Effect of dietary supplementation with a fish oil concentrate on the alkenylacyl class of ethanolamine phospholipid in human platelets

It has been demonstrated that the alkenylacyl class of ethanolamine phospholipid (PE) represents one of the major forms of eicosapentaenoic acid (EPA)-containing phospholipid in the circulating platelets isolated from human subjects consuming a fish oil concentrate. Since the alkenylacyl PE from human platelets is enriched in the eicosanoid precursor arachidonic acid (AA) and the n-6 polyunsaturate adrenic acid (AdA), it was of interest to study changes in alkenylacyl PE fatty acid composition upon fish oil supplementation. Healthy volunteers were given 20 capsules of MaxEPA daily (3.6 g of EPA plus 2.4 g of docosahexaenoic acid, DHA) for 6 weeks followed by a 6-week recovery period. Washed platelet suspensions were prepared and the fatty acid compositions of the phospholipid components were evaluated by thin-layer and gas-liquid chromatography at weeks 0, 3, 6, 9, and 12. Fatty acid composition changes were more pronounced in the alkenylacyl PE than in other platelet phospholipids as a result of fish oil consumption. The alkenylacyl PE exhibited a greater drop (by 20.3 mol%, i.e., from 72.0 to 51.7 mol%) in AA than diacyl PE (by 1.6 mol%) or total (predominantly diacyl) choline phospholipids (PC) (by 4.5 mol%). In alkenylacyl PE, the predominant reservoir of AdA in human platelet phospholipid, a dramatic reduction in the level of AdA also resulted with MaxEPA supplementation (from 7.9 to 3.1 mol%); diacyl PE and total PC decreased by 0.6 and 0.3 mol%, respectively. With respect to the n-3 fatty acids, EPA rose by 12.5 mol% in alkenylacyl PE, compared to only 3.8 and 2.5 mol% in diacyl PE and total PC, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.     

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.     

Preferential synthesis of diacyl and alkenylacyl ethanolamine and choline glycerophospholipids in rabbit platelet membranes

In rabbit platelet membranes, the contents of alkenylacyl phospholipids (plasmalogen) were 56% of phosphatidylethanolamine and 3% of phosphatidylcholine. This uneven distribution of plasmalogens in each phospholipid class could be attributed to the different substrate specificity of ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2). The properties of the enzymes were studied, using endogenous diglycerides and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. The newly formed phospholipids were mainly diacyl and alkenylacyl and only rarely alkylacyl type. The ratios of the labeled alkenylacyl to diacyl type of phospholipids clearly varied with the concentrations of CDP-ethanolamine or CDP-choline. When 1, 10, and 30 microM CDP-[3H]ethanolamine were used, the labeled phospholipids contained 53, 37, and 27% of the alkenylacyl type, respectively. The apparent Km for CDP-ethanolamine to synthesize alkenylacyl and diacyl types were 2.2 and 8.1 microM. On the other hand, when 1, 10, and 30 microM CDP-[14C]choline were used, the labeled lipids contained 10, 17, and 24% alkenylacyl type, respectively. The apparent Km for CDP-choline to synthesize alkenylacyl and diacyl types were 24 and 4.3 microM. Further, the syntheses of diacyl type of phosphatidylethanolamine and the alkenylacyl type of phosphatidylcholine were markedly inhibited by unlabeled CDP-choline and CDP-ethanolamine, respectively. The two enzymes had opposite substrate specificities, and ethanolaminephosphotransferase showed a high preference to plasmalogen synthesis, especially in the presence of CDP-choline.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.     

Fatty chains of alkenylacyl, alkylacyl and diacyl phospholipids in sea urchin spermatozoa.

1. Alkenylacyl, alkylacyl and diacyl phospholipids were analyzed in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. 2. Choline phosphoglycerides (CPG) contained alkylacyl component (19%) in addition to the diacyl component (81%), and alkenylacyl analog was present in a trace amount. The ethanolamine phosphoglycerides (EPG) contained alkenylacyl (51%), alkylacyl (2%) and diacyl (47%) components and the serine phosphoglycerides (SPG), alkylacyl (9%) and diacyl (91%) derivatives. 3. Analysis by gas-liquid chromatography indicated that the fatty chain at the 1-position in alkenylacyl, alkylacyl and diacyl compounds of CPG, EPG and SPG was mainly composed of saturated and monoenoic types (16:0, 18:0, 18:1 and 20:1). In contrast, considerable amounts of polyunsaturated types (20:4 and 20:5) were noted at the 2-position.     

Selective synthesis of the hexaenoic molecular species of ether-linked glycerophosphoethanolamine of Ehrlich ascites tumor cells

In a previous study [Waku, K. and Nakazawa, Y. (1978) Eur. J. Biochem. 88, 489-494], we observed the rapid turnover rate of the molecular species of alkylacyl glycerophosphoethanolamine (Gro-P-Etn) containing docosahexaenoic acid and the high selectivity for this molecular species of ethanolamine phosphotransferase was suggested. To clarify this point, the incorporation of [14C]ethanolamine and [14C]CDP-ethanolamine into the individual molecular species of alkenylacyl, alkylacyl and diacyl Gro-P-Etn has been determined in Ehrlich ascites tumor cells. [14C]Ethanolamine was highly incorporated into the pentaenoic + hexaenoic species of alkenylacyl, alkylacyl and diacyl Gro-P-Etn, whereas incorporation of [14C]ethanolamine into molecular species other than the pentaenoic + hexaenoic species was quite low. The selectivity of ethanolamine phosphotransferase to form the molecular species of alkylacyl and diacyl Gro-P-Etn was examined by incubation of [14C]CDP-ethanolamine and microsomes of Ehrlich ascites tumor cells. The incorporation of [14C]CDP-ethanolamine was found to occur most into the pentaenoic + hexaenoic species of both alkylacyl and diacyl Gro-P-Etn. The present results suggest that the pentaenoic + hexaenoic species are preferentially synthesized among the various kinds of molecular species of alkylacyl and diacyl Gro-P-Etn by the ethanolamine phosphotransferase in Ehrlich ascites tumor cells.     

Incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells

The present experiments characterized the incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells. Confluent cultures were either continuously labeled or pulse labeled with [14C]arachidonic acid. Major lipid classes and ether-linked subclasses of phosphatidyl-ethanolamine (PE) and phosphatidylcholine (PC) were separated by high-performance liquid chromatography and thin-layer chromatography. During continuous labeling, total incorporation of arachidonic acid reached a peak at 8 h and was essentially constant up to 24 h. After 8 h, net label in total PC declined, whereas that in total PE continued to rise. In pulse labeling experiments radioactivity in diacyl PC continuously declined with concomitant increases in both diacyl- and alkenylacyl PE. The data demonstrate that transfer of arachidonic acid from diacyl PC to both diacyl- and alkenylacyl PE occurs in endothelial cells. In contrast to previous observations in platelets, transfer of arachidonic acid to alkenylacyl PE did not require agonist stimulation. This pathway may contribute to the enrichment of endothelial cell PE with arachidonic acid with the potential for subsequent metabolism to prostacyclin.     

Remodeling of arachidonate-containing phosphoglycerides within the human neutrophil

Initial incorporation and subsequent remodeling of 16 phosphoglyceride molecular species containing arachidonate in the human neutrophil have been studied. Neutrophils were pulse-labeled with [3H]arachidonic acid (AA) for 5 min, then phospholipids were analyzed either at this time point or after a subsequent 120-min incubation. [3H]AA was found to be incorporated into phosphoglycerides phosphatidylinositol (PI) greater than phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) by 5 min. Incorporation of [3H]AA was not related to pool size, but reflected an increase in phosphoglyceride turnover. Following the 120-min incubation, only PE gained a significant amount of labeled arachidonate. Specific activity analysis revealed that PI contained the highest labeled/unlabeled ratio at both 5 min and 120 min. After the initial 5-min pulse, the majority of [3H]arachidonate was incorporated into 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-PC, -PE, and -PI showing no preference for fatty acyl chains at the sn-1 position. However, [3H]AA was remodeled into 1-alkyl-acyl-and 1-alk-1-enyl-acyl-sn-glycero-3-PC and -PE molecular species in those neutrophils incubated for the additional 120 min. Specific activities of [3H]AA within all diacyl molecular species were initially higher relative to those alkyl-acyl and alk-1-enyl-acyl molecular species, but for PC and PE became more uniform as label shifted into ether and plasmalogen pools during the additional 120-min incubation. In contrast, the specific activity of 1-stearoyl-2-arachidonoyl-sn-glycero-3-PI remained constant throughout the 120-min incubation.     

Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.     

Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes

Abstract— Three groups of six mice each were killed 1, 4 and 7 days after an intracerebral injection of [1,2-¹⁴C]ethanolamine. The specific radioactivities of the acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and of the acid-stable ethanolamine phosphoglycerides (diacyl and alkyl acyl glycerophosphoryletholamines) from myelin and microsomal fractions were determined. All of these brain ethanolamine phosphoglycerides turn over rapidly with an apparent half-life of less than 3 days. The biosynthesis of alkenyl acyl glycerophosphorylethanolamines from diacyl glycerophosphorylethanolamines in mouse brain myelin or microsomes is unlikely.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Diacyl,Alkylacyl, and Alkenylacyl Phospholipids of Meloidogyne javanica Females

The phospholipid composition and acyl, alkyl, and alkenyl group compositions of diacyl, alkylacyl, and alkenylacyl phosphoglycerides of M. javanica were investigated. Phospholipid was comprised of 61.7% choline phosphoglyceride, 22.0% ethanolamine phosphoglyceride, and smaller quantities of six other lipids. Phospholipid fatty acid was more unsaturated than neutral lipid fatty acid and contained 61.3% octadecenoic (18:1) acid. Fatty acid at the 1-position of diacyl phospholipids was shorter and more saturated than that at the 2-position. Compared to choline phosphoglyceride, ethanolantine phosphoglyceride contained less 18:1 and 20:5 and more 18:0 and 20:0 acid. Alkenylacyl and alkylacyl compounds comprised 34.6% and 9.3%, respectively, of the ethanolamine phosphoglyceride but only 0.5% and 0.6% of the choline phosphoglyceride. Alkenylacyl and alkylacyl ethanolamine phosphoglycerides contained a smaller percentage of 20-carbon polyunsaturated acid at their 2-positions than did their diacyl analogue. At least 95% of the alkenyl and alkyl groups were 18:0 compounds. Tomato roots did not contain alkenylacyl or alkylacyl phosphoglycerides; their occurrence in M. javanica is a significant biochemical difference between the nematode and its host.     

Distribution of arachidonic acid in choline- and ethanolamine-containing phosphoglycerides in subfractionated human neutrophils

Human neutrophils were fractionated on Percoll gradients and the various subcellular fractions were analyzed for phospholipid and fatty acid composition. The results showed that plasma membranes and azurophilic granules were enriched with ethanolamine-(PE) relative to choline-(PC) containing phosphoglycerides. A remarkable degree of uniformity existed throughout the gradient with respect to the subclass composition of the subcellular PC and PE components. In each fraction 50-60% of the PC was diacyl, 40-45% was 1-O-alkyl-2-acyl (ether linked), and 2-5% was 1-O-alk-1'-enyl-2-acyl (plasmalogenic). For PE, 20-25% was diacyl, 7-12% ether linked, and 64-76% plasmalogenic. When neutrophils were incubated for 15 min with [1-14C]arachidonic acid and subfractionated most of the PC-associated label was intracellularly localized. A similar result was observed in PE, however, when the cells were allowed to stand for 2 h in fatty acid-free buffer following the 15 min of labeling and then subfractionated there was a sizable migration of [14C]arachidonate into plasma membrane PE. In all cases the diacyl subclass was labeled most heavily after 15 min but after an additional 2 h of incubation in fatty acid-free buffer there was a direct transfer of label to the ether- and plasmalogenic-linked PC and PE subclasses. It was also found that arachidonoyl-coenzyme A 1-acyl-lysophosphatide acyltransferase activity was inherent in all three major membrane types but was enriched in the endoplasmic reticulum/secondary granule fraction. Arachidonate consistently accounted for roughly 5% of the PC and 17% of the PE fatty chain composition in each subcellular fraction. These findings demonstrate that, despite the uniform arachidonate and PC and PE subclass composition within the various neutrophil subcellular fractions, the bulk of the PC- and PE-associated arachidonate is localized in intracellular membranes.     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

Gender-specific fatty acid profiles in platelet phosphatidyl-choline and -ethanolamine

Previous studies suggested that women synthesise docosahexaenoic acid (DHA) more efficiently from their precursors than men. This study investigated the relationship between diet, platelet phospholipids fatty acids and gender. Dietary intake and platelet phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) fatty acids were determined in Caucasian 40 men and 34 women. Absolute and %energy intakes of arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA, and the ratios of total n-6/n-3 PUFA and linoleic/alpha-linolenic acids did not differ between the sexes. However, women had higher DHA in PC (1.19 vs 1.05 wt%, p<0.05) and PE (3.62 vs 3.21 wt%, p<0.05) than men. Also EPA (1.10 vs 0.93 wt%, p<0.05) was higher in women's PE. Conversely, men had elevated AA and total n-6 fatty acids in PC. The higher platelet DHA levels and lower platelet AA/EPA and AA/DHA ratios in women of child-bearing age compared with men, may lead to less platelet aggregation and vaso-occlusion.     

Direct effects of temperature on phospholipid and polyunsaturated fatty acid metabolism in isolated brain cells from rainbow trout, Oncorhynchus mykiss.

1. The direct effects of temperature on the metabolism of [1-14C]18:2(n-6), [1-14C]18:3(n-3), [1-14C]20:4(n-6) and [1-14C]20:5(n-3) were studied in isolated brain cells from rainbow trout, Oncorhynchus mykiss. 2. Recovery of radioactivity from all the polyunsaturated fatty acids (PUFA) in total lipid was significantly greater at 5 and 15 degrees C than at 25 degrees C. 3. The lower incubation temperatures decreased the relative net incorporation of all the 14C-labelled PUFA into phosphatidylcholine (PC) and increased the relative incorporation of the PUFA into the other phosphoglycerides, especially phosphatidylethanolamine (PE). 4. The effects on PC were generally more significant between 25 and 15 degrees C, whereas the effects on PE were generally significant both between 25 and 15 degrees C and between 15 and 5 degrees C. 5. This suggests that the lysophospholipid acyltransferases responsible for the incorporation of PUFA into different phosphoglycerides may have differential sensitivities to temperature. 6. In contrast, the acyltransferase activities showed fatty acyl preferences that were independent of temperature. 7. Although a trend towards decreased activity at 5 degrees C was apparent, temperature generally had little significant effect on the relative percentages of the PUFA metabolized via the desaturase pathways.