprehensive increase in serum phospholipids than did DHA. DHA was retroconverted to EPA, whereas EPA was elongated to docosapentaenoic acid (DPA). The postprandial triglyceridemia was suppressed by 19 and 49% after prolonged intake of EPA and DHA, respectively, indicating that prolonged intake of DHA is equivalent to or even more efficient than that of EPA in lowering postprandial triglyceridemia. This study indicates that there are metabolic differences between EPA and DHA which may have implications for the use of n−3 fatty acids in preventive and clinical medicine.     

Application of water mimics on preparation of eicosapentaenoic and docosahexaenoic acids containing glycerolipids

To obtain enhanced incorporation of highly unsaturated fatty acids and recovery of glycerolipid products, organic solvents with high dielectric constants (water mimics) were substituted for part of the essential water for lipase activation to study their effect on acidolysis and transesterification. In acidolysis/transesterification of fish oil triglycerides and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Lipozyme IM-60 with ethylene glycol as a water mimic enhanced the incorporation of EPA and suppressed the hydrolysis of synthesized glycerolipid. On the other hand, transesterification between soy phosphatidylcholine and EPA was enhanced by a water and propylene glycol combination. In a nonaqueous medium that contained appropriate amounts of water and organic solvents (water mimics), Lipozyme IM-60 increased transesterification of EPA into soy phosphatidylcholine. Simultaneously, the recovered glycerolipid products showed decreased hydrolysis of newly synthesized EPA- and DHA-containing glycerolipids.     

Incorporation of eicosapentaenoic and docosahexaenoic acids into groundnut oil by lipase-catalyzed ester interchange

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were incorporated into groundnut oil by interesterification with a 1,3-specific lipase fromMucor miehei. The resultant EPA and DHA concentrations of the groundnut oil were 9.5 and 8.0%, respectively.     

Effects of diets enriched in eicosapentaenoic or docosahexaenoic acids on prostanoid metabolism in the rat

To clarify the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on prostaglandin biosynthesis, diets supplemented with oils rich in one fatty acid or the other were fed to rats over a 4-wk period. Animals fed the Max EPA diet showed a significant decrease in plasma and tissue phospholipid arachidonic acid content. While plasma levels of DHA increased on a shark liver oil diet enriched in DHA, the liver and kidney phospholipid contents of DHA were not altered. In addiition, the DHA-enriched diet did not decrease the arachidonic acid content of either liver or kidney phospholipids. Whole blood thromboxane and vascular prostacyclin synthesis were decreased by 65% and 36%, respectively, in animals fed the Max EPA diet. No such decrease was seen in the rats fed DHA-enriched diets. We conclude from these results that in the rat DHA is not likely to have a significant effect on prostaglandin synthesis when given as a dietary supplement.     

Incorporation of linolenic-1-C14 acid into eicosapentaenoic and docosahexaenoic acids in fish

Following intraperitoneal injection of methyl linolenate-1-C¹⁴ into kelp bass,Paralablax clathratus, the highly polyunsaturated fatty acids of their body fats were concentrated by low temperature crystallization from acetone, and eicosapentaenoic and docosahexaenoic acids were isolated from the concentrate by reversed-phase chromatography and hydrogenated. The resulting arachidic and behenic acids were degraded stepwise to margaric acid, and the distribution of activity was determined. The results indicate that the injected linolenic acid was converted to eicosapentaenoic acid and the latter incorporated into docosahexaenoic acid. A probable conversion pathway is linolenic acid→6,9,12,15-octadecatetraenoic acid→8,11,14,17-eicosatetraenoic acid→5,8,11,14,17-eicosapentaenoic acid→7,10,13,16,-19-docosapentaenoic acid→4,7,10,13,16,19-docosahexaenoic acid. Supported by a training grant from the National Heart Institute, Bethesda, Md. This paper based partially on work performed under Contract AT(04-1)GEN-12 between the Atomic Energy Commission and the University of California.     

Characterization of enzymatically synthesized structured lipids containing eicosapentaenoic, docosahexaenoic, and caprylic acids

Structured lipids (SL) containing n-3 polyunsaturated (eicosapentaenoic or docosahexaenoic) and mediumchain (caprylic) fatty acids were synthesized in gram quantities and characterized. Tricaprylin was mixed with n-3-rich polyunsaturated fatty acids in a 1:2 molar ratio and transesterified by incubating at 55°C in hexane with SP 435 lipase (10% by wt of total substrates) in a 125-mL Erlenmeyer flask as the bioreactor. After several batches of reaction, the products were pooled and hexane was evaporated. Short-path distillation was used for purification of synthesized SL. The distillation conditions were 1.1 Torr and 170°C at a feed flow rate of 3 mL/min. Up to 240 g of SL was isolated and deacidified by alkaline extraction or ethanol-water solvents. The fatty acid profile, free fatty acid value, saponification number, iodine value, peroxide value, thiobarbituric acid, and conjugated diene contents were determined. Oxidation stability, with α-tocopherol as antioxidant, and the oxidative stability index were also determined.     

Unusually high levels of eicosatetraenoic,eicosapentaenoic, and docosahexaenoic fatty acids in palestinian freshwater sponges

The fatty acid composition of three freshwater sponges—Ephydatia syriaca, Nudospongilla sp., andCortispongilla barroisi—were studied. Twenty principal fatty acids, and unusually high levels of eicosatetraenoic (5,8,11,14–20:4 up to 10.1% of the total acid mixture), eicosapentaenoic (5,8,11,14,17–20:5 up to 11.6%), and docosahexaenoic acids (4,7,10,13,16,19–22:6 up to 11.8%) were detected. The only demospongic acid found was 5,9,17-hexacosatrienoic acid (1.8–3.7%).     

Low doses of eicosapentaenoic acid, docosahexaenoic acid, and hypolipidemic eicosapentaenoic acid derivatives have no effect on lipid peroxidation in plasma

It was of interest to investigate the influence of both high doses of eicosapentaenoic acid (EPA) and low doses of 2-or 3-methylated EPA on the antioxidant status, as they all cause hypolipidemia, but the dose required is quite different. We fed low doses (250 mg/d/kg body wt) of different EPA derivatives or high doses (1500 mg/d/kg body wt) of EPA and DHA to rats for 5 and 7 d, respectively. The most potent hypolipidemic EPA derivative, 2,2-dimethyl-EPA, did not change the malondialdehyde content in liver or plasma. Plasma vitamin E decreased only after supplementation of those EPA derivatives that caused the greatest increase in the fatty acyl-CoA oxidase activity. Fatty acyl-CoA oxidase activity increased after administration of both EPA and DHA at high doses. High doses of EPA and DHA decreased plasma vitamin E content, whereas only DHA elevated lipid peroxidation. In liver, however, both EPA and DHA increased lipid peroxidation, but the hepatic level of vitamin E was unchanged. The glutathione-requiring enzymes and the glutathione level were unaffected, and no significant changes in the activities of xanthine oxidase and superoxide dismutase were observed in either low-or high-dose experiments. In conclusion, increased peroxisomal β-oxidation in combination with high amounts of polyunsaturated fatty acids caused elevated lipid peroxidation. At low doses of polyunsaturated fatty acids, lipid peroxidation was unchanged, in spite of increased peroxisomal β-oxidation, indicating that polyunsaturation is the most important factor for lipid peroxidation.     

Enzymatic modification of triacylglycerols of high eicosapentaenoic and docosahexaenoic acids content to produce structured lipids

Immobilized lipase, IM60, from Rhizomucor miehei was used as a biocatalyst for the incorporation of capric acid (C10:0) into fish oil originally containing 40.9 mol% eicosapentaenoic (20:5n-3) and 33.0 mol% docosahexaenoic (22:6n-3) acid. Acidolysis was performed with and without organic solvent. Pancreatic lipase-catalyzed sn-2 positional analysis was performed after enzymatic modification. Tocopherol analysis was performed before and after enzymatic modification. Products were analyzed by gas-liquid chromatography. After a 24-h incubation in hexane, there was an average of 43.0±1.6 mol% incorporation of C10:0 into fish oil, while 20:5 and 22:6 decreased to 27.8±2.2 and 23.5±1.3 mol%, respectively. The solvent-free reaction produced an average of 31.8±8.5 mol% C10:0 incorporation, while 20:5 and 22:6 decreased to 33.2±3.3 and 28.3±3.9 mol%, respectively. The effect of incubation time, substrate molar ratio, enzyme load, and added water were also studied. In general, as the enzyme load, molar ratio, and incubation time increased, mol% C10:0 incorporation also increased. The optimal mol% C10:0 incorporation was 41.2% at 48 h for the reaction in hexane and 46.4% at 72 h for the solvent-free reaction. The highest C10:0 incorporation (65.4 mol%) occurred at a molar ratio of 1:8 (fish oil triacylglycerols/capric acid) in hexane. For the solvent-free reaction, the optimal mol% C10:0 incorporation (56.1 mol%) occurred at a molar ratio of 1:6. An enzyme load of 10% gave the highest mol% C10:0 incorporation (41.4 mol%) in hexane; the highest incorporation (38.3 mol%) for the solvent-free reaction occurred at 15% enzyme load. Mol% incorporation of C10:0 declined with increasing amounts of added water. The optimal mol% C10:0 incorporation occurred at 1% added water (47.9 mol%) for the reaction in hexane, and at zero added water for the solvent-free reaction (21.8 mol%). Fish oil containing capric acid was successfully produced and may be nutritionally more beneficial than unmodified oil.     

The role of consumption of alpha-linolenic, eicosapentaenoic and docosahexaenoic acids in human metabolic syndrome and type 2 diabetes--a mini-review

The human metabolic syndrome and its frequent sequela, type 2 diabetes are epidemic around the world. Alpha-linolenic acid (ALA, 18:3 n-3), eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) consumption ameliorates some of these epidemics' features thus leading one to question if consumption of EPA and DHA, and their metabolic precursor ALA reduce the conversion of metabolic syndrome to type 2 diabetes and reduce the major cause of death in the metabolic syndrome and type 2 diabetes-myocardial infarction. Contributing to myocardial infarction are metabolic syndrome's features of dyslipidemia (including elevated total cholesterol and LDL-c), oxidation, inflammation, hypertension, glucose intolerance, overweight and obesity. Inflammation, glucose and lipid levels are variously influenced by disturbances in various adipocytokines which are in turn positively impacted by n-3 polyunsaturated fatty acid consumption. Type 2 diabetes has all these features though elevated total cholesterol and LDL-c are rarer. It is concluded that EPA and DHA consumption significantly benefits metabolic syndrome and type 2 diabetes primarily in terms of dyslipidemia (particularly hypertriglyceridemia) and platelet aggregation with their impact on blood pressure, glucose control, inflammation and oxidation being less established. There is some evidence that EPA and/or DHA consumption, but no published evidence that ALA reduces conversion of metabolic syndrome to type 2 diabetes and reduces death rates due to metabolic syndrome and type 2 diabetes. ALA's only published significance appears to be platelet aggregation reduction in type 2 diabetes.     

Occurrence of geometrical isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids of rats fed heated linseed oil

Monotrans geometrical isomers of 20∶5 n−3 and 22∶6 n−3 were detected in liver lipid of rats fed heated linseed oil. The isomers were identified as being 20∶5 δ5c, 8c, 11c, 14c, 17t and 22∶6 δ4c, 7c, 10c, 13c, 16c, 19t. These fatty acids were isolated as methyl esters by preparative high-performance liquid chromatography (HPLC) on reversed phase columns followed by silver nitrate thin layer chromatography (AgNO₃-TLC). The structures were identified using partial hydrazine reduction, AgNO₃-TLC of the resulting monoenes, oxidative ozonolysis of each monoene band, and gas-liquid chromatography (GLC) of the resulting dimethyl esters and monomethyl esters. Fourier-transform-infrared spectrometry confirmed thetrans geometry in isolated 20∶5 and 22∶6 isomers. The isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids probably resulted from desaturation and elongation of 18∶3 δ9c, 12c, 15t, a geometrical isomer of linolenic acid present in the heated dietary oil.     

Oxidation of synthetic triacylglycerols containing eicosapentaenoic and docosahexaenoic acids: Effect of oxidation system and triacylglycerol structure

Thirteen synthetic triacylglycerols (TAG) containing eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were oxidized in the presence of 2,2′-azobis(2,4-dimethyl-valeronitrile) (AMVN) and 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) as aqueous and nonaqueous radical initiators to investigate the influence of TAG structure and oxidation system on the oxidative stability of TAG that contain highly unsaturated fatty acids (HUFA). A 2:1 (mol/mol) mixture of trieicosapentaenoylglycerol and tripalmitoylglycerol was most susceptible to the AMVN-initiated oxidation among three types of TAG that contained EPA and palmitic acid (2:1, mol/mol). Compared with 1,2 (or 2,3)-dieicosapentaenoyl-3(or 1)-palmitoylglycerol (EEP) and 1,3-dieicosapentaenoyl-2-palmitoylglycerol (EPE), the oxidative rate of EEP was somewhat higher. A similar result was obtained for DHA-containing TAG. The oxidative rate of TAG that contained EPA and palmitic acid (1:2, mol/mol) showed a positive correlation with the amount of EPA in a single TAG molecule. Moreover, in the nonaqueous system, the oxidative rate of EPA-containing TAG was affected by unsaturation and carbon chainlength of constituent fatty acids. In the AAPH-initiated oxidation in the aqueous system, the oxidative rate of TAG with EPA and palmitic acid was higher with the increased quantity of EPA in a single TAG molecule. Also, constituent fatty acids modified the oxidative rate of EPA-containing TAG in an aqueous system. The glycerol position of EPA and DHA also affected the oxidative rate of the TAG. EPA and DHA located at the 1,2 (or 2,3)-position of glycerol were more oxidizable than those at the 1,3-position during AAPH-initiated oxidation. Thus, 1,2(or2,3)-dipalmitoyl-3(or 1)-eicosapentaenoylglycerol was oxidized faster than 1,3-dipalmitoyl-2-eicosapentaenoylglycerol. These observations suggest that the oxidative stability of TAG that contain HUFA could be modulated by the oxidation system and TAG structure.     

Enrichment of eicosapentaenoic acid and docosahexaenoic acid in saponified menhaden oil

Eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3) in free fatty acids (FFA) derived from saponified menhaden oil were concentrated by the solubility differences of FFA-salts in organic solvent. FFA-salts were formed by adding NaOH to a solution containing FFA. A Buchner funnel was used to separate solid phases from liquids containing FFA-salts. FFA that are rich in EPA and DHA can be recovered from the liquid phase by the addition of 12 N HCl. The effects of reaction time, the amount of NaOH, and solvent used on the concentration of EPA and DHA were systematically investigated. With a total volume of 112 mL, made up of 1.85% 15 N NaOH, 88.1% acetone, and 10.0% FFA, a reaction temperature of 30°C, and a reaction time of 1 h, the resulting liquid phase contained 65.4 wt% EPA and DHA, with a corresponding yield of 41.5%. By replacing the acetone with a mixture of 45% acetone and 55% acetonitrile and then storing the liquid phase at −70°C overnight, the content and yield of EPA and DHA in the final liquid phase were 61.4 wt% and 66.2%, respectively.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts

To better understand the mode of action of ω3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 μM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which >95% was associated with phospholipids. In addition, the concentration of 22∶5n−3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P<0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P<0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P<0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5′-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P<0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n−3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.     

Differential utilization of eicosapentaenoic acid and docosahexaenoic acid in human plasma

It has recently been shown that the ω3 fatty acid status in humans can be predicted by the concentration of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma phospholipids [Bjerve, K.S., Brubakk, A.M., Fougner, K.J., Johnsen, H., Midjthell, K., and Vik, T. (1993)Am. J. Clin. Nutr., in press]. In countries with low intake of ω3 fatty acids, the level of EPA in plasma phospholipids is often only about one-fifth the concentration of DHA. The purpose of this study was to investigate whether this difference in the concentration of these two fatty acids was due to a selective loss of EPA relative to DHA or to a lower dietary intake of EPA. Seven female volunteers ingested four grams of MaxEPA daily for 2 wk and in the following 4 wk they ate a diet almost completely devoid of the long-chain ω3 fatty acids. The concentrations of the ω3 fatty acids in the plasma cholesteryl esters, triglycerides and phospholipids and the high density lipoprotein phospholipids were examined at weekly intervals throughout the study. There was a more rapid rise in the concentration of EPA than in DHA levels in the supplementation period in all lipid fractions, but there was a disproportionate rise in DHA relative to EPA in the plasma lipids compared with the ratio in the supplement. In the depletion phase there was a rapid disappearance of EPA from all fractions, such that pre-trial levels were reached by one week post-supplementation. The disappearance of DHA was slower, particularly for the plasma phospholipids: at 4 wk post-supplementation, the DHA concentration in this fraction was still 40% above the pre-trial value. It is suggested that the low plasma EPA values relative to DHA are the result of increased β-oxidation of EPA and/or low dietary intake, rather than a rapid conversion of EPA to DHA. One practical result of this experiment is that, compared with DHA, the maintenance of increased EPA levels in plasma (and therefore tissues) would require constant inputs of EPA due to its more rapid loss from the plasma.     

Geometrical isomerisation of eicosapentaenoic and docosahexaenoic acid at high temperatures

Concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were heated at 140–240 °C for 2–8 h under nitrogen. The trans isomers were analysed by gas chromatography‐mass spectrometry on a BPX‐70 cyanopropyl column. All geometrical isomers of EPA and DHA with one trans double bond were observed. The rate constants (k) for the isomerisation of the all‐cis isomers were calculated and found to be higher than previously reported for linoleic acid and α‐linolenic acid. Arrhenius plots showed a linear relationship between ln k and the reciprocal absolute temperature above 180 °C. The distribution patterns of isomers with one trans double bond are approximately constant up to a degree of isomerisation of 25%. The degree of isomerisation can therefore be estimated from selected trans peaks.     

Preparation of highly purified fatty acids via liquid-liquid partition chromatography

The application of reversed-phase, liquid-liquid partition chromatography to the preparation of highly purified methyl esters of fatty acids is described. The parameters of fractionation of methyl esters by this method are demonstrated with model mixtures of these compounds. Model mixtures are also used to demonstrate the use of adsorption chromatography on columns of silicic acid, impregnated with silver nitrate, in conjunction with liquid-liquid partition chromatography to eliminate fractional distillation in the preparation of polyunsaturated methyl esters. The formation of artefacts of 4, 5 and 6 double bond methyl esters during fractional distillation and their fractionation is described. The use of liquid-liquid partition chromatography for preparative purposes on a large laboratory scale is demonstrated by the preparation of pure methyl linolenate from linseed oil esters. Methyl arachidonate, eicosapentaenoate, docosapentaenoate and docosahexaenoate are also prepared in high purity. Supported in part by the U. S. Public Health Service, NIH grant A-5018, and in part by a contract with the U. S. Dept. of Interior, Fish and Wildlife Service, Bureau of Commercial Fisheries. Presented at the AOCS Meeting in Toronto, Canada, 1962.