Glyceryl ethers in insects: identification of alkyl and alk-1-enyl glyceryl ether phospholipids

Alkyl and alk-1-enyl glyceryl ethers have been identified in the phospholipids of three insect species, the American cockroach (Periplaneta americana), the tobacco budworm (Heliothis virescens), and the boll weevil (Anthonomus grandis). Glyceryl ethers were not detected in the neutral lipids. The ethers were found in the phospholipid fraction of whole insects and in isolated fat body tissue. The ether content varied among the three insect species, and fluctuated during various developmental stages. Gas liquid chromatographic analysis of the alkyl glyceryl ethers and aldehydes derived from the alk-1-enyl glyceryl ethers of the cockroach and budworm whowed striking differences in chain length. However, the hydrocarbon side-chain of the two ether fractions were similar in length for each species. Preliminary evidence indicates that 1-¹⁴C-acetate can be incorporated into alkyl ethers but not into alk-1-enyl ethers ofHeliothis pupae. Presented at the AOCS-AACC joint meeting, Washington, D.C., 1968. Under contract with the U.S. Atomic Energy Commission.     

Quantitative determination of phospholipids in sunflower oil

Phospholipids from sunflower oil samples were enriched by using solid-phase extraction (SPE) cartridges and subsequently separated and analyzed by high-performance liquid chromatography (HPLC) with an ultraviolet detector. The recovery of individual phospholipids at different total concentrations in model oils and the repeatability of the method were investigated. The results demonstrated the utility of SPE-HPLC for quantitative analysis of phospholipids in sunflower oil and the effectiveness for concentrating, separating, identifying, and quantitating phospholipids in samples with phosphatide contents as low as 0.1%. Samples of sunflower oil at different stages of processing were analyzed, and phospholipid profiles in hexane-extracted oil, hot-pressed oil, and water-degummed oils were compared.     

Liberation of aldehydes from alk-1-enyl glyceryl ethers by acid hydrolysis

Labeled alk-1-enyl glyceryl ethers were used in conjunction with thin layer chromatography to study the liberation of aldehydes from alk-1-enyl glyceryl ethers by two acid hydrolysis procedures. Both methods gave similar results, but neither liberated the aldehydes quantitatively. Only 75–85% of the alk-1-enyl glyceryl ether radioactivity was liberated as free aldehydes. Several nonaldehyde products were detected and one appeared to be a cyclic acetal. Under contract with the U.S. Atomic Energy Commission.     

Enrichment of phospholipids from neutral lipids in peanut oil by high-performance liquid chromatography

Phospholipids from crude peanut oil were enriched on a 2-cm silica column and subsequently separated from neutral lipids within the chromatographic system without prior concentration. Hexane effectively removed the bulk neutral lipids, leaving the adsorbed phospholipids on the silica precolumn. Individual phospholipids were separated from the remaining neutral lipids and from each other by using two mixed solvents and a gradient program. This method separates the phospholipids in approximately 27 min after the desired enrichment level has been reached. The research reported in this paper was a cooperative effort by the Agricultural Research Service of the United States Department of Agriculture and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7625.     

Glyceryl ethers in insects: Biosynthesis of ethanolamine phosphoglycerides containing alkyl and alk-1-enyl glyceryl ether linkages

When¹⁴C-labeled acetate, fatty acids or fatty alcohols were injected into or fed to the tobacco budworm, acyl, alkyl and alk-1-enyl moieties of the phospholipids incorporated radioactivity. Fatty acids were the principal precursor in acyl bond formation and fatty alcohols in the synthesis of alkyl and alk-1-enyl glyceryl ethers. Detailed analysis of the etherlinked phosphoglycerides revealed that most of the radioactivity was in the ethanolamine phosphoglycerides, and very little¹⁴C was found in the choline phosphoglycerides. In experiments of a short duration, the alkyl glyceryl ethers incorporated more radioactivity than the alk-1-enyl glyceryl ethers. The reverse was found with long term experiments, when the alk-1-enyl ethers had higher radioactivity. In addition to demonstrating the synthesis of ether-linked ethanolamine phosphoglycerides, the data suggested that fatty alcohols and acids were interconverted by insects and that the alk-1-enyl ethers were derived from the alkyl ethers. Presented at the AOCS Meeting, Houston, May 1971. The following abbreviations and terminology will be used: PE, PC, PI and PS for the generic terms ethanolamine, choline, inositol and serine phosphoglycerides, respectfully. Alkyl glyceryl ether for 1-alkyl-2-acyl-sn-glycerol-3-phosphoryl-, and alk-1-enyl glyceryl ether for 1-alk-1′-enyl-2-acyl-sn-glycerol-3-phosphoryl-(commonly called plasmalogen). These are adapted from the tentative rules published inJ. Lipid Res. 8:522–528 (1967).     

Fatty acid composition of the neutral lipids and individual phospholipids of muscle of cold-stressed arctic mice

Fatty acids of the individual phospholipids and total neutral lipid fractions in skeletal muscle of three species of Arctic mice were identified and quantitated after the mice had been classified as control, cold-sensitive or cold-resistant. The results indicate that some species increase the percentage of unsaturated fatty acids as an apparent result of cold exposure and some species do not. A common finding for all cold-sensitive mice was a significant increase in 14∶0 in phosphatidic acid when compared to cold-resistant and control animals. Hypotheses are presented in an attempt to explain this finding.     

Effect of excessive fatty acid ingestion upon composition of neutral lipids and phospholipids of snailHelix pomatia L.

The effect of an excessive intake of oleic acid on the lipids of the Roman snail (Helix pomatia L.) was studied. The total lipid content increased by 30% which was fully attributable to a marked elevation in the neutral esters and free fatty acids, as phospholipid and free sterol contents remained unaffected. The fatty acid composition of the phospholipids, characterized by high amounts of stearic, linoleic, homolinoleic, and, particularly, arachidonic acids, appeared to be nearly insensitive to this excessive oleic acid ingestion. By contrast, the effect of oleic acid upon the depot lipids was striking: active intestinal resorption of the acid from the dietary supply was shown by the fourfold level of lleic acid in the free fatty acid fraction, whereas a fivefold level of this acid in the glyceride and sterol ester fraction was proof of a substantial esterification. These data support the view that the composition of the structural lipids is specifically species oriented, whereas both the content and the composition of the depot lipids are highly governed by dietary fat intake.     

Reactions of fatty aldehydes with fatty alcohols: Formation of acetals,hemiacetals and alk-1-enyl alkyl ethers

A convenient method for the synthesis of acetals of fatty aldehydes and fatty alcohols by transacetatlation between fatty aldehyde dimethyl acetals and fatty alcohols is described. The acetals undergo decomposition to the alk-1-enyl alkyl ethers during GLC. Equimolar mixtures of fatty aldehydes and fatty alcohols show hemiacetal structure as evidenced by IR spectra in KBr discs but are dissociated completely into their components in solution and during TLC. They do not undergo dehydration and conversion to alk-1-enyl alkyl ethers during GLC under conditions of dealcoholization of acetals but are dissociated into aldehydes and alcohols. Presented at the AOCS Meeting, San Francisco, April 1969.     

Quantitative determination of lipids and their constituents by the Chromarod TLC-FID system

The methanolysis products of neutral sphingolipid and archaebacterial neutral glycolipid were separated on Chromarods S-II (silica gel) with a double developing system. The lipid constituents separated on the rods were scanned automatically with a hydrogen flame ionization detector (Iatroscan). The molar ratios of the constituents determined by this system were very close to the theoretical values of the lipid.     

Comparative evaluation of solvent extraction methods for the determination of neutral and polar lipids in beef

Samples of lean (< 5% fat), medium (13–15%) and high-fat (> 20%) ground beef were extracted for total lipid by 4 methods of wet extraction employing chloroform/methanol (CM), n-hexane/iso-propanol (HIP) and ethyl alcohol/ethyl ether (AE), and by 3 methods of soxhlet extraction of freeze-dried material by petroleum ether (PE) or eithyl ether (EE), CM and methylene chloride/ methanol (MM). The purified lipid was fractionated into neutral and polar lipid fractions by silicic acid chromatography and the frac-tions were analyzed for fatty acid distribution by gas liquid chroma-tography (GLC). The soxhlet procedure employing either PE or EE extracted less than 75% of total lipid, 89% of triglycérides and 15% of polar lipids from lean beef as compared to other methods, and as the fat content increased from 3 to 20%, extracted amounts of polar lipid which increased to 40% of that extracted by other methods. The fatty acid distribution of the fractionated triglycerides and polar lipids was generally within experimental error for each frac-tion, irrespective of the method of extraction. The percentages of 16:0 and 18:1 were significantly less in polar lipids than in trigly-cerides. In addition to significantly higher percentage of 18:2, the polar lipids contained up to 20% of long-chain fatty acids not detected in triglycerides. The soxhlet procedures with CM or MM were as effective as wet extraction procedures in extracting neutral and polar lipids. Presented at the 73rd AOCS annual meeting, Toronto, 1982. Contribution No. 512, Food Research Institute, Agriculture Canada.     

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats

The differential uptake and targeting of intravenously infused [1-¹⁴C]palmitic ([1-¹⁴C] 16∶0) and [1-¹⁴C]arachidonic ([1-¹⁴C]20∶4n−6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 μCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-¹⁴C]20∶4n−6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-¹⁴C]16∶0. The unilateral incorporation coefficient, k ^*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-¹⁴C]20∶4n−6 than for [1-¹⁴C]16∶0. In contrast, [1-¹⁴C]16∶0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-¹⁴C]20∶4n−6 was primarily targeted for esterification into phospholipids, while [1-¹⁴C]16∶0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.     

1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36–38% of 1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1′-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1′-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n−3, 18∶1a/20∶5n−3 and 18∶1a/22∶6n−3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n−6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses. Molecular species of 1-O-alk-1′-enyl-2-acyl-GPE are abbreviated as follows:e.g., 16∶0a/18∶1 GPE is 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The corresponding diacyl species, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, is abbreviated as 16∶0/18∶1.     

Fatty acid composition of phospholipids and neutral lipids during embryonic and early larval development in Atlantic herring (Clupea harengus,L.)

The fatty acid compositions of total polar and total neutral lipids of Atlantic herring eggs and larvae were determined immediately before fertilization, after fertilization and at various times during subsequent embryonic and early larval development. Within 3 hr after fertilization the percentage of total PUFA in neutral lipid decreased from 33% to 20%, with a reciprocal increase in monoenes. Thereafter the percentage of PUFA in the neutral lipids increased progressively, attaining the original level in ripe eggs by the time of yolk sac absorption. During the larval stages the percentage of PUFA continued to increase in the neutral lipid, reaching almost 44% of the total by day 32 after fertilization, although it was reduced to 32% by day 36. The percentage of monoenes in the neutral lipid displayed a progressive decrease during the whole period of development from 3 hr after fertilization. Throughout all the developmental periods the fatty acid composition of total polar lipids remained essentially constant. The polar lipids of the yolk sac displayed virtually the same fatty acid composition as the larval bodies, but the neutral lipids of the yolk sac were low in PUFA compared to the larval bodies. The results are discussed with reference to changes in lipid class composition during development. The conservation of high levels of PUFA in lipids during embryogenesis and early larval development reflects the importance of these fatty acids during development.     

Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots

Separation of polar lipids by two-dimensional thin layer chromatography providing resolution of all the lipid classes commonly encountered in animal cells and a sensitive, rapid, reproducible procedure for determination of phospholipids by phosphorus analysis of spots are described. Values obtained for brain and mitochondrial inner membrane phospholipids are presented.     

Microbial subterminal oxidation of alkanes and alk-1-enes

Oxidation ofn-alkanes and alk-1-enes by aPenicillium species andPseudomonas aeruginosa resulted in the formation of intermediates arising from the oxidation of methylene groups. A catabolic pathway involving oxidation of the hydrocarbon to a secondary alcohol and the corresponding ketone, followed by the formation and subsequent cleavage of an ester intermediate is presented. One of five papers being published from the Symposium “Biochemistry of Hydrocarbon Degradation,” presented at the AOCS Meeting, Chicago, September 1970.     

The role of neutral glycolipids and phospholipids in myxovirus-induced membrane fusion

Myxoviruses (influenza virus and paramyxovirus) enter host cells by two successive steps consisting of attachment and fusion between viral and cellular membranes. The initial attachment is known to occur through specific binding of the viruses with the neuraminic acid-containing receptors of cellular membranes. Evidence is presented here that, in the following step of membrane fusion, neutral glycolipids terminating in galactose and certain phospholipids (primarily lecithin and sphingomyelin) interact with the viral envelopes and that this interaction may be fundamental to the fusion process.     

Experimental nephrotic syndrome induced in the rat by puromycin aminonucleoside: Hepatic synthesis of neutral lipids and phospholipids from3H-water and3H-palmitate

Experimental nephrotic syndrome (ascites, proteinuria, hypoalbuminemia, and hyperlipidemia) was induced in male Wistar rats by seven daily subcutaneous injections of puromycin aminonucleoside (20 mg/kg). Hepatic lipogenesis from³H-water and³H-palmitate was investigated in nephrotic and pair fed control rats by using liver slices. Total incorporation of³H-water into neutral lipids was higher in nephrotic than in control rats (413±124 vs. 229±46 nmoles/g/hr, p<.01). Among neutral lipids, the major increase was observed for triacylglycerols (106±26 vs. 72±21 nmoles/g/hr, p<.05), cholesteryl esters (3.7±2.1 vs. 1.4±.7 nmoles/g/hr, p<.05) and, above all, for cholesterol (123 ±48 vs. 36±18 nmoles/g/hr, p<.0025). Total incorporation of³H-water into phospholipids as well as incorporation of³H-water into individual phospholipids were not significantly increased. Incorporation of³H-palmitate into neutral lipids was increased (312±84 vs. 221±28 nmoles/g/hr, p<.05). Among neutral lipids, a significant increase was observed for 1,3-diacylglycerols (19±3 vs. 13±3 nmoles/g/hr, p<.025), triacylglycerols (228±50 vs. 163±14 nmoles/g/hr, p<.05) and cholesteryl esters (18±5 vs. 10±1 nmoles/g/hr, p<.01). Incorporation of³H-palmitate into phospholipids was not significantly affected. The difference in hepatic lipogenesis between nephrotic and control rats was even more pronounced if the data were corrected for the total liver weight which was significantly increased in the nephrotic rats (11.3±.3 vs. 8.5±.1 g, p<.001). These findings indicate that the synthesis of neutral lipids from both³H-water and³H-palmitate is elevated in rat with aminonucleoside-induced nephrotic syndrome. The possible role of the increased hepatic lipogenesis in the pathogenesis of the nephortic hyperlipidemia is discussed.     

Degumming of soybean oil: Quantitative analysis of phospholipids in crude and degummed oil1

Phospholipids of crude and degummed soybean oils were isolated and separated by column chromatography and thin layer chromatography. Standards and specific spray reagents were used to identify the phospholipids. Phospholipids identified in the crude and degummed oils were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, lysophosphatidylcholine and lysophosphatidylethanolamine. Several unknown phosphorus-containing compounds were present. Samples of crude and degummed oil were collected from four soybean oil processors over four consecutive days. Total and individual phospholipids were quantitated by determining the phosphorus content. The total phosphorus content of crude oil varied among companies, with the average ranging from 453 to 676 ppm. The average total phosphorus content of the degummed oil of the four processors ranged from 12 to 84 ppm. Processors removed an average of 86-98% of the phosphorus present in the crude oil during the degumming process. There was also a daily variation in phosphorus removal within the individual companies. During the degumming process, the proportion of phosphatidylcholine decreased and the proportion of unknown, nonpolar phosphorus compounds increased in samples from all companies. Significantly higher proportions of phosphatidic acid and lyso compounds were found in the degummed oil of some but not all companies. Presented at the 72nd AOCS annual meeting, New Orleans, 1981. Paper No. 7056, Journal Series, Nebraska Agricultural Experiment Station.     

Fatty acid composition of melon seed oil lipids and phospholipids

Fatty acid compositions of crude melon seed oil from two different sources were compared. Melon seeds fromCitrullus vulgaris (syn.C. lanatus) contained phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and phosphatidylserine (PS), whereas melon seeds fromCitrullus colocynthis contained only PC and LPC, but not PS. Analysis of the total lipids revealed that the major fatty acid of the oils was 18:2n-6.Citrullus vulgaris seed oil contained 71.3% andC. colocynthis contained 63.4% of 18:2n-6. The predominant fatty acids in theC. vulgaris PC were 18:2n-6 (32.2%), 18:1n-9 (26.4%) and 16:0 (22.2%), whereas theC. colocynthis PC contained 44.6% of 18:1n-9 as the major fatty acid. The level of monoenes in theC. colocynthis variety (46.2%) was different from theC. vulgaris (27.3%). The major fatty acid in the LPC was 18:1n-9 for both varieties. Notably, theC. colocynthis variety did not contain any PS. The major fatty acids in theC. vulgaris PS were 18:1n-9 (37.9%) and 18:2n-6 (33.7%). Of all the phospholipids, LPC contained the greatest amount of monoenes, 48.6–52.4%.     

Study of neutral lipids ofLupinus mutabilis meal and isolates

Two types of protein isolates have been obtained from defattedLupinus mutabilis meal. The isolates, MA and MB, were obtained by alkaline extraction with 0.2% NaOH and 0.25% sodium bisulfite, respectively, followed by precipitation at the isoelectric point (pH 4.8). Total associated lipids were extracted with 86% ethanol. Neutral lipids were separated in a Florisil column. The lipids in the isolates were similar to those found in the original meal. The following types of compounds were separated, identified, and quantitated: hydrocarbons, waxes, methyl esters, triacylglycerols, free fatty acids, diacylglycerols, and free sterols.