Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.     

Biosynthesis of oleic, arachidonic and docosahexaenoic acids from their C18 precursors in the yolk sac membrane of the avian embryo

The yolk sac membrane (YSM) of the chicken embryo is known to express δ-9 and δ-6 desaturase activities, suggesting that biosynthesis of the unsaturated fatty acids 18:1n-9, 20:4n-6 and 22:6n-3 might occur during the transfer of yolk lipids across the YSM. If so, this biosynthesis could help to satisfy the demands of the embryonic tissues for these unsaturates. To assess the ability of the YSM to perform these conversions, pieces of the tissue were incubated in vitro with the precursor fatty acids, ¹⁴C-18:0, ¹⁴C-18:2n-6 or ¹⁴C-18:3n-3, and the recovery of radioactivity in the respective products, 18:1n-9, 20:4n-6 and 22:6n-3, was determined. After 4 h of continuous incubation, radioactivity from these precursors was incorporated primarily into triacylglycerol and phospholipid of the tissue pieces. Only small proportions (0.3–4.7%) of this incorporated radioactivity were, however, recovered as 18:1n-9, 20:4n-6 or 22:6n-3. The majority of the incorporated label was retained in the form of the precursor fatty acids. After a 1-h pulse incubation with the ¹⁴C precursors, followed by a 3-h chase incubation in the absence of exogenous label, the conversion of incorporated radioactivity to the end product unsaturates was again relatively low (0.5–8.1%). Thus, although conversions of the precursors to the end product fatty acids were detectable in this system, the biosynthesis of these unsaturates is apparently a quantitatively minor pathway in the YSM. Nevertheless, since the amount of 18:2n-6 in the yolk lipids far exceeds that of 20:4n-6, the conversion of even a small proportion of the former to the latter fatty acid could significantly increase the supply of 20:4n-6 to the embryonic tissues.     

Composition, accumulation and utilization of yolk lipids in teleost fish

Lipid reserves in teleost eggs are stored in lipoprotein yolk and, in some species, a discrete oil globule. Lipoprotein yolk lipids are primarily polar lipids, especially phosphatidylcholine and phosphatidylethanolamine (PE), and are rich in (n–3) polyunsaturated fatty acids, especially 22:6(n–3) (docosahexaenoic acid, DHA). Oil consists of neutral lipids and is rich in monounsaturated fatty acids (MUFA). Egg lipids are derived from dietary fatty acid, fatty acid mobilized from reserves and possibly fatty acid synthesized de novo. There is selective incorporation of essential fatty acids, particularly DHA, into yolk lipids and discrimination against incorporation of 22:1(n–11). Lipid is delivered to the oocyte by vitellogenin, which is rich in polar lipids, and likely also by other lipoproteins, especially very low density lipoprotein, which is rich in triacylglycerol (TAG). All classes of lipid may be used as fuel during embryonic and larval development and MUFA are preferred fatty acids for catabolism by embryos. Catabolism of oil globules is frequently delayed until latter stages of development. In some species, DHA derived from hydrolysis of phospholipid may be conserved by transfer to the neutral lipid. Recent work has expanded knowledge of the role of DHA in membrane structure, especially in neural tissue, and molecular species analysis has indicated that PE containing sn-1 oleic acid is a prime contributor to membrane fluidity. The results of this type of study provide an explanation for the selection pressures that influence yolk lipid composition. Future work ought to expand knowledge of specific roles of individual fatty acids in embryos along with knowledge of the ecological physiology of ovarian recrudescence, environmental influences on vitellogenin and yolk lipid composition, and the control of yolk lipid accumulation and utilization.     

Metabolism of n -9, n -6 and n -3 fatty acids in hepatoma Morris 7777 cells. Preferential accumulation of linoleic acid in cardiolipin

The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids.The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Glyceride metabolism in cultured cells dissociated from rat cerebral cortex

Abstract— [1-¹⁴C]stearic acid and [2-³H]glycerol were rapidly taken up and esterified into triacylglycerol and phospholipids by rat brain cells cultivated in monolayers. Expressed in terms of pool size, the incorporation of glycerol and stearate into triacylglycerol was 6- and 8-fold, respectively, higher than the incorporation into the choline phosphoglycerides. Tritium-labelled glycerol in both triacylglycerol and glycerophosphatides was diluted more rapidly than the [¹⁴C] labelled fatty acids. Chase experiments indicated a transfer of fatty acid from one lipid class to another, mainly from triacylglycerol to phospholipids, with no apparent loss of radioactivity. The accumulation of triacylglycerol in the brain cells was a function of both the presence of exogenous fatty acids in the culture medium and the metabolic needs of the cells; as long as the cells were involved in active formation of membranes the proportion of triacylglycerol was relatively small; its concentration increased while cell division slowed down in older, fully monolayered cultures.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.     

Selective retention of n-3 and n-6 fatty acids in human milk lipids in the face of increasing proportions of medium chain-length (C10-14) fatty acids

This paper reports the results of our analysis of the impact high levels of de novo fatty acids have on the proportions of essential and non-essential fatty acids in human milk lipids. The data for seven fatty acids (linoleic, alpha-linolenic, arachidonic (AA), docosahexaenoic (DHA), palmitic, stearic and oleic) were derived from several studies conducted in Nigeria. The proportion by weight of each of these fatty acids was plotted versus the proportion of C10-14 fatty acids. As the proportion of C10-14 fatty acids increased from 15 to 65%, there was not a proportional decrease in the percentages of all seven fatty acids, but, instead, preferential incorporation of the essential fatty acids, AA and DHA into the triacylglycerol component of the milk. At the same time, the proportions of stearic and oleic acid declined by 69% and 86%, respectively. However, the proportions of linoleic acid, palmitic acid, DHA, AA and alpha-linolenic acid, in milk lipids decreased by only 44%, 40%, 39%, 28% and 2.3%, respectively. These observations indicate that as the contribution of C10-14 fatty acids increases, essential fatty acids are preferentially incorporated into milk triacylglycerols at the expense of oleic acid and stearic acid.     

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro

Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.     

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.     

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.