Mass Spectrometry of the Lithium Adducts of Diacylglycerols Containing Hydroxy FA in Castor Oil and Two Normal FA

Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass spectrometry of the lithium adducts. They were RR (diricinolein, R is ricinoleate), RL, RS, R‐diOH18:0, R‐diOH18:1, R‐diOH18:2, R‐triOH18:0, R‐triOH18:1, R‐triOH18:2, diOH18:0‐diOH18:1, diOH18:1‐diOH18:1 and diOH18:1‐diOH18:2. The MS² fragment ions, [M + Li ? FA]⁺ and [FA + Li]⁺, from the lithium adducts of DAG containing hydroxy FA (one or two hydroxy FA), were used for the identification. The additional fragment ions from the neutral losses of FA lithium salts [M + Li ? FALi]⁺ were used for the identification of eleven DAG containing two normal FA in a soybean oil bioconversion product. The MS² fragment ions from the neutral losses of FA lithium salts [M + Li ? FALi]⁺ were not detected from the DAG containing hydroxy FA. The DAG containing FA with more hydroxyl groups than the other FA on the same DAG molecule tended to have a prominent fragment ion [FA + Li]⁺ and an undetectable fragment ion [M + Li ? FA]⁺ while the FA was the more hydroxylated FA. Also the less hydroxylated FA of a DAG tended to have a prominent fragment ion [M + Li ? FA]⁺ and an undetectable fragment ion [FA + Li]⁺ while the FA was the less hydroxylated FA.     

Effect of dietary fish oil on the rate of very low density lipoprotein triacylglycerol formation and on the metabolism of chylomicrons

The mechanism by which ω3 fatty acids lower plasma triacylglycerol levels was investigated. Rats were fed fish oil, olive oil (10% fat by weight) or a nonpurified diet 4% fat by weight) for 15 days. Lipoprotein lipase was inhibited by intra-arterial administration of Triton WR 1339 to estimate hepatic triacylglycerol output. Rats fed the olive oil diet showed a higher rate of triacylglycerol formation than rats fed the ω3 fatty acid diet or the low-fat diet. All three groups showed identical rates of removal from plasma of intraarterially administered artificial chylomicrons that had simultaneously been labeled with cholesteryl [1-¹⁴C]oleate and [9,10(n)-³H]triolein. Liver radioactivity and total fat content were lowest in rats fed the fish oil diet, indicating that ω3 fatty acids were preferentially metabolized in liver. Chylomicrons obtained from donor rats fed either fish oil containg [¹⁴C]cholesterol or olive oil containing [³H]cholesterol were removed at similar rates when infused together intraarterially into recipient animals. A slower formation of plasma very low density lipoprotein triacylglycerols in rats fed fish oil is probably due to a faster rate of oxidation of the fatty acid chains in the liver resulting in decreased plasma triacylglycerol concentrations.     

Characterization of α- and γ-linolenic acid oils by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Triacylglycerols of oils rich in α- and/or γ-linolenic acids were analyzed by reversed-phase high-performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. The (APCI)MS spectra of most triacylglycerols exhibited [M + H]⁺ and [M - RCOO]⁺ ions, which defined the molecular weight and the molecular association of fatty acyl residues, respectively. Reversed-phase HPLC resulted in, at least, partial separation of triacylglycerols containing α- and/or γ-linolenic acid moieties. Molecules containing α-linolenic acid residues exhibited a relatively weaker retention by the stationary phase than the corresponding molecules containing γ-linolenic acid residues. Good separation of triacylglycerols of cloudberry seed oil, evening primrose oil, borage oil, and black-currant seed oil was achieved. The molecular species identification of separated components was based on the (APCI)MS data as well as on the elution properties of triacylglycerols on reversed-phase HPLC. This study demonstrated the efficiency of HPLC-(APCI)MS in determining the molecular species compositions of triacylglycerols in complex natural mixtures. Good quality mass spectra could be extracted even from minor chromatographic peaks representing 0.2% or less of the total triacylglycerols.     

Structured Estolides: Control of Length and Sequence

Using ester-forming reactions such as carbodiimide coupling and a modified Yamaguchi symmetrical anhydride method, a variety of estolides based on 17-hydroxy oleic and 17-hydroxy stearic acid have been prepared. These hydroxy fatty acids are produced in good yields from hydrolysis of sophorolipids, which are in turn derived from fermentation of fats and oils. Since the estolides are formed one unit, or ester bond, at a time, their length and sequence can be precisely controlled. The key to this control is the use of protecting groups at either the carboxylic or hydroxy end of the starting hydroxy fatty acids. Two mono-protected dimers, for example, when combined in a fragment-condensation approach, give a tetramer with no “contamination” from estolides of other lengths. This methodology opens the way to functionalized estolides, and several variants were prepared: hybrid estolides, containing non-fatty acid moieties such as amino acids; polymerizable estolides, containing a norbornene unit; and non-linear estolides that extend from a branched core such as glycerol or pentaerythritol. With the benzoyl chloride-mediated symmetrical anhydride method, yields for individual coupling steps ranged from 75 to 93%. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Asymmetric distribution of decanoate in triacylglycerol synthesized in vitro by mammary glands of lactating mice

Slices, prepared from the mammary glands of lactating mice, were incubated with either [1-¹⁴C]acetate, [U-¹⁴C]glucose, or [1-¹⁴C]decanoate. From all 3 substrates, radioactivity in the synthesized lipids was found mainly in triacylglycerols (TG). When acetate or glucose served as substrate, decanoate (C₁₀) accounted for 24% of the fatty acids in TG. Hydrolysis of the TG by pancreatic lipase yielded [¹⁴C] fatty acids which had relatively more C₁₀ (38%) than did either of the other hydrolysis products mono- or diacylglycerol (14–17%). However, when TG produced by slices from C₁₀ were hydrolyzed, the acid was found to be esterified equally at the C-1, C-2 and C-3 of glycerol. Thus, when fatty acids are synthesized de novo and are converted to TG by gland slices, C₁₀ is predominantly located in the C-3 position, a finding in accord with the situation in milk TG, although such preferential incorporation does not occur when the free acid is presented to the tissue slices.     

Accumulation of neutral lipids by human skin fibroblasts: Differential effects of saturated and unsaturated fatty acids

The accumulation of neutral lipids by human skin fibroblasts grown in medium supplemented with fatty acids has been investigated. GM-10 cells incorporated exogenous fatty acids into both phospholipids and neutral lipids. More [¹⁴C] oleate, linoleate, or linolenate was incorporated into triacylglycerol than was [¹⁴C] palmitate or stearate. Supplementation of medium containing delipidized serum with unsaturated fatty acids resulted in far more stimulation of [¹⁴C] glycerol incorporation into triacylglycerol than did supplementation with saturated fatty acids. Palmitate- and stearate-fed cells incorporated sizable amounts of [¹⁴C] fatty acids and [¹⁴C] glycerol into diacylglycerol as well as triacylglycerol, especially at higher fatty acid concentrations. Increased oleate supplementation from 10–300 μM resulted in increased triacylglycerol synthesis and accumulation of discrete cytoplasmic lipid droplets; palmitate concentrations above 70 μm were toxic. Micrographs of the palmitate-fed cells showed electron translucent slits, suggesting solid depositions of saturated fat, rather than the discrete osmiophilic droplets found in oleate-fed cells. Although GM-10 cells can synthesize fully saturated triacylglycerols, these data suggest that in cells fed saturated fatty acids, solid depositions of neutral lipids may sequester diacylglycerols and thus limit triacylglycerol synthesis.     

Lipogenesis in the developing brain from intracranially administered [1-14C] acetate and [U-14C] glucose

Fifteen-day-old rats divided into two groups were given [1-¹⁴C]acetate or [U-¹⁴C] glucose by intracranial injection and were sacrificed after 1 hr. Analysis of lipids from the two groups showed differences in the incorporation of radioactivity in the polar lipids and cholesterol. Analysis of brain fatty acid showed that whereas radioactivity from acetate was incorporated into saturated, monoand polyunsaturated fatty acids, the radioactivity from [U-¹⁴C] glucose was found only in 16∶0, 18∶0, and 18∶1. No radioactivity was found in polyunsaturated fatty acids even after concentration of this fraction by AgNO₃:SiO₂ thin layer chromatographic method. This difference is discussed in hypothetical terms of nonhomogeneous acetyl CoA pool, formation of acetyl CoA from glucose exclusively inside the mitochondria, and activation of injected acetate to acetyl CoA.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. I. Disaturated monoenoic triacylglycerols

The triacylglycerols of winter butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver ion high-performance liquid chromatography (Ag-HPLC). The acyl carbon number distribution of the triacylglycerols in each fraction was elucidated by reversed-phase HPLC and mass spectrometry (MS). The MS analysis of each fraction gave deprotonated triacylglycerol [M - H]⁻ ions which were produced by chemical ionization with ammonia. The daughter spectrum of each of the [M - H]⁻ ions provided information on its fatty acid constituents. Successful fractionation of triacylglycerols differing in the configuration of one fatty acyl residue by Ag-HPLC was important because geometrical isomers could not be distinguished by the MS system used. In addition to the fatty acid compositions, reversed-phase HPLC analysis demonstrated the purity of the collected fractions: molecules having acis-trans difference were separated nearly to the baseline. Triacylglycerols differing in the configuration of one fatty acyl residue were not equally distributed in relation to their acyl carbon numbers. This indicates that during the biosynthesis of triacylglycerols,cis- andtrans-fatty acids are processed differently. Although the fatty acid compositions of the corresponding molecular weight species of disaturatedtrans- and disaturatedcis-monoenoic triacylglycerols were similar, there may be differences in the amounts of different fatty acid combinations or in the distribution of fatty acids between the primary and secondary glycerol positions. In addition to the main components, it was possible to analyze minor triacylglycerols, such as molecules containing one odd-chain fatty acid, by the MS system used.     

Metabolism of long-chain fatty acids,alcohols and alkylglycerols in the fish parasiteParatenuisentis ambiguus (Acanthocephala)

Specific differences between the acyl composition of lipids on the helminthParatenuisentis ambiguus and its host eel, as shown previously, prompted us to study the lipid metabolism in this intestinal fish parasite. Adults and larvae ofP. ambiguus were fed various lipid precursors, e.g., fatty acids, long-chain alcohols and 1-O-alkylglycerols, which may occur as common nutrients of intestinal parasites. Incorporation of [1-¹⁴C]palmitic acid into neutral and polar lipids was found to be similar under aerobic and near-anaerobic conditions. In adult parasites maintained in culture medium supplemented with glucose, [1-¹⁴C]palmitic acid was incorporated mainly into triacylglycerols and phosphatidylcholines, whereas [1-¹⁴C]oleic acid was incorporated preferentially into triacylglycerols. In fasted adults, as well as in larvae, [1-¹⁴C]oleic acid was mainly transferred to phosphatidylcholines. Lipolytic activity was detected in adult parasites that had been incubated with radioactive trioleoylglycerol. [1-¹⁴C]Hexadecan-1-ol was oxidized inP. ambiguus at a high rate to labeled palmitic acid, which was incorporated into various lipid classes ofP. ambiguus. Small but significant proportions of radioactivity from hexadecan-1-ol were incorporated into ether glycerolipids of the parasite. A more direct precursor in ether glycerolipid metabolism, i.e.,rac-1-O-[1′-¹⁴C] hexadecylglycerol, was incorporated into alkyl and 1′-alkenyl moieties of choline and etha-nolamine etherglycerophospholipids ofP. ambiguus in high yield. High proportions of labeled diacylglycerols, triacylglycerols and steryl esters were detected in surface lipids as well as lipid extracts of the culture media after incubation ofP. ambiguus with [1-¹⁴C]palmitic or [1-¹⁴C]oleic acids. The results suggest that palmitic acid and oleic acid are incorporated into neutral and polar lipids ofP. ambiguus maintained in glucose medium quite differently with oleic acid showing a strong preference for triacylglycerols. However, the incorporation of palmitic acid in glucose-fed parasites was similar to that of oleic acid in fasted parasites, as well as in larvae. This may be explained by partial fatty acid depletion in fasted worms and rapid cell division in larvae, respectively.     

Evidence for acyl transfer reactions between neutral glycerolipids in dogfish (Squalus acanthias) serum

Radioactively labeled triacylglycerols, 1,3-dioleoyl-2-[1-¹⁴C]-palmitoylglycerol and 1,3-[9,10-³H]-dioleoyl-2-palmitoylglycerol, were incubated with dogfish (Squalus acanthias) serum for periods of up to 10.0 hr. Changes in the positional distributions of carbon-14 and tritium within the triacylglycerols in 5.0 hr and 10.0 hr indicate that intermolecular and intramolecular shifts occur among the fatty acids. In addition, a maximum of 8.3% of the carbon-14 and 5.9% of the tritium was incorporated into 1-alkyl-2,3-diacylglycerols; essentially all of this incorporated radioactivity was associated with the acyl chains in the 1.5 and 5.0 hr incubations. In the 10.0 hr incubations, however, 25% of the tritium incorporated into the 1-alkyl-2,3-diacylglycerols was associated with the 0-alkyl chains. Radioactivity was not incorporated significantly into free fatty acids in the 1.5 and 5.0 hr incubations. These results indicate that acyl transfer reactions take place among molecular species of triacylglycerols, as well as between triacylglycerols and 1-alkyl-2,3-diacylglycerols. In the latter case, the conversions appear to be operative in the virtual absence of net biosynthesis.     

Incorporation of [1-14C] acetate into lipids of soybean cell suspensions

Suspension cultures of soybean cells incorporated [1-¹⁴C] acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 min; more than 75% of this label was found to be in the oleic acid of the phosphatidylcholine. After longer periods of incubation, the proportion of¹⁴C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively,bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglycerols, and the glycolipid fraction. Contribution no. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977.     

Identification of the less common isologous short-chain triacylglycerols in the most volatile 2.5% molecular distillate of butter oil

The isologous short-chain triacylglycerols of the most volatile 2.5% distillate of butter oil were resolved by reversed-phase high-performance liquid chromatography (HPLC) with mass spectrometry. The molecular species were identified by means of the [MH]⁺ and the [MH-RCOOH]⁺ ions in positive chemical ionization mode. A set of empirically determined incremental elution factors was found that could be used to calculate the accurate elution order of natural butterfat triacylglycerols when analyzed by reversed-phase HPLC. The triacylglycerols were also resolved by temperature-programmed gas-liquid chromatography on capillary columns coated with polar liquid phases. The high polarity of the columns provided separation of triacylglycerols on the basis of the degree of unsaturation, as well as on the nature of the shortest acyl chain, with the isologous species having the shortest chainlength eluting last. Both saturated and unsaturated triacylglycerols containing normal and branched-chain odd-carbon fatty acids in combination with short-chain acids were identified, and over 150 molecular species were quantitated.     

Formation of trierucoylglycerol (trierucin) from 1,2-Dierucoylglycerol by a homogenate of microspore-derived embryos ofBrassica napus L

Homogenates of microspore-derived embryos of rape (Brassica napus L.) incubated with [1-¹⁴C]erucoyl-CoA and 1,2-dierucoylglycerol are able to assemble trierucoyl-glycerol (trierucin). In addition, radioactive triacylglycerols are formed by transferring [1-¹⁴C]-erucoyl moieties to endogenous lipid precursors. Stereospecific analysis of radioactive triacylglycerols revealed that labeled erucoyl moieties had been incorporated exclusively into thesn-1,3 positions with more than 95% of radioactivity in thesn-3 position. No incorporation of labeled erucic acid into thesn-2 position has been observed. All data agree with the involvement of 1,2-diacylglycerol acyltransferase (E.C. 2.3.1.20), which utilized 1,2-dierucoylglycerol as well as endogenous 1,2-diacylglycerols as acceptors of erucoyl moieties. This result is of particular interest for the genetic modification of rape and other Cruciferae for producing trierucin in their seed oils. NRCC No. 33513.     

Ratios of Regioisomers of Minor Acylglycerols Less Polar than Triricinolein in Castor Oil Estimated by Mass Spectrometry

We have recently reported the identification of forty new minor molecular species of acylglycerols containing hydroxy fatty acids less polar than triricinolein by electrospray ionization mass spectrometry of the lithium adducts. The ratios of regioisomers of triacylglycerols (ABC and AAB types) and tetraacylglycerols (AAAB type) identified were estimated by the relative abundances of the fragment ions from the neutral losses of fatty acids as α,β-unsaturated fatty acids at the sn-2 position. The order of the contents of regioisomers of triacylglycerols with the fatty acids at the sn-2 position are: nonhydroxy fatty acids > monohydroxy fatty acids > dihydroxy fatty acids > trihydroxy fatty acids. For tetraacylglycerols (AAAB type) such as ricinoleoylricinoleoyl–ricinoleoyl–oleoyl–glycerol (RRRO), ricinoleoylricinoleoyl chain was predominately at the sn-2 position, while ricinoleate was not detected at the sn-2 position.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Structure and Biological Significance of Triacylglycerols Containing Cyclopropene Acyl Moieties

Cyclopropene fatty acids are constituents of seed lipids of various plants, including sources of edible fats, such as cottonseed and kapok. Physiological and toxicological properties of lipids containing cyclopropene acyl moieties are reviewed and the difficulties encountered in the elucidation of the structures of triacylglycerols containing cyclopropene acyl moieties are discussed. A novel method for the analysis of such triacylglycerols is presented.     

The effect of penicillin on fatty acid synthesis and excretion inStreptococcus mutans BHT

Treatment of exponentially growing cultures ofStreptococcus mutans BHT with growth-inhibitory concentrations (0.2 μg/ml) of benzylpenicillin stimulates the incorporation of [2-¹⁴C] acetate into lipids excreted by the cells by as much as 69-fold, but does not change the amount of¹⁴C incorporated into intracellular lipids. At this concentration of penicillin cellular lysis does not occur. The radioactive label is incorporated exclusively into the fatty acid moieties of the glycerolipids. The increase in the radioactive content of the extracellular lipids reflects an actual net increase in the total fatty acid content as determined by a chemical assay. During a 4-hr incubation in the presence of penicillin, the extracellular fatty acid ester concentration (per mg cell dry weight) increases 1.5 fold, even though there is no growth or cellular lysis. No change is observed in the intracellular fatty acid ester content. An indication of the relative rate of fatty acid synthesis was most readily obtained by placingS. mutans BHT in a buffer containing¹⁴C-acetate. Under these nongrowing conditions free fatty acids are the only lipids labeled, a factor which simplifies the assay. The addition of glycerol to the buffer causes all of the nonesterified fatty acids to be incorporated into glycerolipid. The cells excrete much of the lipid whether glycerol is present or not. Addition of penicillin to the nongrowth supporting buffer system does not stimulate the incorporation of [¹⁴C]-acetate into fatty acids. However, if cells are exposed to penicillin in a growth-supporting medium and then are transferred to the nongrowing buffer system containing no penicillin, the previously exposed cells retain the ability to incorporate [¹⁴C]-acetate into fatty acid at a higher rate than untreated cells over a prolonged period of time. The stimulation of [¹⁴C]-acetate into fatty acids in this system parallels but is not dependent on the stimulation by penicillin of the incorporation of [¹⁴C]-glycerol into glycerolipid and lipoteichoic acid synthesis previously demonstrated by our laboratory. The material of this paper is part of a thesis to be submitted by J.L.B. in partial fulfillment of the requirements for the Ph.D. degree from the Department of Biochemistry, Temple University.     

Utilization of extracellular lipids by HT29/219 cancer cells in culture

Uptake and incorporation of long-chain fatty acids were studied in a human colorectal cancer cell line (HT29/219) grown in culture medium supplemented with either fetal calf serum (FSC) or horse serum (HS). The cells were grown for 120 h with no change of medium; the two major cellular lipid classes, the phospholipids and the triacylglycerols, were analyzed at regular time-points. We observed significant changes in the concentration of most fatty acids throughout culture, and differences in their composition when different sera were used to supplement the medium. Minimal levels of free fatty acids were found in the cells, indicating a very small “free fatty acid pool”. A major difference between the cells grown in media supplemented with different sera was the changes observed in concentrations of cellular polyunsaturated fatty acids during growth. In cells grown with FCS (in which 20∶4n−6 is present), the levels of this acid in the phsopholipid and triacylglycerol fractions declined rapidly during cell growth, suggesting further metabolism. In cells grown in medium supplemented with HS, 18∶2n−6 was the major polyunsaturated acid present. There was clear evidence that this acid accumulated in the cellular triacylglycerol and phospholipid fractions. Furthermore, its concentration did not decline during growth in culture, suggesting minimal conversion to other polyunsaturated n−6 acids. Our results suggest that fatty acids from additional sources in the medium, for example triacylglycerols and phospholipids associated with the lipoproteins, are taken up by the cells. There is also indication of cellular fatty acid synthesis, particularly of monounsaturated and saturated acids during the culture period. HT29/219 cells were shown to take up and incorporate radioactivity when trace amounts of [1-¹⁴C]-labeled arachidonic, linoleic or oleic acids were added to the culture medium. Most (80%) of the label was detected in cellular phospholipids and triacylglycerols, although the specific activities of these various fatty acids were different in the two lipid fractions.     

Composition of triacylglycerols containing cyclopropene fatty acids in seed lipids of Munguba (Bombax munguba Mart.)

The seed lipids of Munguba (Bombax munguba Mart., Bombacaceae) were found to contain substantial proportions (ca. 30%) of cyclopropene acyl moieties (sterculoyl and malvaloyl). In a novel approach to the analysis of glycerolipids containing cyclopropene acyl moieties, the triacylglycerols of Munguba seed were first treated with silver nitrate in acetonitrile-acetone (1:1, v/v), in order to convert the cyclopropene groups to the correspondingα,β-unsaturated ketones. The resulting triacylglycerols were fractionated by thin layer chromatography into molecular species containing none, one and two keto (corresponding to none, one and two cyclopropene) acyl moieties per molecule. Each of these molecular species was further fractionated according to carbon numbers by gas chromatography, and the positional distribution of the acyl moieties in each species was determined. Both sterculoyl and malvaloyl moieties were found predominantly at thesn-2 position of the triacylglycerols. The major triacylglycerols of Munguba seed were found to be 1,3-dipalmitoyl-2-oleoylglycerol, 1,3-dipalmitoyl-2-linoleoylglycerol and 1,3-dipalmitoyl-2-sterculoylglycerol.