Esterified lipids of the freshwater dinoflagellatePeridinium lomnickii

Steryl esters, phytyl esters and triacylglycerols of a naturally occurring freshwater dinoflagellate,Peridinium lomnickii, were identified using capillary gas chromatography-mass spectrometry (GC-MS). Steryl esters differing in degree of unsaturation were separated, prior to analysis, by argentation thin layer chromatography. 5α(H)-Cholestanol was more dominant, relative to 4α-methylstanols, in steryl esters than in the free sterols, but the same sterol moieties occurred in both fractions. Monoenoic fatty acids were enriched in the steryl esters relative to the free fatty acids. Major acyl groups in steryl esters were 16∶0 or 20∶1, with smaller amounts of 14∶0 and 18∶1. In triacylglycerols the acyl moieties were 14∶0, 16∶0, 18∶1, 16∶1 and 12∶0, in order of decreasing abundance. Phytyl esters, previously inferred to occur in a marine dinoflagellate only by analysis of transesterified products, were identified by GC-MS comparison with authentic compounds. Direct analysis of these esterified lipids has not been reported for freshwater phytoplankton. The 4α-methylstanyl esters, 5α(H)-cholestan-3β-yl esters and phytyl esters occurring inP. lomnickii are further features in common with marine dinoflagellates, additional to the 4α-methylsterols reported previously.     

Lipids of cultured hepatoma cells: II. Effect of media lipids on cellular phospholipids

Minimal deviation hepatoma cells were cultured in a modified Swim's 77 medium supplemented with decreasing amounts of serum, lipid-free serum, and lipid-free serum containing added palmitic or linoleic acids. Cellular phospholipids were extracted and the class distribution determined quantitatively. The fatty acid composition of each phospholipid class was determined, and the percentages from cells grown on each of the various media were compared. Cellular phospholipid class and fatty acid compositions differed from media compositions, indicating that intact serum phospholipids are not incorporated into cellular structures. Phosphatidylcholine percentages decreased as the media serum and lipid levels decreased, while phosphatidylinositol and phosphatidylethanolamine percentages increased. Sphingomyelin of cells grown in medium containing added linoleic acids contained a high level of a 24∶2 acid. All classes, except sphingomyelin, contained elevated levels of 18∶1 acid and decreased levels of polyunsaturated fatty acids, relative to normal rat liver. Cells cultured on lipid-free medium did not contain increased concentrations of 20∶3 acid, suggesting that this hepatoma cell cannot desaturate monoenoic acids. Phosphoglycerides of cells, grown on lipid-free medium, had the highest monoene fatty acid concentration, whereas those cells grown on media containing added linoleic acid had the lowest concentrations, suggesting that linoleate may inhibit or regulate monoenoic acid biosynthesis in this cell. These mass data also demonstrate that monoenoic fatty acid biosynthesis in this cultured hepatoma cell responds to dietary changes.     

Lipids of cultured hepatoma cells: I. Effect of serum lipid levels on cell and media lipids

Minimal deviation hepatoma cells were cultured as monolayers to confluency in roller flasks containing modified Swim's medium, supplemented with decreasing amounts of serum, lipid-free serum, and lipid-free serum containing added fatty acids. Good cell growth was observed until serum levels fell below 5% of the medium. Media containing lipid-free serum or lipid-free serum plus linoleic or palmitic acids did not support good growth. Lipids were extracted from cells; media, obtained during the first and last half of the incubation period, resolved into neutral and phospholipid fractions; fatty acid composition of each fraction analyzed by gas liquid chromatography; and lipid class distributions compared by thin layer chromatography. The data showed that the media contained more neutral lipids and phospholipids after incubation than initially, indicating that minimal deviation hepatoma cells excreted lipids into the media. The class composition of the excreted lipids resembled that of the serum. A comparison of media, cells, and serum fatty acid compositions indicated that the lipids secreted into the media were of cellular origin. Although some differences were noted, in general, cells grown on the nine different media had the same ca. neutral lipid and phospholipid class and fatty acid compositions. In contrast, dramatic differences were observed in the class and fatty acid compositions of the serums from that of the cells and media. These results indicate that exogenous serum lipids had little influence on cellular class and fatty acid compositions of the minimal deviation hepatoma cells. This neoplasm did not contain detectable levels of glyceryl ether diesters, indicating that this compound is not characteristic of all tumors. Lipid class profiles and fatty acid compositions of cells grown on various media suggest that the minimal deviation hepatoma cells can synthesize most, if not all, neutral lipid and phosphoglyceride classes found in liver. Presented at the AOCS Meeting, New Orleans, April 1973.     

Lipids of cultured hepatoma cells: IV. Effect of serum and lipid upon cellular and media neutral lipids

Minimal deviation hepatoma 7288C cells were cultured in a modified Swim's medium supplemented with decreasing levels of serum, lipid-free serum, lipid-free serum plus fatty acids, and other additives. Cellular and media neutral lipid classes were quantitated, the fatty acids of triglycerides and sterol esters analyzed, and the carbon number distribution of triglycerides determined. Cellular triglyceride biosynthesis virtually was inhibited when the medium was supplemented with bovine serum alone. This inhibition was not observed when the medium was supplemented with fetal calf serum alone or mixtures of fetal calf serum and bovine serum. Cells cultivated on medium supplemented with lipid-free serum plus palmitic or linoleic acids had much lower levels of free and esterified cholesterol. The fatty acid composition of cellular triglycerides and cholesterol esters differed dramatically from the corresponding media lipid classes. Except when linoleic acid was added to the medium, changes in the media serum and lipid levels had only marginal effects upon the fatty acid composition of cellular triglycerides and cholesterol esters. These data, in conjunction with earlier data that showed the media neutral lipid levels did not decrease during cell growth, indicate that these hepatoma cells utilize little or no serum triglycerides and cholesterol esters. Linoleic acid added to the medium dramatically reduced the level of 18∶1 acids in cellular triglycerides and cholesterol esters. Palmitic acid added to the medium did not change the fatty acid compositions significantly. Comparison of experimentally determined and calculated triglyceride carbon number percentages indicated a random distribution of fatty acids in this glyceride. The fatty acid composition of cellular triglycerides was similar to the composition of the cholesterol esters. The lack of characteristic and distinguishable compositions of these two classes that occur in most normal tissues suggests a loss of specificity in the lipid metabolism of this neoplasm at the class level.     

Lipid components and enzymatic hydrolysis of lipids in muscle of Chinese freshwater fish

The lipid and fatty acid composition of muscle of 10 species of freshwater fish obtained from a market of Shanghai City was examined. Total lipids (TL) ranged over 0.9–4.7% of muscle for all samples. The content of triacylglycerol (TG) in muscle ranged over 0.2–3.4% and that of polar lipids (PL) was 0.5–1.3%. Differences of TL content were dependent on TG contents. The predominant important fatty acids (>10% of the total fatty acids in TL) were 16∶0 and 18∶1n−9 with some 16∶1n−7, 18∶2n−6, and 22∶6n−3. The polyunsaturated fatty acids (PUFA) content was 10.2–43.4%, and especially Chinese sea bass contained above 20% of 22∶6n−3 in the total fatty acids. There were higher levels of PUFA such as 20∶5n−3 and 22∶6n−3 in PL than in neutral lipids. Muscle of the silver carp was stored at 20°C, and changes of lipid classes during storage were examined. Free fatty acids increased, and PL decreased during storage. This phenomenon was inhibited by heating the muscle, suggesting that lipid hydrolysis by phospholipase occurred in silver carp muscle.     

Tumor lipids: Comparison of glyceryl ethers of mouse and insect tumors

Examination of the lipids of two tumor-bearing mutant strains of fruit fly reveal that they do not have the elevated content of glyceryl ethers typical of many neoplasms of higher animals. Glyceryl ether diesters were absent, and the amounts of alkyl and alk-1-enyl glyceryl ethers of tumorous and normal tissues were similar.     

Comparison of Methods for Separating Polar Lipids from Oat Oil

Polar lipids of crude oat oil were investigated. Oat oil was separated from oats by extraction with isopropanol. Polar lipids were fractionated from crude oil by supercritical CO₂-extraction, by ultrafiltration in hexane and by water degumming. The polar fraction from CO₂-extraction had the highest phospholipid and lowest tocopherol content. The polar fractions made by different methods possessed similar antioxidative properties. However, the polar lipids from oats were more powerful antioxidants than those made from soybean or rapeseed oil.     

Lipids ofCrassostrea virginica. I. Preliminary investigations of aldehyde and phosphorous containing lipids in oyster tissue

Oyster tissue contained 2.4% lipid, 0.14 μmole aldehyde per milligram lipid and at least 10 μg phosphorous per milligram lipid. The neutral lipid represented 58%, the glycolipid 6%, and the polar lipid 36% of the total lipid recovered after silicic acid column chromatography. Aldehydes were found in all fractions, but the presence of plasmalogen was verified in only the neutral and polar lipid fractions. At least 68% of the plasmalogen in oyster lipid was found in the polar lipid fraction. At least 13% of the phosphorous in oyster lipids was present as phosphonolipid. The distribution of phosphate and phosphonate lipids was: diacyl phospholipid 38.1%, plasmalogen phospholipid 21.8%, sphingophosphonolipid 13.5%, glyceryl ether phospholipid 8.3%, sphingophospholipid 6.9%, plasmalogen phosphonolipid 6.4%, diacyl phosphonolipid 2.6%, and glyceryl ether phosphonolipid 2.4%. When the per cent of phosphorous as phosphonolipid within the plasmalogen and glyceryl ether classes was calculated, similar values were obtained. These results support the hypothesis that there is a product precursor relationship between these two classes of lipids. Some of the data taken from a thesis to be submitted to the Graduate School, University of Maryland, by Leslye Johnson in partial fulfillment of the requirements for the M.S. degree in biochemistry.     

Boreal freshwater fish diet modifies the plasma lipids and prostanoids and membrane fatty acids in man

The effect of fish diet on 43 healthy male students was studied. They ate a fish-containing meal for 15 weeks on an average of 3.7 times per week. Twenty-one of them voluntarily restricted their lipid intake while the rest ate normally. Controls continued their usual eating habits (19 students). The meals consisted of Finnish freshwater fish (87%) (vendace, pike, perch and rainbow trout) and brackish water fish (13%) (Baltic herring) that provided about 1 g of ω-3 polyunsaturated fatty acids per day (0.25 g eicosapentaenoic acid and 0.55 g docosahexaenoic acid). During the diet, ω-3 fatty acids increased in erythrocyte ghosts and platelets at the expense of ω-6 fatty acids. The concentration of serum cholesterol diminished in those fish consumers who lowered their lipid intake. Apolipo-protein A₁ and B were lowered in both fish-consuming groups. Triglyceride levels also showed a tendency to decrease. The formation of thromboxane B₂ during incubation of whole blood decreased in both fish-consuming groups. The decrease of plasma 6-keto-PGF_1α was not statistically significant, if compared with the controls. The results obtained indicate that a moderate intake of fish-containing meals has some beneficial effects on the plasma lipid and prostanoid metabolism, when coronary heart disease risk factors are considered.     

Lipids of the cottonseed. III. Perchloric acid ashing of lipids for the determination of phosphorus

1. Cottonseed oil is effectively oxidized with a mixture of perchloric and nitric acids in connection with the determination of phosphorus by the Fisk and Subbarow method.
2. The development of the molybdenum blue in the procedure is independent of the acid content in the reducing medium up to 0.5 normal.
3. The presence of perchloric acid does not interfere with the development of the molybdenum blue.
4. The phosphorus content of cottonseed extracts depends on the nature of the solvent. The quantities obtained varied from 0.0071 to 0.117%.
5. A phospholipid containing 3.73% phosphorus was isolated from cottonseed.

     

Enzymatic Synthesis of Feruloylated Lipids: Comparison of the Efficiency of Vinyl Ferulate and Ethyl Ferulate as Substrates

A facile and efficient enzymatic synthesis approach to synthesize feruloylated lipids, which are composed of 1(3)-feruloyl-monooleyl-glycerol and 1(3)-feruloyl-dioleyl-glycerol, through lipase-catalyzed transesterification using vinyl ferulate (VF) and ethyl ferulate (EF) as substrate, respectively, with triolein was developed. When VF was used as substrate, a maximum of conversion yield of 91.1% was obtained at 55 °C, 20 mg/mL enzyme content, water activity (a _w) = 0.07, 62 h. This was greater than that when EF was used as substrate (69.6%, 50 °C, 33.3 mg/mL enzyme content, a _w = 0.07, 96 h). Candida antarctica lipase (Novozym 435) can be reused for 13 runs without evident loss in activity and stability when VF was used as substrate. The results demonstrate that VF has greater synthetic efficiency and it provides another effective approach to prepare feruloylated lipids under normal pressure, making industry application feasible.     

Comparison between extraction of lipids and fatty acids from microalgal biomass

Seven solvent mixtures have been used to extract the lipid fraction of lyophilized biomass ofIsochrysis galbana. Six of them were composed of biocompatible solvents. Each method was carried out under relaxed operating conditions (i.e., one hour at room temperature) with extraction in a nitrogen atmosphere to prevent autooxidation and degradation of polyunsaturated fatty acids (PUFAs). Apart from the well-established Bligh and Dyer method [Can. J. Biochem. Physiol. 37:911 (1959)] (Cl₃CH/MeOH/H₂O, 1∶2∶0.8, vol/vol/vol), which rendered the highest yield of lipids (93.8%), ethanol (96%) and hexane/ethanol (96%), 1∶2.5 vol/vol produced the best results (84.4 and 79.6%, respectively). To obtain free fatty acids, KOH was added to the solvent mixtures used to extract the total lipids, except for Cl₃CH/MeOH/H₂O, and direct saponification was carried out at 60°C for 1 h or at room temperature for 8 h. The highest yields obtained by direct saponicification were 81% with hexane/ethanol (96%), 1∶2.5, vol/vol and 79.8% with ethanol (96%). Partial yields of the mainn-3 PUFAs found inI. galbana, stearidonic acid (SA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were calculated for both extraction methods. For lipid extraction with ethanol (96%), yields of 91, 82 and 83% were obtained for SA, EPA and DHA, respectively. When direct saponification was used, hexane/ethanol (96%; 1∶2.5, vol/vol) produced the best yields of (91, 79 and 69% for SA, EPA and DHA, respectively).     

Structured and Specialty Lipids in Continuous Packed Column Reactors: Comparison of Production Using One and Two Enzyme Beds

In this study, speciality lipids based on fish oil (FO) and capric acid (CA) were produced in packed bed bioreactors using immobilized Lipozyme IM from Rhizomucor miehei in a solvent-free environment. Our goal was to compare the product quality and yield among reactors consisting of one or two enzyme columns. Response surface methodology (RSM) was used to optimize process variables for maximum incorporation (Inc) of CA for each reactor configuration. The studied process variables were substrate molar ratio (1:1–3:1 CA/FO), temperature (35–55 °C) and flow rate (0.5–1.5 mL/min). All experiments were conducted based on a face-centered cube design. The maximum predicted Inc of CA into FO (31.7 mol%) using one column was obtained when substrate molar ratio, temperature and flow rate of substrates were 2.70:1 (CA/FO), 55 °C and 0.5 mL/min. The corresponding optimal Inc of CA into FO (22.7 mol%) using two columns was predicted at 2.95:1 (CA/FO), 55 °C and 0.86 mL/min. Analysis of variance (ANOVA) showed that Inc of CA into the one-bed design was significantly influenced by all experimental conditions, with substrate molar ratio having the greatest impact. In the two-bed design, only temperature and flow rate had an effect; the molar ratio of substrates was not significant. Coefficients of determination were low for both designs, indicating a poor fit of our data to the model. However, the main purpose of this study was to assess the effect of process factors on Inc of CA into FO, rather than generate a model useful for prediction of responses under conditions not examined with this design. Therefore, the significant ANOVA results are much more important, stressing the real relationship between parameters and response, than the low coefficients of determination.     

Quantitative gas chromatographic analysis of lipids: Comparison of gas density balance and flame ionization detector

Accuracy of available detectors for gas chromatography is a subject of continuing research in analytical chemistry. The quantitative deficiency of the flame ionization detector, as well as of other detectors, has been widely recognized, and empirical correction factors have been required. By contrast, the gas density balance, the forgotten ideal detector, should not require calibration. A gas density balance, now available in a commercial chromatograph, and a flame ionization detector were compared for quantitative analyses of lipids. Wt percents of known methyl ester mixtures were determined, as well as mole percents of aldehyde fragments from certain ozonized octadecenoate isomers. Percentages were calculated from area response without correction factors for the gas density balance and with correction factors, based upon the number of ionizable carbon-atoms, for the flame ionization detector. Accuracy, as measured by percentage deviation from either known or theoretical values, was better for gas density balance data than for flame ionization detector data. Aldehyde and aldehydic ester fragments formed by reductive ozonolysis of octadecenoate isomers from partially hydrogenated methyl linolenate also were determined with each detector. Theoretically, ozonolysis of these monoenes should yield an aldehyde and an aldehydic ester in equal mole percents. Experimentally, the average of the ratios of aldehyde to aldehydic ester from each of the Δ5-Δ13 monoenes was 1.29 for the FID data (corrected) and 1.01 for GDB data (uncorrected). This difference in averages approaches significance at the 95% confidence level. For the Δ14 and Δ15 monoenes from which C₄ and C₃ aldehydes are formed, ionizable carbon-atom corrections proved even less adequate. Presented at the AOCS Fall Meeting, Chicago, September 1973. ARS, USDA.     

Inhibited Oxidation of Lipids I: Complex Estimation and Comparison of the Antioxidative Properties of Some Natural and Synthetic Antioxidants

The activity A of antioxidants, representing a ratio between the stabilization factor F and the decrease in oxidation rate after the introduction of an inhibitor DOR (A = F/DOR) is proposed for a complex estimation of the effect of antioxidants. The specific changes in F, DOR and A are discussed in association with the participation of the inhibitor molecule and the radical formed from it in the different reactions of initiation and propagation of the chains. Several types of antioxidants (α-tocopherol, ferulic acid, BHT and BHA) in concentrations ranging from 0.02 – 0.20 wt% are investigated during the oxidation of lard at 100°C. It is established that at concentrations of 0.02–0.05 wt% the antioxidative activity increases in the sequence ferulic acid < BHT < BHA <α-tocopherol. At high concentrations (above 0.10 wt%) the activity increase follows the sequence α-tocopherol < BHT < BHA < ferulic acid. Enhancement of the antioxidant concentration above 0.02 wt% is expedient only in the case of ferulic acid. The molecules of α-tocopherol, BHA and BHT participate not only in chain termination but also in first order rate side reactions with respect to the inhibitor, which explains the decrease in their efficiency with rising concentration. The molecule of ferulic acid does not participate in side reactions. The aroxyl radical formed from BHT does not participate in chain propagation, whereas the radicals of BHA and ferulic acid take part in such reactions. The aroxyl radical of α-tocopherol joins in more than one side reactions.     

Dietary Structured Lipids and Phytosteryl Esters: Blood Lipids and Cardiovascular Status in Spontaneously Hypertensive Rats

This study examined the dietary effects of enzymatically modified sesame oil with caprylic acid (structured lipids, SL) and phytosteryl esters (PE) on blood lipid profiles and cardiovascular parameters of spontaneously hypertensive rats (SHR) fed high-fat and high-cholesterol (HFHC) diets. The dietary groups were: normal diet (control), sesame oil (SO), SL, SO fortified with PE (SOP), and SL fortified with PE (SLP). After 9 weeks of feeding, the body weights, liver weights, and liver weight/body weight ratios in all HFHC-fed groups were higher than controls. Plasma total and LDL cholesterol levels in all HFHC-fed groups were similar to one another but higher than those in controls. Plasma HDL cholesterol levels in rats fed SOP and SLP were higher than those in controls or rats fed SO and SL. Plasma HDL/total cholesterol ratios in rats fed SOP and SLP were similar to those in controls and were higher than those in rats fed SO and SL. There was no difference in plasma lipid profiles between rats fed SO and SL. Arterial blood pressures (BP) in conscious HFHC-fed rats were similar to those in controls whereas heart rates (HR) in all HFHC-fed groups were similar to one another but were higher than that in controls. These findings demonstrate that (1) the dietary effects of SL on plasma lipid profiles and resting BP and HR are similar to those of SO, (2) PE had positive effects on plasma lipid profiles, and (3) 9-week intake of SL and PE did not have pronounced effects on resting BP but induced tachycardia in SHR.     

Inhibited Oxidation of Lipids II: Comparison of the Antioxidative Properties of Some Hydroxy Derivatives of Benzoic and Cinnamic Acids

The antioxidative properties of p-hydroxy-benzoic acid, vanillic acid, syringic acid, 3,4-dihydroxy-benzoic acid, p-coumaric acid, ferulic acid, sinapic acid and caffeic acid in lard autoxidation at 100°C are compared. The effect of phenolic acids is investigated within the concentration range 0.02 – 0.20 wt %. It is proved that the derivatives of the benzoic acid have weaker inhibiting properties than is the case of the corresponding analogues of the cinnamic acid due to the more active participation of the inhibitor in the elementary reactions of propagation and initiation of the radical chain process. The activity (a complex parameter demonstrating the efficiency and strength of the antioxidant) of the phenolic acids investigated decreases in the sequence: caffeic acid >3,4-dihydroxy-benzoic acid > sinapic acid > syringic acid>ferulic acid>p-coumaric acid>vanillic acid. The increase in concentration of the acids investigated is most advisable in the case of sinapic, syringic and ferulic acids which are not consumed in side reactions.     

Comparison of fatty acids and lipids of smolting hatchery-fed and wild atlantic salmonSalmo salar

In Atlantic Canada the Atlantic salmonSalmo salar change from the parr stage to the smolt stage while still in fresh water, preparatory to migration to salt water. In some stocks this takes place during the second overwintering. In several hatcheries where the water temperature drops to 0–0.5 C and the ponds ice over, there is a high incidence of erosion of the dorsal and pectoral fins and sometimes of the caudal fin. No disease organism has been identified, and the lesions heal over in most cases. Dietary fatty acids were thought, to be a factor. A detailed study of lipid recoveries and classes has shown that in the skins of abnormal fish the total lipid, is 7.8% compared to 4.7% in control fish. Unexpectedly, an analysis of one lot of healthy smoltstage wild fish showed that whole bodies have only a quarter of the lipid of comparble hatchery fish. Comparison of fatty acids showed that wild fish lipids include a higher proportion of arachidonic acid than those of the hatchery fish. in the latter, linoleic acid is provided readily by diet but the elongation to arachidonic acid evidently does not proceed. These results suggest that the smolt lipid, is involved intimately with either the cause of the dermal lesion or is a defense mechanism, possibly mediated through oxygenase activity.     

Lipids of gelatinous antarctic zooplankton: Cnidaria and Ctenophora

Antarctic gelatinous zooplankton, including Cnidaria (Calycopsis borchgrevinki, Diphyes antarctica, Stygiomedusa gigantea, Atolla wyvillei, Dimophyes arctica) and Ctenophora (Beroe cucumis, B. forskalii, Pleurobrachia pileus, Bolinopsis infundibulum) were collected near Elephant Island, South Shetland Islands, during January and February 1997 and 1998. Total lipid was low in all zooplankton (0.1–5 mg g⁻¹ wet mass) and included primarily polar lipids (59–96% of total lipid). Triacylglycerols were 0–26% of total lipids, and wax esters were 0–11% in all species. Cholesterol was the major sterol in all Cnidaria (50–63% of total sterols) whereas in most ctenophores it was lower at 26–45%. These cholesterol levels are consistent with a combined carnivorous and phytoplanktivorous diet in the ctenophores, with the carnivorous diet more dominant in the Cnidaria. Other sterols included primarily trans-dehydrocholesterol, desmosterol, 24-methylcholest-5,22E-dien-3β-ol, 24-nordehydrocholesterol, and 24-methylenecholesterol. Total stanols were 0–6% in all zooplankton. Eicosapentaenoic acid and docosahexaenoic acid were the major polyunsaturated fatty acids (PUFA) in all samples (7–25% of total fatty acids) except for A. wyvillei in which docosapentaenoic acid was 10% of total fatty acids. The PUFA 18∶5n−3 was not detected in 1997 samples, but constituted 0.2–0.8% in most 1998 samples. Monounsaturated fatty acids included primarily 18∶1n−9c, 16∶1n−7c, and 18∶1n−7c. The principal saturated fatty acids in all samples were 16∶0, 18∶0, and 14∶0. These data are the first for many of these zooplankton species and the first sterol data for most species. The use of the signature lipid approach has enabled examination of aspects of trophodynamics not obtainable by conventional techniques.