Fatty acid metabolism in the calanoid copepodParacalanus parvus: 1. Polyunsaturated fatty acids

The metabolic fate of radioactive linoleate and α-linolenate administered to the South Atlantic copepodParacalanus parvus was studied. The wild copepod was able to incorporate the labeled acids dissolved in seawater. The radioactive linoleate was elongated to 20∶2ω6 and 22∶2ω6 and desaturated by a Δ6 desaturase to 18∶3ω6. α-Linolenate was also desaturated by a Δ6 desaturase to 18∶4ω3 and elongated to 20∶3ω3. The copepod was able to convert α-18∶3 to 20∶5ω3 and 22∶6ω3. Member of the Carrera del Investigador Científico of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Member of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Cientificas y Tècnicas.     

Adsorption analysis of lipids. III. Synthetic mixtures of ethyl stearate,oleate, linoleate,and linolenate

Summary The adsorption analysis of six binary mixtures composed of ethyl stearate, ethyl oleate, ethyl linoleate, and ethyl linolenate has been studied on alumina columns. Distillation methods have been shown by other workers to fractionate the stearate-oleate system but not the oleate-linoleate system. Application of the adsorption analysis technique resulted in fractionation of all six systems including the oleate-linoleate mixture. Measures of the performance of the columns were obtained by dividing the eluate into two portions at the point in the fractionation at which the starting composition was reached. The first component to be eluted from each of the six pairs was obtained in the first fraction with an average purity of between 68 and 83%. The second component was obtained in the second fraction with average purity of 61 to 76%. The first component to be eluted was obtained in higher purity than the second. The efficiency of separation of pairs appeared to depend on the difference in degree of unsaturation of the components. Overall recoveries of esters from the columns ranged from 71.8 to 87.5%. Presented at the 21st fall meeting of the American Oil Chemists' Society, Oct. 20–22, 1947, Chicago, Illinois. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Kinetics of the reaction of isopropenyl acetate and stearic acid to form isopropenyl stearate

A mathematical kinetics model has been developed for the reaction of stearic acid with isopropenyl acetate to form isopropenyl stearate. Though the reaction is reversible, the kinetics can be described mathematically most simply by a pseudo first order irreversible reaction model. The model predicts the rate of formation of isopropenyl stearate as a function of time, temperature, catalyst concentration and equilibrium concentration of isopropenyl stearate. Equilibrium is determined from an empirical equation as a function of the ratio of the original concentration of the reactants. Temperature has little effect on the equilibrium. E. Market. and Nutr. Res. Div., ARS, USDA.     

Fatty acid metabolism inDrosophila melanogaster: II. Metabolic origin of monoenes

Evidence is presented thatDrosophila larvae produce monounsaturated fatty acids by two independent pathways. One of these pathways, involving the direct desaturation of long chain precursors, is sensitive to inhibition by linoleate. The other pathway is resistant to linoleate inhibition and probably has tetradecnoate-Δ⁵ cis and tetradecnoate-Δ⁷ cis as intermediates in the synthesis of palmitoleate and oleate, respectively. The use of radioactive precursors of varying chain length and labeled at different positions in the carbon chains provided evidence for the isolation and structure of intermediates involved in the linoleate resistant pathway.     

High-pressure phase equilibrium measurements and thermodynamic modeling for the systems involving CO2, ethyl esters (oleate,stearate, palmitate) and acetone

The aim of this work was to study the phase behavior of systems involving carbon dioxide (CO₂), fatty acid ethyl esters (ethyl oleate, ethyl stearate and ethyl palmitate) and acetone at high pressures. The phase behavior involving these components is an important step regarding the design and optimization of industrial processes based on supercritical conditions, such as biodiesel production and fatty esters fractionation involving supercritical and/or pressurized solvents. In addition, supercritical CO₂ can offer an interesting alternative for glycerol separation in water-free biodiesel purification processes. The binary systems investigated in this work were CO₂ + ethyl oleate, and CO₂ + ethyl stearate and these were compared with the CO₂ + ethyl palmitate system. The ternary CO₂ + ethyl palmitate + acetone was also investigated at two different ethyl palmitate to acetone molar ratios of (1:1) and (1:3). The static synthetic method using a variable-volume view cell was employed to obtain the experimental data in the temperature range of 303.15–353.15 K. Vapor–liquid (VL), liquid–liquid (LL) and vapor–liquid–liquid (VLL) phase transitions were observed in these systems. In the binary systems, the solubility increased with the presence of unsaturation and decreased with the number of carbon atoms in the fatty ester chain. Addition of acetone as well as ethanol eliminated the liquid–liquid immiscibility and reduced the pressure transitions, therefore increasing the solubility of the ester in supercritical CO₂. The experimental data sets for the binary and ternary systems were successfully modeled using the Peng–Robinson equation of state with the classical van der Waals quadratic mixing rule (PR-vdW2) and Wong-Sandler (PR-WS) mixing rule. Both models showed good performance in the phase equilibrium correlations and in predictions for the binary and ternary systems.     

Terpolymers. III. Isochronal viscoelastic parameters for terpolymers of vinyl stearate,vinyl acetate,and vinyl chloride

Isochronal viscoelastic parameters were collected for many of the copolymers, terpolymers, and diluent mixtures whose mechanical properties at ambient temperatures were reported in the preceding paper. In the polymeric systems, vinyl stearate, acting as the primary internal plasticizer, was introduced into terpolymers by displacing vinyl acetate from base copolymers of vinyl acetate and vinyl chloride, across the range of composition. In the diluent mixtures, poly(vinyl chloride) was plasticized by di-2-ethylhexyl phthalate across the range of compositions. For direct comparison with the mixtures, vinyl chloride was plasticized by copolymerization with vinyl stearate across the same range of compositions. Moduli for the co- and terpolymers reached the low values characteristic of soft materials at room temperature only through a short range of vinyl stearate composition. At higher internal plasticizer compositions, side-chain crystallization stiffened the samples and raised their moduli. In contrast, moduli for the mixtures decreased steadily with increase in diluent at ambient temperature. The effective use temperature ranges were narrow for the co- and terpolymers but broad for the mixtures. Curve broadening was similar for both types of systems, but reached a maximum at about 40 weight-% plasticizer for the diluent mixtures. The slopes of the glassy modulus with decreasing temperature at 50°C below T_g for the vinyl stearate copolymers were relatively large. However, moduli close to that of poly(vinyl chloride) were reached only near the temperature range associated with the γ-transition. Consequently, this behavior was attributed to motions of the side chains in the glassy matrix. Room temperature moduli, which could be obtained before the onset of melting, were correlated with the fractional side-chain crystallinity for polymers having a high vinyl stearate content. From this relation, the modulus for the hexagonal crystal form of the side-chain crystallites of poly(vinyl stearate) was estimated to be 1.2×10¹⁰ dynes/cm². Moduli for the glassy amorphous phase of this same polymer appeared to have one sixth of this value at 40°C below the glass transition. The glass transition temperature occurred about 10° below the inflection temperature at 10⁹ dynes/cm², as an average for all of the systems studied.     

Fatty acid metabolism in young oysters,Crassostrea gigas: Polyunsaturated fatty acids

Tetraselmis suecica andDunaliella tertiolecta were grown for 24 hr in the presence of¹⁴C sodium bicarbonate and then fed separately to batches of juvenile oysters,Crassostrea gigas, for 3 days.D. tertiolecta contained fatty acids no longer than C₁₈; 22∶6ω3 was absent inT. suecica. Analysis of the oyster fatty acids by radio gas chromatography (GC) showed that oysters were able to incorporate some of the dietary¹⁴C label into long-chain fatty acids not supplied in the diet, e.g., C₂₀ and C₂₂ mono- and polyunsaturated fatty acids, and particularly 20∶5ω3. However, the low¹⁴C incorporation into fatty acids longer or more unsaturated than those supplied in the diet suggests that elongation and desaturation activity in young oysters is not sufficient to sustain optimum growth.     

Terpolymers. II. Mechanical properties and transition temperatures of terpolymers of vinyl stearate,vinyl acetate,and vinyl chloride

Vinyl stearate was studied as a major internal plasticizer in terpolymers containing vinyl acetate and vinyl chloride. The terpolymers were prepared by systematically replacing vinyl acetate by close increments of vinyl stearate starting with combinations of vinyl acetate and vinyl chloride, in increments, over all compositions. For comparison of properties, a complete range of copolymers of vinyl stearate and vinyl chloride, as well as mixtures of poly(vinyl chloride) and di-2-ethylhexyl phthalate (DOP) were also made. The external plasticizer was more efficient in reducing the glass temperature than was vinyl stearate. The decline in T_g with weight fraction of plasticizer was linear for the copolymers and terpolymers but concave downward with the liquid diluent. The linear decline was shown to involve mere additivity of the free volume contributed by each side-chain methylene (or methyl) group in both vinyl esters to reducing T_g. The mechanism of the diluent system was more complex. However, the magnitude of the reduction of tensile modulus at a given weight fraction of DOP could be equaled or exceeded by the same amount of vinyl stearate, by increasing the vinyl acetate content of the base copolymer to 40 mole-% or more. Unfortunately, the ultimate strengths and elongations of internally plasticized systems were reduced more than those of the mixtures at comparable compositions. Vinyl stearate was found to markedly retard photolytic degradation compared to both vinyl acetate and the external plasticizer in unstabilized samples having nearly the same thermal treatment. The effect was greater than could be ascribed to dilution by the long alkyl group. The production of a stearoyl radical more stable than the radicals initiating dehydrochlorination is suggested as a possible mechanism.     

The oxidation of methyl oleate. II. A reaction between methyl hydroperoxido oleate and oleic acid

Oleic acid is intermolecularly oxidized by methyl hydroperoxido leate at 90°C, through the addition of oxygen at the olefinic bond with the formation of opexy and dihydroxy compounds. Only the lowmelting isomeric form of dihydroxystearic acid could be isolated from the reaction mixture. The oxidation of olefinic linkages by hydroperoxides appears to account, at least in part, for the observed reduction of unsaturation in autoxidizing fats. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Evaluation of butyl stearate,butyl oleate,butyl ricmoleate,and methyl lmoleate as poly(vinyl chloride) plasticizers

An investigation of butyl stearate, butyl oleate, butyl rieinoleate, and methyl lmoleate was made to determine the extent of their compatibility with poly(vinyl chloride) and their plasticizing effects on the physical, properties of the resulting molded sheets. These monoesters were evaluated as the sole plasticizer and in combination with DOP. The results showed the materials to have limited compatibility in general, being less than 10% of the total mix. In combination with DOP these compounds were found to improve the plasticizer efficiency, color, low temperature flexibility, and heat stability of the molded poly (vinyl chloride) sheets over those containing DOP as the sole plasticizer. Tensile strength, per cent elongation, and light stability properties were essentially comparable to those found for pure DOP.     

Fatty acid metabolism and cell proliferation. VII. Antioxidant effects of tocopherols and their quinones

The antioxidant capacities of α- and γ-tocopherols (α-E and γ-E) and their quinones (α-EQ and γ-EQ) were determined in non-biological and biological systems. The non-biological system consisted of arachidonic acid [20∶4 (n−6)], the oxidant cumene hydroperoxide, and a Fe³⁺ catalyst to facilitate malondialdehyde (MDA) formation from lipid peroxides. α-E and γ-E had similar antioxidant capacities in this system. α-EQ also functioned as an antioxidant, while γ-EQ exhibited a crossover effect by functioning as an antioxidant at low concentrations and a prooxidant at high concentrations. Biological lipid peroxidation in smooth muscle cells challenged with 20∶4 (n−6) was measured both by MDA formation in confluent cultures and by cell growth in proliferating cultures. α-E, γ-E and α-EQ had similar antioxidant capacities, but γ-EQ was highly cytotoxic for cells in both confluent and proliferating cultures. Cellular retention of antioxidants was estimated indirectly from MDA formation when cells were loaded with an antioxidant (preincubation) and then incubated for varying periods of time in fresh media containing 20∶4 (n−6). Cellular retention also was measured directly with tritiated α-E and tritiated αEQ. These studies showed that cellular retention decreased in the sequence γ-E>α-E>α-EQ. Thus, cellular retention does not explain the enhanced antioxidant capacity of α-E compared to γ-E that has been reported for animal systems. The antioxidant capacity of αE evidently is enhanced by its metabolism to a quinone which, unlike the quinone from γ-E, functions as a biological antioxidant.     

Fatty acid metabolism and cell proliferation: IV. Effect of prostanoid biosynthesis from endogenous fatty acid release with cyclosporin-A

Cyclosporin-A (Cyc-A) stimulates prostanoid (PGI₂) synthesis in confluent smooth muscle cells from guinea pig aorta through the release of endogenous fatty acid. Cyc-A, like other stimulatory agents for prostanoids, promotes smooth muscle cell proliferation and prostanoid synthesis in these proliferating cells. Indomethacin, a cyclooxygenase inhibitor, and exogenous arachidonic acid block the Cyc-A effect on cell proliferation.     

Fatty acid metabolism in the chloroplast lipids of green and blue-green algae

The pattern of uptake of radioactivity into chloroplast lipids when a green alga (Chlorella vulgaris) was incubated with sodium 2-¹⁴C-acetate differed appreciably from that obtained when two blue-green algae (Anabaena cylindrica andAnacystis nidulans) were incubated under similar conditions. The fatty acids of the digalactosyl diglyceride and sulphoquinovosyl diglyceride fractions from the blue-green algae were labeled more rapidly than were those of the corresponding fractions fromC. vulgaris, whereas the activity in the acids of the phosphatidyl glycerol fraction fromA. cylindrica andA. nidulans was relatively lower than that in the green alga. The results indicate that the metabolic behavior of chloroplast lipids may vary considerably according to the class of alga concerned. In all three alga, the evidence points to an intermediary function for the chloroplast lipids in fatty acid synthesis. Only limited exchange of acyl groups between the different chloroplast lipids seemed to occur during photoautotrophic growth.     

Fatty acid metabolism and cell proliferation. V. Evaluation of pathways for the generation of lipid peroxides

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Confluent cells at passage level 4–6 were challenged with arachidonic acid and treated with a number of antioxidants and inhibitors of specific lipid peroxidation pathways. Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) and the isolation of hydroperoxy fatty acids (HPETE) by high performance liquid chromatography (HPLC). Prostanoids were measured by radioimmunoassay and the separation of labeled compounds by HPLC. MDA, 6-keto-PGF_1α, and PGE₂ were formed when cells were challenged with arachidonic acid and these cells synthesized small amounts of one HPETE isomer, 15-HPETE. The HPETE isomers characteristic of the lipoxygenase pathway, 12-HPETE and 5-HPETE, were not detected. Furthermore, the lipoxygenase inhibitors, eicosatetraynoic acid (ETYA) and 6,7-dihydroxycoumarin (Esculetin), did not block MDA formation. These data show that MDA is not generated in the cells by a lipoxygenase pathway. The cyclooxygenase inhibitors, indomethacin and ETYA, did not block MDA formation but these agents blocked the formation of 15-HPETE. These data show both that 15-HPETE is generated by a cooxidation pathway and that 15-HPETE and cooxidation are not involved in MDA formation. Three inhibitors of cytochrome P₄₅₀ linked lipid peroxidation, 2-amino-3-ethoxycarbonyl-6-benzyl-4, 5,6,7-tetrahydrothieno-[2,3-C]-pyridine (Tinoridine), 3-methyl-1,2-di-3-pyridyl-1-propanone (Metyrapone) and phenobarbital, did not block MDA formation. These data support earlier studies that indicated that MDA is not generated by a P₄₅₀ pathway. Cells contained a bound precursor that decomposed to MDA when cells were treated with Fe³⁺. The cells exhibited autofluorescence and concentric lamellae in lipid droplets that are characteristic of ceroid-lipofuscin. These observations are consistent with lipid peroxidation through increased peroxisomal activity leading to the generation of MDA and the accumulation of ceroid-lipofuscin. The natural antioxidants, vitamin E and vitamin E quinone (EQ), and the synthetic antioxidants, butylated hydroxytoluene and nordihydroguaiaretic acid (NDGA), α-naphthol (α-N) and propyl gallate (PrGa), all blocked MDA formation in confluent smooth muscle cells, showing that these antioxidants did not function solely as specific inhibitors of lipoxygenase, cooxidation or P₄₅₀ pathways. Cell proliferation was measured in cells challenged with arachidonic acid and treated with antioxidants and other inhibitors. The least cytotoxic and most potent antioxidant, EQ, blocked MDA formation in confluent cells and promoted growth in proliferating cells when it was present in either system in the same concentration range. The synthetic antioxidants, NDGA, α-N and PrGa, blocked prostanoid synthesis and promoted growth in proliferating cells. The cyclooxygenase inhibitors, indomethacin, ETYA and Esculetin, did not enhance cell proliferation even though they were highly effective inhibitors of prostanoid synthesis. These data suggest, but do not prove, the hypothesis that cell proliferation is controlled in part by general peroxidation reactions rather than the specific peroxidation reaction involved in prostanoid synthesis.     

Lipid metabolism of the yellow clam,Mesodesma mactroides: 2-Polyunsaturated fatty acid metabolism

The fate of labeled linoleic, α-linolenic, and higher homologs of α-linolenic acid administered to the yellow clam,Mesodesma mactroides, was investigated. It was found that the clam incorporated the acids dissolved in sea water and converted 18∶2 (n−6) into 20∶2 (n−6) and 18∶3 (n−3) into 18∶4 (n−3) and 20∶3 (n−3). The addition of casein hydrolysate to the sea water increased the desaturation capacity of the clam and allowed the conversion of 18∶2 (n−6) into 18∶3 (n−6) to be demonstrated. An enhanced desaturation of 18∶3 (n−3) into 18∶4 (n−3) was also demonstrated. After 12 hr administration of the acid, no radioactivity was found in arachidonic, 20∶5 (n−3), or 22∶6 (n−3). Feeding the clams a culture ofPhaeodactylum tricornutum previously incubated with 1-¹⁴C-α-linolenic acid demonstrated that all the homologs of the α-linolenic series were found in the clam without any important changes. Six hour administration of labeled linolenic acid resulted in the incorporation of the acid into diglycerides and phospholipids. Member of the carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientificas y Tecnicas     

Lipid metabolism of the yellow clam,Mesodesma mactroides: 3-Saturated fatty acids and acetate metabolism

The fate of labeled palmitate, stearate, and acetate administered to the yellow clam,Mesodesma mactroides, was investigated. 1-¹⁴C palmitic and 1-¹⁴C stearic acids were oxidized to CO₂ to a limited extent. They were mainly incorporated in diacylglycerols and triacylglycerols and were converted to higher homologs. After administration, palmitic acid was converted to stearic and oleic acids, whereas administered stearic acid was converted to 18∶1, 18∶2, 20∶1, and 20∶2 acids. Labeled acetate was readily included by the clam in 12∶0, 14∶0, 14∶1, 15∶0, 16∶1, 16∶1, 16∶2, 18∶2, 18∶1, 18∶2, 20∶1, 20∶2, and 20∶3 acids.     

Adulterated butterfat: Fatty acid composition of triglycerides and 2-monoglycerides

Beef tallow and cottonseed oil were mixed with a pure butterfat in the ratios of 2%, 4% and 6% to obtain admixtures of beef tallow with butterfat and cottonseed oil with butterfat. The hydrolysis of individual triglycerides was carried out using the lipase to obtain 2-monoglycerides. The results indicated that butterfat had a higher percentage of C14:0 and C16:0 acids than found in the triglycerides and 2-monoglycerides of beef tallow and cottonseed oil. Beef tallow contained a higher proportion of C18:0 and C18:1 acids than butterfat and cottonseed oil triglycerides or 2-monoglycerides. Cottonseed oil had a higher percentage of C18:2 acid located in triglyceride or 2-monoglyceride than found in butterfat or beef tallow triglycerides and 2-monoglycerides. The analysis of the samples of butterfat containing 2%, 4% and 6% beef tallow revealed that the addition of beef tallow to butterfat affected the fatty acid composition of butterfat triglycerides and 2-monoglycerides with C18:0 and C18:1 acids; the effect was increased with increasing percentages of beef tallow. The addition of cottonseed oil to butterfat in the ratios of 2%, 4% and 6% affected the fatty acid composition of butterfat triglycerides and 2-monoglycerides. It was found that both C18:1 and C18:2 increased as the added cottonseed oil percentages increased.     

Fatty acid synthesis from [2-14C]acetate in normal and peroxisome-deficient (Zellweger) fibroblasts

The incorporation of [2-¹⁴C]acetate into the lipids of normal and peroxisome-deficient (Zellweger's syndrome) skin fibroblasts was examined. Most of the label was incorporated into triacyglycerol fatty acids in normal as well as Zellweger's syndrome cells. Triacylglycerols and cholesteryl esters in Zellweger's syndrome cells contained increased levels of labelled saturated and monounsaturated very long-chain fatty acids (VLCFA, that is fatty acids with more than 22 carbon atoms), in particular hexacosanoic (26∶0) and hexacosaenoic (26∶1) acids. As traces of labelled VLCFA with up to 32 carbon atoms were detected in triacylglycerols even in control cells it is probable that these fatty acids are formed naturally during the elongation process. Our data suggest that peroxisomes are involved in the chain shortening of the saturated and monounsaturated VLCFA.