Nonequilibrium transitions in thermotropic phases of eicosenoic acid methyl esters

Methyl esters ofcis-5-eicosenoic (5-EAME) andcis-11-eicosenoic (11-EAME) acids from the seed oil ofLimnanthes alba (Meadowfoam) exhibit a degree of thermotropic polymorphism unobserved with shorter and longer chainlength monoenic methyl esters. 5-EAME freezes at 264 K and melts at 266 K if cooled no lower than 215 K. 11-EAME freezes at 239 K and melts at 255 K if cooled at no lower than 240 K. Solids cooled to lower temperatures undergo phase transformation to highermelting polymorphs (274 K, 5-EAME; 262 K, 11-EAME), and samples often exhibit double melting endotherms. Quantities of each high-melting phase vary with time at temperatures below characteristic initiation temperatures. Highly temperature-sensitive phase conversions suggest low temperature nucleation, followed by crystal growth and conversion, as reheating allows molecular motion. Once formed, both high-melting phases melt with essentially the same melting entropy. Thermodynamic and kinetic analyses imply that differences exhibited by the isomeric esters derive from aliphatic configuration distal to the double bond.     

The eicosenoic acid of cameline seed oil

Summary The eicosenoic acid previously discovered in cameline seed oil (1) was isolated and identified. It melts at 22.5°C. and yields an elaidinated acid melting at 43.5°C. and a dioxy derivative melting at 132°C. By its hydrogenation to arachidic acid and destructive oxidation to undecanedioic acid it was shown to be normal Δ¹¹-eicosenoic acid-1 like the acid in jojoba wax.     

The effect of dietary erucic acid on cardiac triglycerides and free fatty acid levels in rats

Male Sprague-Dawley rats, 3 weeks of age, were fed semisynthetic diets containing test oils at 20% by weight for 3 days, 1 week, and 16 weeks. The test oils contained up to 22.3% erucic acid. Growth retardation was evident in rats fed rapeseed oil high in erucic acid, and soybean oil and Tower rapeseed oil diets containing about 5% erucic acid. Cardiac triglyceride accumulation was found in rats fed diets containing about 5% erucic acid but not in rats fed Tower rapeseed oil which contains 0.2% of this acid. The cardiac free fatty acid levels were low, 50–100 μg/g of wet heart tissue, and were not affected by feeding diets containing about 5% erucic acid. Feeding a diet containing a high erucic acid rapeseed oil did result in higher free fatty acid levels but only at 3 days and 1 week; the level at 16 weeks was similar to the other oils. The fatty acid analysis of cardiac triglycerides and free fatty acids showed high percentages of erucic acid at 3 days and 1 week; at 16 weeks these levels had declined significantly. The results indicate that the accumulated erucic and eicosenoic acids, at 3 days and 1 week, accounted for the increase in cardiac free fatty acids when rats were fed the high erucic acid rapeseed oil. There appears to be no evidence that the early cardiac triglyceride or free fatty acid accumulation is related to the formation of the long term myocardial lesions. Contribution No. 739 Animal Research Institute.     

Effects of dietary saturated fat on erucic acid induced myocardial lipidosis in rats

Male Sprague-Dawley rats were fed for one week diets containing 20% by weight fat/oil mixtures with different levels of erucic acid (22∶1n−9) (∼2.5 or 9%) and total saturated fatty acids (∼8 or 35%). Corn oil and high erucic acid rapeseed (HEAR) oil were fed as controls. The same hearts were evaluated histologically using oil red O staining and chemically for cardiac triacylglycerol (TAG) and 22∶1n−9 content in cardiac TAG to compare the three methods for assessing lipid accumulation in rat hearts. Rats fed corn oil showed trace myocardial lipidosis by staining, and a cardiac TAG content of 3.6 mg/g wet weight in the absence of dietary 22∶1n−9. An increase in dietary 22∶1n−9 resulted in significantly increased myocardial lipidosis as assessed histologically and by an accumulation of 22∶1n−9 in heart lipids; there was no increase in cardiac TAG except when HEAR oil was fed. An increase in saturated fatty acids showed no changes in myocardial lipid content assessed histologically, the content of cardiac TAG or the 22∶1n−9 content of TAG at either 2.5 or 9% dietary 22∶1n−9. The histological staining method was more significantly correlated to 22∶1n−9 in cardiac TAG (r=0.49;P<0.001) than to total cardiac TAG (r=0.40;P<0.05). The 22∶1n−9 content was highest in cardiac TAG and free fatty acids. Among the cardiac phospholipids, the highest incorporation was observed into phosphatidylserine, followed by sphingomyelin. With the addition of saturated fat, the fatty acid composition showed decreased accumulation of 22∶1n−9 and increased levels of arachidonic and docosahexaenoic acids in most cardiac phospholipids, despite decreased dietary concentrations of their precursor fatty acids, linoleic and linolenic acids.     

Polymorphic behavior of erucic acid

Polymorphic behavior of erucic acid was examined by differential scanning calorimetry (DSC), X-ray diffractometry (XRD) and optical microscopy. Four individual polymorphs were observed, α₁, γ₁, α and γ. The single crystals of α and α₁ were obtained from acetonitrile solutions. DSC and XRD studies exhibited reversible transformations between α and γ, and between α₁ and γ₁. The two transformations were characterized by an order (γ and γ₁)-disorder (α and α₁) transformation due to melting of the aliphatic chain between acis double bond and a methyl end group, as revealed in α and γ of oleic acid. The thermodynamic stability among the four polymorphs was determined based on the transformation features and the solubility data. α₁ is most stable above 25.9 C, melting at 34.0 C, α is most stable in a range of temperature between −1.0 and 25.9 C, transforming in a crystalline state to α₁ at 31.2 C on heating. γ is most stable below −1.0 C. It can be concluded that the polymorphism of erucic acid is different from that of oleic acid with respect to the thermal and structural behaviors, although some similarities are revealed. Presented at the AOCS-JOCS joint meeting in Hawaii in 1986 and at the AOCS Annual Meeting in New Orleans in 1987.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Synthesis and properties of erucic acid triacylglycerols

The chemical synthesis of high-erucic acid triacylglycerols by a direct esterification approach was investigated in this study. A two-stage process was adopted in which reactants (without any catalyst) were heated at 160±5°C for 4 h, followed by heating at 250±10°C for 8 h. Purification of the esterified product was achieved by alkali refining, followed by alumina column chromatography. A greater than 85% yield of pure triacylglycerols was obtained, which contained ≈90% erucic acid, by using a 5% molar excess of erucic acid in the reaction. Oils containing erucic acid ranging from 45 to 91% were prepared by chemically interesterifying native high-erucic acid rapeseed (HEAR) oil and the synthetic high-erucic acid triacylglycerols. The melting point, cloud point, pour point, titer and viscosity of all oils exhibited positive correlations with the erucic acid content, whereas saponification value, iodine value and refractive index showed negative correlations. Randomization of native HEAR oil resulted in an increase in the melting point, cloud point and pour point.     

Purification of erucic acid by low-temperature crystallization

Purification of crucic acid for laboratory use by low-temperature crystallization from aqueous acetone, ethanol, and methanol has been investigated. Two crystallizations of technical grade commercial acid (76–86%) from acetone-water (5∶1) provided products of 94–98% purity, depending on the original composition; residual impurities were primarily C₂₀ monoene and C₂₂ saturated acids. A laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, USDA.     

Brassidic acid: Preparation from erucic acid and mechanism of elaidinization

Brassidic acid was prepared by elaidinization of 95% erucic acid with 4 mole % nitrous acid at 70 C for 30 min, followed by crystallization from 95% ethanol. Yield was 70%, and purity was 96–97% by gas liquid chromatography and thin layer chromatography. The isomerization reaction was monitored by IRtrans absorption for optimal reaction rate and yield. There was no migration of the double bond. The NMR spectrum of thetrans protons was wide and complex with a chemical shift of 5.34 δ. The nitrous acid elaidinization, generally explained as a free radical process, is believed to be induced initially by the nitrogen dioxide anion (nitrite) and followed immediately by complex formation between the excited triplet anion and the olefin. The complex rotates to the opposite geometric configuration driven by a spin-orbital coupling process. N. Market. Nutr. Res. Div., ARS, USDA.     

Crismer values and erucic acid contents of rapeseed oils

Crismer Values of rapeseed oil extracted from different Canadian varieties are reported. Seventeen samples of oil containing up to 4.1% erucic acid gave an average value of 68.45±0.92 C with a range of 67.10 to 69.29. Crismer Values of high erucic acid oils (20~45% erucic acid) ranged from 76 to 82 C. Contribution No. 317 from the Food Research Institute, Agriculture Canada, Ottawa, Ontario K1A OC6     

Antimicrobial properties of some erucic acid-glycolic acid derivatives

Twelve fatty acid derivatives of glycolic acid containing erucic acid, or other selected vegetable oil fatty acids, were synthesized. These were screened for antimicrobial activity against a gram-positive bacterium,Staphylococcus aureus; a gram-negative bacterium,Escherichia coli; a mold,Penicillium notatum, and a yeast,Candida utilis. All of the compounds inhibited at least one of these organisms. These compounds were derivatives of glycolic acid prepared by esterification of the carboxyl function of glycolic acid with a long-chain fatty alcohol and reaction of the hydroxyl function of glycolic acid with a long-chain fatty acyl group.     

Preparation and characterization of surface active erucic acid derivatives

Nonionic, cationic and anionic surfactants, derivatives ofcis-13-docosenoic acid (erucic acid), have been prepared and characterized, and their performance has been evaluated and compared with the corresponding derivatives of fatty acids with shorter alkyl chain length. Nonionic erucic acid ethoxylates give a solution behavior anticipated from the hydrophilic-lipophilic balance of the molecule; however, the increased molecular size as compared to ordinary surfactants results, e.g., in higher temperature stability of the surfactant aggregates. Anomalous solution behavior was found and investigated for anionic surfactants, triethanolammonium salts of erucic acid, and some shorter homologues. The effects are discussed in terms of the acid-base equilibria of the alkanolammonium counterion and the acid, together with effects due to the molecular size of the counterion.     

Metabolism of14C-labelled oleic acid,erucic acid and nervonic acid in rats

1-¹⁴C-Oleic acid, 2-¹⁴C-erucic acid and 2-¹⁴C-nervonic acid were administered to rats by tail-vein and the distribution of radioactivity in liver lipids was determined at intervals from 15 min to 6 hr after injection. High levels of activity were found after short time intervals which were mainly associated with triglycerides in the case of oleic acid and with free fatty acids in the case of erucic acid and nervonic acid. The activity in these lipids decreased with time and was later exceeded by that in more polar lipids. In rats given erucic acid or nervonic acid, sphingolipids were more highly labelled than glycerophosphatides. Nervonic acid showed little tendency to form a complex with serum albumin and erucic acid complexed less readily than palmitic acid. Presented at the AOCS Meeting in Houston, April 1965.     

Processing of crambe for oil and isolation of erucic acid

Crambe seed (Crambe abyssinica) is an excellent, recently established source of high-erucic acid oil. Erucic acid has a number of important and potential applications. To develop this potential, a rapid bench-scale method was desired whereby purified erucic acid in up to several 100-g quantities could be produced from crambe seed. Using the method developed, oil was expressed from dried, intact seed; clarified, degummed, and bleached; and saponified and acidified to obtain the free fatty acids. Analysis by inductively coupled plasma of the free fatty acids showed negligible levels of phosphorus and most minerals. Erucic acid was twice crystallized from 95% ethanol at −14°C, resulting in a purity of 87.1%. This process yielded 365 g erucic acid crystals per kg bleached oil. Nuclear magnetic resonance analysis showed that the prepared erucic acid had an excellent pattern of correlation with a commercial standard. The time needed to convert 1 kg of crambe seed to erucic acid is about 48 h. Crystal filtration and drying stages under the current process conditions require 30% of the overall time. The method is suitable for producing adequate quantities of erucic acid for use in studies of its bench-scale conversion. There is obviously, still, a fruitful field of work to be explored in the formalization of refining procedures for crambe oil. It seems that crambe is destined to continue expansion into the high-erucic acid oil markets.     

Relationship between erucic acid and myocardial changes in male rats

The back and belly fat of pigs fed a diet containing 20% by wt rapeseed oil (22% erucic acid) for 16 weeks was rendered into oil. This rendered pig fat, which contained 5.6% erucic acid, was fed to male rats in three separate experiments at 20% by wt of the diet for 16 weeks. In experiment I rendered pig fat was compared only toBrassica campestris var. Span rapeseed oil containing 4.8% erucic acid. In experiments II and III, rendered pig fat was compared to commerical lard containing 0.2% docosenoic acid, commercial lard to which 5.4% free erucic acid was added, and Span rapessed oil. There was no significant (P<0.01) differences observed in the level of erucic acid in the hearts of rats fed diets of rendered pig fat, Span rapeseed oil, or commercial lard plus erucic acid. However, the incidence (P<0.001) and severity (P<0.01) of cardiac lesions were significantly higher in Span rapeseed oil fed rats compared to rats fed control diets. The number of rats affected or the severity of lesions in the rendered pig fat fed group was not significantly different from controls. The results of this study indicate that the myocardial lesions associated with feeding 20% rapeseed oil diets are not related to the content of erucic acid per se. The possible reasons why rapeseed oil causes cardiac lesions in rats are discussed. It is suggested that a triglyceride imbalance in the oil might play an important role in causing these lesions in rats. Contribution No. 585, Animal Research Institute, Agriculture Canada, Ottawa, Canada, K1A 0C6.     

Enrichment of eicosenoic and docosadienoic acids fromLimnanthes oil

The long chain (>C₁₈) monoene and diene acids ofLimnanthes oil are useful in synthesizing diene and tetraene wax ester intermediates for prospective lubricant additives and PVC plasticizers. Low-temperature crystallization of theL. alba acids (61% eicosenoic, 19% docosadienoic, and 20% others) in acetone (initial concentration=0.05 g/ml) at −50 C enriches eicosenoic acid (74%) in the precipitated fraction while concentrating docosadienoic (70%) in the supernatant fraction. This simple and efficient process is well suited for large-scale laboratory separations.     

Gas-liquid chromatography of triglycerides from erucic acid oils and fish oils

By critically selecting optimum operating conditions, quantitative gas-liquid chromatography of triglycerides has been extended to molecules containing substantial amounts of C₂₀, C₂₂, and C₂₄ fatty acids. The triglycerides of four erucic acid oils (water cress, rapessed, nasturtium, andLunaria annua) and two fully hydrogenated fish oils (menhaden and tuna) have been quantitatively analyzed by this technique. The average fatty acid chain length calculated from the triglyceride composition of each oil agreed closely with that determined by GLC of its respective methyl esters. Several conclusions about the triglyceride composition of the fats analyzed are discussed. Winner, AOCS Bond Award. Presented at the AOCS Meeting in Cincinnati, October 1965.     

Lipid composition and erucic acid in rat liver cells in culture

Erucic acid (Δ¹³-docosenoic acid) was added to fetal calf serum, then fed to rat liver epithelial cells in culture, and uptake measured at intervals over 24 hr. During the first 6 hr. of incubation, uptake of the docosenoic acid was 21 nmoles/hr/mg protein in 7-day cells, and 15 mmoles/hr/mg protein in 14-day cells. Of¹⁴C-labeled erucic acid taken up by the cells in 24 hr, radioactivity measurements showed 60% of the total lipid¹⁴C activity derived from [1-¹⁴C] 22∶1 in neutral lipid (NL) and 40% in phospholipid (PL); whereas 55% of lipid¹⁴C activity was in NL and 45% in PL when the substrate was [14-¹⁴C] 22∶1. Within the NL fraction, 75% of¹⁴C activity derived from [1-¹⁴C] 22∶1 was in triglyceride (TG) and 11% in cholesterol (CHL), while 79% was in TG and 6.5% in CHL when the substrate was [14-¹⁴C] 22∶1. Triglycerides and cholesteryl esters accumulated in the cells during incubation with erucic acid. Among phospholipids separated by thin layer chromatography, 75% of¹⁴C activity was in lecithin (PC), 10% in phosphatidylethanolamine (PE), 5% in sphingomyelin (SPH), and 1% or less in cardiolipin (DPG). The highest specific activity (SA) was in PC, followed by SPH and PE. Incubation with erucic acid altered fatty acid composition of PC, PE, and SPH, although amounts of phospholipids were unaffected. Gas liquid chromatography analyses detected 18% erucic acid in PC, 2% in PE, and 4–5% in SPH.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.