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.     

Capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry of γ- and α-linolenic acid containing triacylglycerols in berry oils

The effect of the γ-linolenic acid (18:3n-6) residue on the elution of triacylglycerols on a 25% cyanopropyl-25% phenyl-50% methylpolysiloxane stationary phase was confirmed by using capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry [cSFC-(APCI)MS]. The general elution rule on this stationary phase is that triacylglycerols having the same ACN+2n value coeluted (ACN-acyl carbon number and n=combined number of double bonds in the acyl chains). The different effect of γ- and α-linolenic acid residues on the retention of triacylglycerols and the use of cSFC-(APCI)MS allowed the study of the number of different linolenic acid residue isomer combinations in triacylglycerols with an identical ACN and degree of unsaturation. Stearidonic acid (18:4n-3) residue was found to have a similar effect on the retention behavior of triacylglycerols as that of γ-linolenic acid residue. The abundance of the [M-RCOO]⁺ ion, formed by the loss of one fatty acid moiety of a triacylglycerol, was found to be clearly higher in the case of γ-isomer of the linolenic acid than that of α-isomer in the identical regiospecific position. This indicates that the distance of the double bonds from the glycerol backbone in the acyl chain affects the stability of a triacylglycerol molecule in the (APCI)MS system. The triacylglycerol composition and the fatty acid combinations of triacylglycerols were found to be almost identical in black currant (Ribes nigrum) and alpine currant (R. alpinum) seed oils.     

Identification of milk fat triacylglycerols by capillary supercritical fluid chromatography-atmospheric pressure chemical lonization mass spectrometry

Identification of milk fat triacylglycerols was accomplished by capillary supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. Supercritical carbon dioxide was the carrier fluid in SFC. Ionization was achieved by introducing vapor of ammonia in methanol into the ionization chamber, which resulted in the formation of abundant [M+18]⁺ and [M-RCOO]⁺ ions of triacylglycerols. These ions defined both the molecular weight and the fatty acid constituents of a triacylglycerol, respectively. SFC on a nonpolar stationary phase provided an efficient separation of triacylglycerols according to the combined number of carbon atoms in the acyl chains of a molecule. In addition to the identification of the major chromatographic peaks representing molecules with 26–54 acyl carbons, minor peaks representing triacylglycerols with an odd number of acyl carbons were separated and identified. Furthermore, compositional information on partially separated isobaric triacylglycerols, which differed substantially in the chain length of the fatty acyl residues, was achieved within some of the peaks. A new finding of the present study was the formation of abundant [M+18]⁺ ions of saturated triacylglycerols in addition to diagnostic fragment ions, being of primary importance in structure elucidation. This extends the applicability of capillary SFC-(APCI)MS in the analysis of both saturated and unsaturated triacylglycerols.     

Fullerenoid lipids: first synthesis of structured triacylglycerols containing an Aza-[60]fullerene unit

Some 1,2- and 1,3-diacyl glycerols (with acyl groups as stearyl, oleyl, linoleyl, or stearolyl) were synthesized by conventional methods. The diacyl glycerols were esterified with 6-bromo-hexanoic acid to give the corresponding bromotriacylglycerols (of the type AAB and ABA containing a bromo group at the distal part of the hexanoate chain). The bromo function was transformed to an azide group by reaction of the bromotriacylglycerols with sodium azide. The resulting azido-triacylglycerols were then reacted with [60]fullerence to give the requisite aza-fullerenoid triacylglycerol of the type ABA or AAB (45–62% yield based on the amount of [60]fullerence reacted). The nitrogen atom attached to the carbon cage formed a “[5,6]-open” type aza substructure, which was confirmed by the appearance of 31–32 signals in the region of δ_C 133–148 (carbon shifts of sp ² carbons of the cage) in the ¹³C nuclear magnetic resonance spectra. The spectroscopic and mass spectrometric properties of these novel fullerenoid triacylglycerols are reported.     

Regioisomers of octanoic acid-containing structured triacylglycerols analyzed by tandem mass spectrometry using ammonia negative ion chemical ionization

Tandem mass spectrometry based on ammonia negative ion chemical ionization and sample introduction via direct exposure probe was applied to analysis of regioisomeric structures of octanoic acid containing structured triacylglycerols (TAG) of type MML, MLM, MLL, and LML (M, medium-chain fatty acid; L, long-chain fatty acid). Collision-induced dissociation of deprotonated parent TAG with argon was used to produce daughter ion spectra with appropriate fragmentation patterns for structure determination. Fatty acids constituting the TAG molecule were identified according to [RCO₂]⁻ ions in the daughter ion spectra. With the standard curve for ratios of [M-H-RCO₂H-100]⁻ ions corresponding to each [RCO₂]⁻ ion, determined with known mixtures of sn-1/3 and sn-2 regioisomers of structured TAG, it was possible to determine the proportions of different regioisomers in unknown samples. The method enabled quantification of MML- and MLM-type structured TAG. In the case of MLL- and LML-type TAG, it was possible to determine the most abundant regioisomer in the unknown mixture and estimate the proportions of regioisomers when there were more than 50% MLL-type isomers in the mixture.     

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.     

Capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry of triacylglycerols in berry oils

A capillary supercritical fluid chromatograph (SFC), combined with a triple-quadrupole mass spectrometer (MS) via a liquid chromatography-atmospheric pressure chemical ionization (LC-APCI) interface, was utilized in the analysis of berry oil triacylglycerols. No modification of the commercially available interface was required. Vapor of different solvents, such as methanol, isopropanol, water, or ammonium hydroxide in methanol, was introduced in the sheath gas flow in the APCI source to achieve adequate ionization of triacylglycerols. The separation of triacylglycerols according to acyl carbon number and degree of unsaturation was accomplished on a 20 m × 50 μm i.d. SB-Cyanopropyl-25 column. The resolution of triacylglycerols in the reconstructed ion chromatogram and the sensitivity of the SFC-(APCI)MS system was comparable to or slightly better than that obtained with a flame ionization detector. No baseline drifting was observed during the SFC density programming. Triacylglycerols formed diagnostic [M + H]⁺ and [M - RCOO]⁺ ions with all tested reactant ion solvents except with ammonium hydroxide in methanol, which formed abundant [M + 18]⁺ ions instead of [M + H]⁺ ions. The abundance of the [M + H]⁺ ion increased with increasing degree of unsaturation of a triacylglycerol, whereas the abundance of the [M - RCOO]⁺ ion depended on the regiospecific distribution of the fatty acid moiety between the sn-1/3 positions and the sn-2 position and on the number of double bonds.     

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.     

Fast atom bombardment and tandem mass spectrometry of phosphatidylserine and phosphatidylcholine

Fast atom bombardment (FAB) desorption of phosphatidylserine and various phosphatidylcholines produces a limited number of very informative negative ions. Especially significant is the formation of (M-H)⁻ ions for phosphatidylserine, a compound which does not yield informative high mass ions by other ionization methods. Phosphatidylcholines of not yield (M-H)⁻ ions but instead produce three characteristic high mass ions, (M-CH ₃ ⁺ _⁻, [M-HN(CH₃) ₃ ⁺ ]⁻ and [M-HN(CH_{3 3} ⁺ -C₂H₂]⁻. Both classes of lipids also yield anions attributed to the carboxylate components of these complex lipids. FAB desorption in combination with collisional activation allows for characterization of fragmentation and determination of structural features. Collisional activation of the carboxylate anion fragments from the complex lipids is especially informative. Structural characterization of the fatty acid chain can be achieved as the released saturated carboxylate anions undergo a highly specific 1,4-elimination of H₂, which results in the losses of the elements of CH₄, C₂H₆, C₃H₈...in a fashion entirely consistent with the chemistry of carboxylate anions desorbed from free fatty acids. These C_nH_2n+2 losses begin at the alkyl terminus and progress along the entire alkyl chain. Modified fatty acids undergo a similar fragmentation; however, the modification affects the series of C_nH_2n+2 losses in a manner which permits determining the type of modification and its location on the fatty acid chain.     

Soybean oil triacylglycerol analysis by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Soybean oil triacylglycerols from genetically modified soybean lines were conclusively identified by reversed-phase high-performance liquid chromatography coupled with mass spectrometry with atmospheric pressure chemical ionization. Atmospheric pressure chemical ionization is a soft ionization technique which gives simple spectra for triacylglycerols. Spectral identification of the triacylglycerols was based on the molecular [M+1]⁺ ion and the 1(2)-, 2(3)- and 1(3)-diacylglycerol fragments. Triacylglycerols identified in high-stearic and high-palmitic soybean varieties were quantitated by reversed-phase high-performance liquid chromatography with flame-ionization detection. There was excellent agreement between the fatty acid composition calculated from the triacylglycerol composition and the fatty acid composition obtained by gas chromatography of the transmethylated oils. The oils of the modified soybean varieties, compared to typical soybean oil, contained increased content of triacylglycerols known to be more oxidatively stable, such as linoleoyloleoylstearoyl, linoleoylpalmitoylstearoyl, and linoleoyldipalmitoyl glycerols, and less triacylglycerols like trilinoleoylglycerol, known to decrease oxidative stability. This study showed that the atmospheric pressure chemical ionization technique is suitable for mass spectral identification of neutral molecules, such as triacylglycerols, which do not contain a chargeable functional group.     

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.     

Lymphatic recovery of exogenous oleic acid in rats on long chain or specific structured triacylglycerol diets

Specific structured triacylglycerols, MLM (M=medium-chain fatty acid, L=long-chain fatty acid), rapidly deliver energy and long-chain fatty acids to the body and are used for longer periods in human enteral feeding. In the present study rats were fed diets of 10 wt% MLM or LLL (L=oleic acid [18∶1n−9], M=caprylic acid [8∶0]) for 2 wk. Then lymph was collected 24 h following administration of a single bolus of ¹³C-labeled MLM or LLL. The total lymphatic recovery of exogenous 18∶1n−9 24 h after administration of a single bolus of MLM or LLL was similar in rats on the LLL diet (43% and 45%, respectively). However, the recovery of exogenous 18∶1n−9 was higher after a single bolus of MLM compared with a bolus of LLL in rats on the MLM diet (40% and 24%, respectively, P=0.009). The recovery of lymphatic 18∶1n−9 of the LLL bolus tended to depend on the diet triacylglycerol structure and composition (P=0.07). This study demonstrated that with a diet containing specific structured triacylglycerol, the lymphatic recovery of 18∶1n−9 after a single bolus of fat was dependent on the triacylglycerol structure of the bolus. This indicates that the lymphatic recovery of long-chain fatty acids from a single meal depends on the overall long-chain fatty acid composition of the habitual diet. This could have implications for enteral feeding for longer periods.     

Analysis of triacylglycerols by silver-ion high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Triacylglycerols of the seed oils rich in α- and/or γ-linolenic acid moieties were separated by silver-ion high-performance liquid chromatography (HPLC) followed by on-line atmospheric pressure chemical ionization-mass spectrometric (APCI-MS) detection. Mass spectra of most triacylglycerols exhibited abundant [M + H]⁺ and [M − RCO₂]⁺ ions, which defined the molecular weight and the molecular association of fatty acyl residues of a triacylglycerol, respectively. Silver ions formed weaker complexes with triacylglycerols containing γ-linolenic acid than with those containing α-linolenic acid, i.e., the elution order of molecules wasXYT _γ>XYT _α’,XT _γ T _α>XT _α T _α>, andT _γ T _γ T _γ>T _γ T _γ T _α>T _γ T _α T _α>T _α T _α T _α, whereT _α=α-linolenic acid,T _γ=γ-linolenic acid, andX, Y=fatty acids different from linolenic acid. Furthermore, silver-ion HPLC resulted in partial separation within equally unsaturated triacylglycerols according to differences in the combined number of acyl carbons. Regioisomeric forms of triacylglycerols were not determined from the seed oil samples, although differences were measured with reference compounds in the relative abundances of [M − RCO₂]⁺ ions formed by a loss of a fatty acyl residue from thesn-2 position and thesn-1/3 positions. Silverion HPLC/APCI-MS provided valuable information for structure elucidation of seed oil triacylglycerols: 43 molecular species were identified from cloudberry seed oil, 39 from evening primrose oil, 79 from borage oil, 44 from alpine currant, and 56 from black currant seed oils. The quantitation requires to be studied further, especially in those cases where several molecular weight species of triacylglycerols eluted in a single chromatographic peak.     

Analysis of molecular species of peroxide adducts of triacylglycerols following treatment of corn oil with tert-butyl hydroperoxide

We recently demonstrated that exposure of synthetic mono-and diunsaturated triacylglycerols to tert-butylhy-droperoxide (TBHP) leads to formation of stable adducts of the oxidizing agent and the unsaturated esters (Sjövall, O., Kuksis, A., and Kallio, H., Reversed Phase High-Performance Liquid Chromatographic Separation of tert-Butyl Hydroperoxide Oxidation Products of Unsaturated Triacylglycerols. J. Chromatogr. A 905, 119–132, 2001). In the present study we isolated and identified the TBHP adducts of corn oil triacylglycerols. The much wider range of molecular species available in the corn oil permitted us to demonstrate that the yield of the adducts varies with the degree of unsaturation of the triacylglycerol. The highest yields were obtained for the linoleate (20% of linoleoyl-containing residual triacylglycerols) and the lowest ones for the oleate (5% of oleoyl-containing residual triacylglycerols) tria-cylglycerols, whereas the saturated triacylglycerols did not give TBHP adducts in readily detectable amounts. Normal-phase thin-layer chromatography along with reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization was used to isolate and separate the major molecular species of polyunsaturated triacylglycerols and corresponding TBHP adducts. As an extreme example, the dilinoleoylmonooleoylglycerol was identified as the mono-, di-, tri-, tetra-, and penta-TBHP adduct. LC/MS with electrospray ionization at elevated capillary exit voltage (pseudo tandem mass spectrometry) was used to confirm structures of the [M?RCOOH]⁺ ions and the absence of TBHP adducts of [M?RCOOH]⁺. It is concluded that stable adduct formation is an unavoidable complication of preparation of oxotriacylglycerols by oxidation with concentrated TBHP solutions and care must be taken to resolve the adducts from the desired oxidation product.     

Purification and properties of an extracellular lipase fromPythium ultimum

An extracellular triacylglycerol lipase (EC 3.1.1.3) fromPythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL-6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co²⁺, Fe²⁺, Sn²⁺ and Mn²⁺ and stimulated by Ca²⁺, Mg²⁺, Na⁺, K⁺ and surfactants such as taurocholic acid, Triton X−100,n-octyl glucoside,n-dodecyl-β-D-maltoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate(CHAPS), and 3-[-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having thecis double bond configuration. AAES publication No. 6-933419.     

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.     

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.     

Analysis of the seed oil of Heisteria silvanii (Olacaceae)—A rich source of a novel C18 acetylenic fatty acid

Besides some usual fatty acids (FA), two conjugated ene-yne acetylenic FA [trans-10-heptadecen-8-ynoic acid (pyrulic acid) (7.4%), and trans-11-octadecen-9-ynoic acid (ximenynic acid) (3.5%)], a novel ene-yne-ene acetylenic FA [cis-7, trans-11-octadecadiene-9-ynoic acid (heisteric acid) (22.6%)], and 9,10-epoxystearic acid (0.6%) could be identified in the seed oil of Heisteria silvanii (Olacaceae). Two further conjugated acetylenic FA [9,11-octadecadiynoic acid (0.1%) and 13-octadecene-9,11-diynoic acid (0.4%)] were identified tentatively by their mass spectra. The FA mixture has been analyzed by gas chromatography/mass spectrometry (GC/MS) of their methyl ester and 4,4-dimethyloxazoline derivatives. The structure of heisteric acid was elucidated after isolation via preparative silver ion thin-layer chromatography and by various spectroscopic methods [ultraviolet; infrared; ¹H, ¹³C nuclear magnetic resonance (NMR); ¹H−¹H and ¹H−¹³C correlation spectroscopy]. To determine the position of the conjugated ene-yne-ene system, the NMR spectra were also measured after addition of the lanthanide shift reagent Resolve-Al EuFOD^TM. Furthermore, the triyglyceride mixture was analyzed by high-temperature GC and high-temperature GC coupled with negative chemical ionization MS. A glass capillary column coated with a methoxy-terminated 50%-diphenyl-50%-dimethylpolysiloxane was used for the separation of the triacylglycerol (TAG) species. No evidence of decomposition of the TAG species containing conjugated ene-yne-ene FA was observed. Twenty-six species of the separated TAG were identified by means of their abundant quasi molecular ion [M−H]⁻ and their corresponding carboxylate anions [RCOO]⁻ of the fatty acids, respectively. The major molecular species of the TAG were found to be 16:0/18:1/18:1, 16:0/18:1/18:3 (heisteric acid), 17:2 (pyrulic acid)/18:1/18:1, 18:1/18:1/18:3 (heisteric acid). The TAG containing acetylenic FA showed an unexpected increase of the retention time in comparison to the TAG containing usual FA, thus making the prediction of the elution order of lipid samples containing acetylenic FA difficult.     

Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.     

Influence of dietary fish oil on the relative synthesis of triacylglycerol and phospholipids in rat liverin vivo

The influence of dietary fish oil containing n−3 polyunsaturated fatty acids on the biosynthesis of triacylglycerol relative to total individual phospholipids was studied in rat liverin vivo. The dietary lipid (10% by weight of diet) was either sunflower oil enriched in linoleic acid (SO group) or MaxEPA fish oil/sunflower oil, 9∶1 by weight (FO group) enriched in eicosapentaenoic acid (EPA, 20∶5n−3) plus docosahexaenoic acid (DHA, 22∶6n−3). After a 3-week feeding period, the triacylglycerol content (in μmmol/g liver) was 44% lower in the FO group relative to the SO animals. Thein vivo incorporation of [³H]glycerol into individual hepatic lipids resulted in triacyl-glycerol/total phospholipid radioactivity ratios of 2.1 and 0.9 for the SO and FO groups, respectively. These results indicate an inhibitory effect of dietary EPA/DHA on triacylglycerol relative to phospholipid synthesis from intermediary 1,2-diacylglycerol in rat liverin vivo. This metabolic alteration was accompanied by a substantially lower amount (in μmol/g liver) of arachidonic acid and higher levels of EPA plus DHA in the triacylglycerol, choline glycerophospholipid (CGP), and ethanolamine glycerophospholipid (EGP) of the FO group. A moderately higher labelling of the EGP from [³H]glycerol was observed in the FO as compared to the SO group (as evidenced by CGP/EGP radioactivity ratios of 1.3∶1 and 1.8∶1, respectively). The present study providesin vivo evidence for a dampening effect of dietary fish oil on the synthesis of liver triacylglycerol relative to phospholipid and a moderate alteration ofde novo synthesis of individual phospholipids. Presented in part at the 80th Annual Meeting of the AOCS in Cincinnati, Ohio (May, 1989).