Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from ¹⁴C-oleate of 1-palmitoyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[¹⁴C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.     

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.     

Incorporation of laurate and hydroxylaurate into phosphatidylcholines and acylglycerols in castor microsomes

Because castor produces oil with a high content of hydroxyl FA (90% ricinoleate), we were interested in determining the flexibility of castor seed microsomes in incorporating other hydroxyl FA into castor oil. To this end, we incubated the [¹⁴C]-labeled 12:0 FA laurate (La), 11-hydroxylaurate, and 12-hydroxylaurate with castor microsomes that were capable of synthesizing castor oil. The molecular species of PC and acylglycerols (AG) incorporating these nonendogenous FA of castor were identified by reversed-phase C₈ and C₁₈ HPLC, respectively. [¹⁴C]Laurate was incorporated into the molecular species of PC and AG at levels of 10 and 4%, respectively, that of [¹⁴C]ricinoleate. Similar to those from the incorporation of six [¹⁴C]FA reported previously [ricinoleate (R), oleate (O), linoleate (L), linolenate (Ln), stearate (S), and palmitate (P)], the molecular species of PC incorporating [¹⁴C]laureate were LLa-PC>PLa-PC>OLa-PC>LnLa-PC>SLa-PC>RLa-PC. The molecular species of AG incorporating [¹⁴C]laurate were RRLa>LaLa>RLa>RLLa>ROLa>LOLa>LLLa>LLa>LLnLa>RSLa>OOLa. The retention times for lipids incorporating laurate were similar to those of lipids incorporating linolenate, because the equivalent carbon numbers of laurate and linolenate are the same. Relative retention times of the molecular species of PC and AG containing laurate are also reported here. The incorporation of 11-hydroxylaurate and 12-hydroxylaurate into PC and AG was not detected.     

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5°C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual subtrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.     

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids

Freshly isolated rat hepatocytes were incubated for 20 min with [U-¹⁴C]glycerol in the presence or absence of unlabeled linoleic (18∶2n-6), arachidonic (20∶4n-6), or docosahexaenoic (22∶6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (≈95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PF), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-¹⁴C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.     

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.     

Ratios of Regioisomers of the Molecular Species of Triacylglycerols in Lesquerella (Physaria fendleri) Oil Estimated by Mass Spectrometry

The ratios of regioisomers of 72 molecular species of triacylglycerols (TAG) in lesquerella oil were estimated using the electrospray ionization mass spectrometry of the lithium adducts of TAG in the HPLC fractions of lesquerella oil. The ratios of ion signal intensities (or relative abundances) of the fragment ions from the neutral losses of fatty acids (FA) as α‐lactones at the sn‐2 position (MS³) of the molecular species of TAG were used as the ratios of the regioisomers. The order of the preference of FA incorporation at the sn‐2 position of the molecular species of TAG in lesquerella was as: normal FA > OH18 (monohydroxy FA with 18 carbon atoms) > diOH18 > OH20 > diOH20, while in castor was as: normal FA > OH18 > OH20 > diOH18 > triOH18. Elongation (from C₁₈ to C₂₀) was more effective than hydroxylation in lesquerella to incorporate hydroxy FA at the sn‐1/3 positions. The block of elongation in lesquerella may be used to increase the content of hydroxy FA, e.g., ricinoleate, at the sn‐2 position of TAG and to produce triricinolein (or castor oil) for industrial uses. The content of normal FA at the sn‐2 position was about 95 %, mainly oleate (38 %), linolenate (31 %) and linoleate (23 %). This high normal FA content (95 %) at the sn‐2 position was a big space for the replacement of ricinoleate to increase the hydroxy FA content in lesquerella oil. The content of hydroxy FA at the sn‐1/3 positions was 91 % mainly lesquerolic acid (85 %) and the content of normal FA was 6.7 % at the sn‐1/3 position in lesquerella oil.     

Platelet-activating factor modulates phospholipid acylation in human neutrophils

The present study showed that platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) rapidly (within 15 sec) stimulated the incorporation of both [1-¹⁴C]arachidonate and [1-¹⁴C]docosahexaenoate into phosphatidylinositol (PI) and phosphatidylcholine (PC) in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation (percentage of control) was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10⁻⁷ M and started to decline at 10⁻⁶ M. Extracellular Ca²⁺ was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporated of extracellular fatty acids into PI and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA.     

Diacylglycerol acyltransferases from Vernonia and Stokesia prefer substrates with vernolic acid

Genetic engineering of common oil crops for industrially valuable epoxy FA production by expressing epoxygenase genes alone had limited success. Identifying other key genes responsible for the selective incorporation of epoxy FA into seed oil in natural high accumulators appears to be an important next step. We investigated the substrate preferences of acyl CoA: diacylglycerol acyltransferases (DGAT) of two natural high accumulators of vernolic acid, Vernonia galamensis and Stokesia laevis, as compared with a common oilseed crop soybean. Developing seed microsomes were fed with either [¹⁴C]oleoyl CoA or [¹⁴C]vernoloyl CoA in combinations with no exogenous DAG or with 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-vernoloyl-sn-glycerol, 1,2-divernoloyl-sn-glycerol, 1,2-dioleoyl-rac-glycerol, or 1,2-divernoloyl-rac-glycerol to determine their relative incorporation into TAG. The results showed that in using sn-1,2-DAG, the highest DGAT activity was from the substrate combination of vernoloyl CoA with 1,2-divernoloyl-sn-glycerol, and the lowest was from vernoloyl CoA or oleoyl CoA with 1,2-dioleoyl-sn-glycerol in both V. galamensis and S. laevis. Soybean DGAT was more active with oleoyl CoA than vernoloyl CoA, and more active with 1,2-dioleoyl-sn-glycerol when oleoyl CoA was fed. DGAT assays without exogenous DAG, or with exogenous sn-1,2-DAG fed individually or simultaneously showed consistent results. In combinations with either oleoyl CoA or vernoloyl CoA, DGAT had much higher activity with rac-1,2-DAG than with their corresponding sn-1,2-DAG, and the substrate selectivity was diminished when rac-1,2-DAG were used instead of sn-1,2-DAG. These studies suggest that DGAT action might be an important step for selective incorporation of vernolic acid into TAG in V. galamensis and S. laevis.     

The metabolism of 1-acyl-PAF in rabbit platelets and its possible interaction with PAF

The metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF), a naturally occurring analogue of platelet activating factor (PAF), was investigated in rabbit platelets. Our studies showed that 1-acyl-[³H]PAF (1-palmitoyl-2-acetyl-sn-glycero-3-phospho[N-methyl-³H]-choline) was converted by platelets into phosphatidyl-[³H]choline ([³H]PC) in a time-dependent fashion. The formation of [³H]PC occurred at a rate similar to that observed when lyso-[³H]PC (palmitoyl-sn-glycero-3-phospho[N-methyl-³H]choline) was used as substrate. In addition, a time-dependent increase in the level of water-soluble radioactivity was observed during the incubation of platelets with either 1-acyl-[³H]PAF or lyso-[³H]PC. This increase was parallel to the formation of [³H]PC and was not observed in the presence of [¹⁴C]PAF (1-octadecyl-2-acetyl-sn-glycerol-3-phospho[N methyl-¹⁴C]choline). Analysis by thin-layer chromatography showed that the soluble radioactivity was mainly associated with glycerophosphocholine (GPC). On the other hand, the preincubation of platelets with phenylmethylsulfonyl fluoride, an inhibitor of the acetylhydrolase, reduced the hydrolysis of 1-acyl-[³H]PAF to [³H]GPC with a concomitant accumulation of radioactivity in 1-acyl-PAF. These findings suggest that 1-acyl-PAF is converted into PC through deacetylation-reacylation with lysoPC as an obligatory intermediate. The findings also indicate that the lysoPC resulting from 1-acyl-PAF is either reacylated to phosphatidylcholine (PC) or hydrolyzed to GPC by lysophospholipase. Finally, we showed that the stimulation of platelets with PAF led to a time- and concentration-dependent increase in the conversion of 1-acyl-[³H]PAF to [³H]PC. The stimulatory effect of PAF was not observed when platelets were lysed before incubation, suggesting that PAF enhances the metabolism of 1-acyl-PAF, probably by accelerating its translocation through the plasma membrane.     

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-¹⁴C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-¹⁴C]LA. [1-¹⁴C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-¹⁴C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-¹⁴C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-¹⁴C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-¹⁴C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

Absorption of synthetic,stereochemically defined acylglycerols in the rat

The stereochemistry of fat digestion and absorption was investigated in rats with thoracic duct fistulas, after feeding synthetic triacylglycerol or alkyldiacylglycerol. After feeding 1,2-dilauroyl-3-oleoyl-sn-glycerol, dilauroyloleoylglycerol and lauroyldioleoylglycerol were the most abundant chyle triacylglycerols. Positional analysis of the fatty acid distribution and the absence of optical activity indicated that the following structures dominated:rac-1,2-dilauroyl-3-oleoylglycerol andrac-1,3-dioleoyl-2-lauroylglycerol. Therefore, the triacylglycerol resynthesized from 2-lauroylglycerol (precursor to 60% of chyle triacylglycerol) and other precursors was essentially racemic. Chyle phospholipids contained largely endogenous fatty acids, and the proportion of lauric acid was very low. A racemic mixture of 1,2-di[³H] oleoyl-3-tetradecyl-sn-glycerol and 1-tetradecyl-2,3-di[¹⁴C] oleoyl-sn-glycerol was absorbed to a lower degree than triacylglycerol. The appearance of oleic acid with different labels in chyle and intestinal lipids did not differ, indicating the absence of stereospecificity in fat digestion. Possible explanations for the low absorption are discussed.     

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [³²P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine to the medium,¹⁴C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,¹⁴C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the¹⁴C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min. Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A₁ and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A₂ was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A₁ and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

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.     

The relative incorporation of arachidonic and eicosapentaenoic acids into human platelet phospholipids

The incorporation of arachidonic acid (AA) as compared to eicosapentaenoic acid (EPA) into human platelet phospholipids was tested by incubating washed platelets with a known mixture of [³H]AA and [¹⁴C]EPA. Following incubation, the platelet lipids were extracted, the individual phospholipids—phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE)—were separated by thin layer chromatography, and their corresponding [³H]/[¹⁴C] ratios were determined. Based on a [³H]/[¹⁴C] ratio of unity for the substrate mixture, the PC, PS, PI and PE exhibited ratios of 0.55, 0.93, 1.12 and 0.74, respectively, which were significantly different from 1.00 in all instances except in the case of PS. These results indicate that PC and PE selectively incorporated EPA, while PI showed preference toward AA. These selectivities may account partly for the differing AA/EPA mass ratios that have been observed among the individual phospholipids of human subjects consuming fish oils.     

Prolonged retention of doubly labeled phosphatidylcholine in human plasma and erythrocytes after oral administration

The plasma kinetics of a preparation of dilinoleoyl phosphatidylcholine (DLPC) specifically labeled with³H in the choline moiety and with¹⁴C in the 2-fatty acid (FA) were evaluated in six healthy volunteers after oral administration. Retention of both isotopes in plasma exceeded expectations, with a half-life in the elimination phase of 172.2 h for³H and 69.7 h for¹⁴C. Up to 60 d after administration, there were still significant levels of radioactivity present in plasma. The relative stability of the [¹⁴C]FA label was demonstrated by the retention for more than 12 h of an isotope ratio close to that of the compound administered. The¹⁴C label of DLPC remained in position-2, as assessed by cleavage of plasma phospholipids with phospholipase A₂. The [³H]choline label showed an early incorporation into high density lipoproteins and subsequently into low density lipoproteins (LDL); conversely, the¹⁴C radioactivity was rapidly incorporated into triacylglycerols that were mainly associated with very low density lipoproteins. Radioactivity measurements revealed that both isotopes remained the longest time in LDL. In red blood cell (RBC) lipids, [³H]choline radioactivity accumulated over time, with a plateau after 48 h, whereas FA radioactivity accumulated more rapidly and was followed by a progressive decay. Analysis of the isotope ratio in these cells suggested an early incorporation of lyso products followed by rapid transfer of FA from plasma. The RBC maintained considerable radioactivity for a prolonged time, thus acting as a possible reservoir for the DLPC administered. Our study showed that dilinoleoyl PC remained in plasma longer than predicted based on earlier studies, and that after absorption the FA label was found in position 2.     

Metabolic transformation of intracraneally injected [1-14C]linoleic and [1-14C]α-linolenic acids in malnourished developing rats

The effect of a low protein diet during pregnancy and lactation on the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from brains of ten-day-old rats was studied. The results indicated that partial deprivation of protein during early development was associated with an increase in the fatty acids of the n−9 family in PC. The fatty acids of the linoleic acid series decreased in PE but were not modified in PC. These minor changes did not affect the double bond index values either in PC or in PE. The effect of protein depletion on thein vivo metabolic transformation of intracraneally injected [1-¹⁴C]linoleic and [1-¹⁴C]α-linolenic acids was also studied. The percentage distribution of the labeled precursors and their derivatives among PC and PE differed from that of mass distribution. These results indicate that the direct uptake of polyunsaturated fatty acids from the blood and/or the low turnover rate of these acids incorporated into PC and PE might be involved in maintaining the fatty acid pattern of these brain lipids.