Further studies on artificial geometrical isomers of α-linolenic acid in edible linolenic acid-containing oils

Fifteen samples of commercial edible soybean and rapeseed oils (and mixtures of these) from Belgium, Great Britain and Germany have been analyzed for theirtrans-polyunsaturated fatty acid content. Only one sample out of the 13 refined samples, and the two cold-pressed samples, contained trace amounts oftrans isomers. Others contained between 1 and 3.3% of their total fatty acids as geometrical isomers of linoleic and linolenic acids. The degree of isomerization (DI) of linolenic acid varied between 10.5 and 26.9%. Combining results obtained in this study together with corresponding data for French oils (totalling 21 samples) indicates that the relative percentages of individual linolenic acid geometrical isomers depend on linolenic acid DI. Relationships linking these parameters could be approximated by straight lines, at least for DIs lying between 9 and 30%. Extrapolation to DI=0 suggests that the relative probabilities of isomerization of double bonds in positions 9, 12, and 15 are 41.7, 6.1 and 52.1%, respectively, at the very beginning of the isomerization reaction. At that time, the probability of a simultaneous isomerization of double bonds in positions 9 and 15 is close to zero. Thet,c,t isomer is apparently formedvia thec,c,t and thet,c,c isomers, the former being somewhat more prone to a second geometrical isomerization than the latter. The relative proportion of thec,t,c isomer is practically independent from the DI, at least between 9 and 30%, which would suggest that this isomer is an “end-product” of thecis-trans isomerization reaction.     

trans-Polyunsaturated fatty acids in French edible rapeseed and soybean oils

The fatty acid compositions of rapeseed and soybean oils marketed in France have been determined by gas liquid chromatography on a fused-silica capillary column coated with a 100% cyanopropyl polysiloxane stationary phase. Under the operating conditions employed, methyl esters of linolenic acid geometrical isomers could be separated and quantitated easily without any other complementary technique. With only one exception, all samples under study (eight salad oils and five food samples) contain geometrical isomers of linolenic acid in measurable, although variable, amounts. Totaltrans-18:3 acids may account for up to 3% of total fatty acids. This value corresponds to a degree of isomerization (percentage oftrans isomers relative to total octadecatrienoic acids) of 30%. Examination of our data indicates that the distribution pattern of linolenic acid geometrical isomers does not depend on the degree of isomerization. The two main isomers always have thec,c,t and thet,c,c configurations. These isomers occur in the almost invariable relative proportions of 47.8±1.7% and 41.1±1.0%, respectively. The third mono-trans isomer is present in lower amounts−6.5±0.7%. The only di-trans isomer that can be quantitated with sufficient accuracy is thet,c,t isomer (4.9±1.5%). Mono-trans isomers of linoleic acid are also present in these oils. However, their maximum percentages are lower than those determined for linolenic acid geometrical isomers. In the oils showing the highest degrees of isomerization,trans isomers of linoleic acid account for 0.5% (rapeseed oils) and 1% (soybean oils) of total fatty acids. Taking into account all data, it would appear that the probability of isomerization of linolenic acid is about 13–14 times that of linoleic acid.     

Thermal degradation of long‐chain polyunsaturated fatty acids during deodorization of fish oil

Long‐chain polyunsaturated fatty acids (LC‐PUFA) of the n‐3 series, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities especially in the functionality of the central nervous system. Due to the occurrence of numerous methylene‐interrupted ethylenic double bonds, these fatty acids are very sensitive to air (oxygen) and temperature. Non‐volatile degradation products, which include polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPA and DHA, were evaluated in fish oil samples obtained by deodorization under vacuum of semi‐refined fish oil at 180, 220 and 250 °C. Polymers are the major degradation products generated at high deodorization temperatures, with 19.5% oligomers being formed in oil deodorized at 250 °C. A significant amount of CFAM was produced during deodorization at temperatures above or equal to 220 °C. In fact, 23.9 and 66.3 mg/g of C20 and C22 CFAM were found in samples deodorized at 220 and 250 °C, respectively. Only minor changes were observed in the EPA and DHA trans isomer content and composition after deodorization at 180 °C. At this temperature, the formation of polar compounds and CFAM was also low. However, the oil deodorized at 220 and 250 °C contained 4.2% and 7.6% geometrical isomers, respectively. Even after a deodorization at 250 °C, the majority of geometrical isomers were mono‐ and di‐trans. These results indicate that deodorization of fish oils should be conducted at a maximal temperature of 180 °C. This temperature seems to be lower than the activation energy required for polymerization (intra and inter) and geometrical isomerization.     

Geometrical and positional isomerization of alkenyl acetates produced by hydrogenation of alkynyl acetates over palladium metal catalysts

Alkenyl acetates containing both geometrical isomers can be produced by a single controlled catalytic hydrogenation of the corresponding alkynyl acetate. The hydrogenation is capable of yielding formulations containing up to 60% trans isomer; the reduction is attended by positional isomerization in both geometrical isomers.NRCC No. 16834.     

Heat-induced geometrical isomerization of α-linolenic acid: Effect of temperature and heating time on the appearance of individual isomers

The formation of linolenic acid geometrical isomers (LAGIs) was studied in linseed oil that was heated under vacuum in sealed ampoules at different temperatures (190–260°C) for several durations (2–16 h). A temperature of about 190°C seems to be necessary to induce the formation of LAGIs. At higher temperatures, disappearance of linolenic acid follows a first-order kinetic. The formation of LAGIs increases with both heating time and temperature, degrees of isomerization of linolenic acid higher than 50–60% could easily be obtained by simply heating the oil under vacuum. Side reactions remain at a low level. The mean probabilities of isomerization of individual ethylenic bonds are similar to those determined in linolenic acid-containing oils marketed in European countries, 41.9, 4.7 and 53.3% for double bonds in positions 9, 12 and 15, respectively. The di-trans t,c,t (trans,cis,trans) isomer is formedvia the mono-trans c,c,t andt,c,c isomers by a two-step reaction. The proportions of thec,c,t andt,c,c isomers (relative to total LAGIs) decrease linearly with the heating time. The proportion of thec,t,c isomer is only slightly affected by this parameter; however, it increases with temperature. The proportion of thet,c,t isomer increases linearly with heating time at each tested temperature, at the expense of thec,c,t andt,c,c isomers. However, there is no simple relationship linking the disappearance of each of the mono-trans isomers and the formation of the di-trans isomer.     

Hydrogenation of Low Erucic Acid Rapeseed Oil

The effects of some process conditions on the overall reaction rate and on trans isomer formation during hydrogenation of low erucic acid rapeseed oil with a commercial nickel catalyst were studied. Experimental data were correlated by an empirical equation which would be used to predict the reaction rate constants for any given process conditions. Temperature had a major effect on rate and trans isomer formation. Pressure and catalyst concentration, however, had only minor effects. It is suggested that apart from these factors, the extent of trans isomer formed is also controlled by the nature and amount of the unsaturation in the oil and by the overall reaction rate. A new parameter, specific isomerization index was introduced to represent the formation of trans isomers during the hydrogenation reaction. The effects of solvents n-hexane and 2-propanol on the reaction rate and trans isomers formation were determined.     

Combined Urea Complexation and Argentated Silica Gel Column Chromatography for Concentration and Separation of PUFAs from Tuna Oil: Based on Improved DPA Level

Eicosapentaenoic acid (EPA, 20:5n‐3), docosapentaenoic acid (DPA) isomers (22:5n‐6 and 22:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) derived from tuna oil were concentrated by three stages of urea fractionation at various crystallization temperatures and different fatty acid/urea ratios. Thereafter, polyunsaturated fatty acids concentrate containing comparatively enriched DPA levels was purified by argentated silica gel column chromatography. A product containing 22.2 ± 0.6 % EPA, 4.6 ± 0.0 % DPAn‐6, 5.9 ± 0.1 % DPAn‐3 and 42.3 ± 1.2 % DHA was obtained at 1:1.6 fatty acid/urea ratio (w/w) by crystallization at ?8 °C for 16 h, ?20 °C for 8 h, and ?8 °C for 16 h. A DPA isomer concentrate containing 26.1 ± 0.5 % DPAn‐6 and 22.3 ± 0.4 % DPAn‐3 was achieved by argentated silica gel chromatography in the 6 % acetone/n‐hexane solvent fraction (v/v), and the recovery of both fatty acids was 66.1 ± 3.2 and 70.7 ± 2.2 %, respectively. Furthermore, 91.9 ± 2.5 % EPA and 99.5 ± 2.1 % DHA with recoveries of 47.8 ± 2.0 and 56.7 ± 3.3 %, respectively, were obtained in various fractions.     

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.     

Deodorization of vegetable oils: Prediction of trans polyunsaturated fatty acid content

Kinetics of the formation of trans linoleic acid and trans linolenic acid were compared. Pilot plant-scale tests on canola oils were carried out to validate the laboratory-scale kinetic model of geometrical isomerization of polyunsaturated fatty acids described in our earlier publication. The reliability of the model was confirmed by statistical calculations. Formation of the individual trans linoleic and linolenic acids was studied, as well as the effect of the degree of isomerization on the distribution of the trans fatty acid isomers. Oil samples were deodorized at temperatures from 204 to 230°C from 2 to 86 h. Results showed an increase in the relative percentage of isomerized linolenic and linoleic acid with an increase in either the deodorization time or the temperature. The percentage of trans linoleic acid (compared to the total) after deodorization ranged from <1 to nearly 6%, whereas the percentage of trans linolenic acid ranged from <1 to >65%. Applying this model, the researchers determined the conditions required to produce a specially isomerized oil for a nutritional study. The practical applications of these trials are as follows: (i) the trans fatty acid level of refined oils can be predicted for given deodorization conditions, (ii) the conditions to meet increasingly strict consumer demands concerning the trans isomer content can be calculated, and (iii) the deodorizer design can be characterized by the deviation from the theoretical trans fatty acid content of the deodorized oil.     

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.     

Synthesis of acetyl, docosahexaenoyl-glycerophosphocholine and its characterization using nuclear magnetic resonance

Docosahexaenoic acid (DHA) circulates in mammals in lipoproteins and bound to serum albumin as a nonesterified fatty acid as well as esterified in lysophosphatidylcholine (lysoPC). 1-Lyso,2-DHA-glycerophosphocholine (GPC) is an unstable isomer because of a primary alcohol at the sn-1 position. To keep DHA at the sn-2 position of lysoPC, its usual position for the corresponding lysoPC to be acylated into PC in tissues, we synthesized 1-acetyl,2-DHA-GPC and confirmed its structure by use of nuclear magnetic resonance (NMR) spectroscopy in comparison with its positional isomer, 1-DHA,2-acetyl-GPC. 1-Lyso,2-DHA-GPC was prepared from 1-stearoyl,2-DHA-GPC by enzymatic hydrolysis and purified by high-performance liquid chromatography. The isomerization of 1-lyso,2-DHA-GPC into 1-DHA,2-lyso-GPC was obtained by keeping the former overnight at room temperature under nitrogen. Both lysoPC isomers were acetylated by acetic anhydride into 1-acetyl,2-DHA-GPC and 1-DHA,2-acetyl-GPC, respectively, and the resulting phospholipids were fully characterized by NMR. In particular, the 1,2 substitution pattern of the acetyl and DHA chains could be easily detected by 2D heteronuclear multibond correlation. We conclude that 1-acetyl,2-DHA-GPC might be considered as a stable form of 1-lyso,2-DHA-GPC for its delivery to tissues, if the latter exhibits acetyl hydrolase activity.     

Retention behavior of trans isomers of eicosapentaenoic and docosahexaenoic acid methyl esters on a polyethylene glycol stationary phase

A polyethylene glycol (PEG) stationary phase was evaluated for the separation of mono‐trans isomers of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) methyl esters. The resolution patterns were compared to patterns achieved with previously applied conditions on a cyanopropyl phase. There were no overlaps between all‐cis EPA/DHA and their mono‐trans isomers on the PEG phase. Because of overlap between 22:0 and 22:1 isomers, the PEG column is not a good choice for analyses of EPA trans isomers in crude fish oils. However, if the saturated and monounsaturated fatty acids are not present in significant amounts, PEG can be a better choice than cyanopropyl columns.     

Higher Serum EPA or DHA, and Lower ARA Compositions with Age Independent Fatty Acid Intake in Japanese Aged 40 to 79

Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are the predominant long-chain polyunsaturated fatty acids (PUFA) among membrane phospholipids in the mammalian brain and neural tissues. This cross-sectional study examined age effects on serum eicosapentaenoic acid (EPA), DHA, and ARA compositions assessed with reference to dietary intakes among 1,014 Japanese men and 1,028 Japanese women aged 40–79 years. Venous blood was collected early in the morning after at least 12-h fasting. Serum fatty acid (FA) compositions were expressed as molar percentages of the total FA (mol% of total). Diet was assessed using a 3-day dietary record that included photographs. Participants were categorized into groups by sex and age (40–49, 50–59, 60–69, and 70–79 years). Intakes of fish, EPA, and DHA tended to increase with age. Significant positive correlations between serum FA composition and the corresponding weight percentage of total FA intake were observed for EPA and DHA in all sex and age groups, and for ARA among females in their 40s. Serum EPA and DHA compositions were higher, while ARA decreased with age, and these associations remained consistent even after adjusting for corresponding FA intake. These results suggest potential effects of age on differences in blood EPA, DHA, and ARA compositions, independent of corresponding FA intake among community-dwelling Japanese men and women.     

Fatty acid and conjugated linoleic acid isomer profiles in human milk fat

The fatty acid composition of 39 mature human milk samples from four Spanish women collected between 2 and 18 weeks during lactation was studied by gas chromatography. The conjugated linoleic acid (CLA) isomer profile was also determined by silver‐ion HPLC (Ag⁺‐HPLC) with three columns in series. The major fatty acid fraction in milk lipids throughout lactation was represented by the monounsaturated fatty acids, with oleic acid being the predominant compound (36–49% of total fatty acids). The saturated fatty acid fraction represented more than 35% of the total fatty acids, and polyunsaturated fatty acids ranged on average between 10 and 13%. Mean values of total CLA varied from 0.12 to 0.15% of total fatty acids. The complex mixture of CLA isomers was separated by Ag⁺‐HPLC. Rumenic acid (RA, cis‐9 trans‐11 C18:2) was the major isomer, representing more than 60% of total CLA. Trans‐9 trans‐11 and 7‐9 (cis‐trans + trans‐cis) C18:2 were the main CLA isomers after RA. Very small amounts of 8‐10 and 10‐12 C18:2 (cis‐trans + trans‐cis) isomers were detected, as were different proportions of cis‐11 trans‐13 and trans‐11 cis‐13 C18:2. Although most of the isomers were present in all samples, their concentrations varied considerably.     

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.     

Rubber substrates and their influence on isomerization of conjugated dienes in pheromone dispensers

Release rate and degree of isomerization of pheromones with conjugated double bonds were studied in dispensers prepared from several rubber substrates. The substrates compared were made of rubber, cured with elemental sulfur or accelerators based on organic sulfur compounds or organic peroxides. Isomerization of the double bonds occurs immediately after impregnation of the substrate, and the degree of isomerization increases during field use and/or storage. The propensity of the isomers to isomerize corresponds to their proportion in the equilibrium mixture. AnE,Z isomer is isomerized faster than theE,E isomer, and finally a near-equilibrium mixture of the four isomers is present. Minimal isomerization was found in non-sulfur-cured substrates which are the material of choice.     

EPA and DHA Levels in Whole Blood Decrease More Rapidly when Stored at −20 °C as Compared with Room Temperature, 4 and −75 °C

High‐throughput n‐3 fatty acid profiling is enabled by collection techniques such as venous whole blood and fingertip prick (FTP) sampling, but the resulting increased sample numbers increases storage demand. Highly unsaturated fatty acids (HUFA) in erythrocytes are susceptible to oxidation, but this tendency is poorly characterized in venous and FTP whole blood. Presently, whole blood samples with low and high n‐3 content collected with ethylenediaminetetraacetic acid were stored on chromatography paper with and without BHT pre‐treatment for up to 180 days at different temperatures (room, 4, ?20, ?75 °C). Whole blood prepared with heparin and BHT and stored in cryovials was also examined. Eicosapentaenoic acid (EPA, 20:5n‐3) + docosahexaenoic acid (DHA, 22:6n‐3) is relatively stable when stored at ?75 °C under various conditions but rapidly decreases in whole blood when stored at ?20 °C. At ?20 °C, BHT + heparin prepared whole blood can prevent decreases in cryovials up to 180 days but BHT only slows the decreases on chromatography paper. Surprisingly, whole blood stored at 4 °C and room temperature was less susceptible to decreases in EPA + DHA as compared with ?20 °C storage. Assessments of n‐3 blood biomarkers indicate the % n‐3 HUFA in total HUFA was more stable as compared with the sum of the relative % of EPA + DHA. In conclusion, FTP and venous whole blood for fatty acid analysis should be stored at ?75 °C whenever possible. In the absence of ?75 °C storage conditions, BHT should be added and 4 °C or room temperature appear to be better alternatives to ?20 °C.     

Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans

The aim of this study was to determine whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), or both, were responsible for the triglyceride (TG)-lowering effects of fish oil. EPA (91% pure) and DHA (83% pure), a fish oil concentrate (FOC; 41% EPA and 23% DHA) and an olive oil (OO) placebo (all ethyl esters) were tested. A total of 49 normolipidemic subjects participated. Each subject was given placebo for 2–3 wk and one of the n-3 supplements for 3 wk in randomized, blinded trials. The target n-3 fatty acid (FA) intake was 3 g/day in all studies. Blood samples were drawn twice at the end of each supplementation phase and analyzed for lipids, lipoproteins, and phospholipid FA composition. In all groups, the phospholipid FA composition changed to reflect the n-3 FA given. On DHA supplementation, EPA levels increased to a small but significant extent, suggesting that some retroconversion may have occurred. EPA supplementation did not raise DHA levels, however, FOC and EPA produced significant decreases in both TG and very low density lipoprotein (VLDL) cholesterol (C) levels (P<0.01) and increases in low density lipoprotein (LDL) cholesterol levels (P<0.05). DHA supplementation did not affect cholesterol, triglyceride, VLDL, LDL, or high density lipoprotein (HDL) levels, but it did cause a significant increase in the HDL₂/HDL₃ cholesterol ratio. We conclude that EPA appears to be primarily responsible for TG-lowering (and LDL-C raising) effects of fish oil.     

Determination of Geometrical Isomers of 9,12-Octadecadienoic Acid Present in Food Fats

The present work presents a method to be used in the determination of different geometrical isomers of 9,12-octadecadienoic acid present in food fats. The method is based on isotope dilution, the radioactive isomers needed being prepared by isomerization of cis,cis-9,12-octadecadienoic acid. Radioactivity is acquired through esterification of the isomers by ¹⁴C-diazomethane. For the identification of the isomers infra-red spectrophotometry, alkali isomerization combined with ultraviolet spectrophotometry, gas chromatography, and ozonolysis are used. The isomer to be determined is separated from other fatty acids by argentation thin layer chromatography. The standard deviation of the method was in 15 parallel analyses 0.04%. The sample of food fat to be examined was milk fat, the cis,cis-9,12-octadecadienoic acid content obtained being 0.83%, which corresponded to about one half of octadecadienoic acids of the sample. When a fatty acid mixture with a known composition was analyzed, the cis,cis-9,12-octadecadienoic acid yield was 99.7% on the average, the standard deviation being 0.04%.     

Fractionation of squid visceral oil ethyl esters by short-path distillation

Squid visceral oil contains high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Its ethyl esters were fractionated by short-path distillation in this study. The elimination temperatures of squid visceral oil ethyl esters (SVOEE) ranged from 50 to 140°C, increasing with the carbon number of ethyl esters. The elimination temperature of cholesterol was higher than those of SVOEE. The SVOEE of Illex argentinus (SVOEE-A) was more advantageous as the raw material (feed) than that of Ommastrephes bartrami (SVOEE-B) for the isolation of EPA and DHA, because SVOEE-A contained less 20∶1 and 22∶1. When SVOEE-A originally containing 9.0% EPA, 14.7% DHA, and 1,121 mg/100 g of cholesterol was distilled from 50 to 150°C with 20°C interval, the 130°C distillate could give 15.5% EPA and 34.7% DHA with 99 mg/100 g of cholesterol, and the yield was 21.8%. The 150°C distillate could give 43.1% DHA with 496 mg/100 g of cholesterol. Furthermore, the distillates collected from 110 to 150°C contained 24.4 to 50.2% of EPA plus DHA, and their total yield was 58.3%. The final residue after 150°C distillation contained 77% of the total cholesterol in the initial SVOEE-A, and the yield was 6.0%.