Different metabolic rates for arachidonoyl molecular species of ethanolamine glycerophospholipids in rat brain

The ethanolamine glycerophospholipids (EGP) contain most of the arachidonate (20:4, n-6) and adrenate (22:4, n-6), potential precursors of biologically potent prostaglandins and related compounds. Much better methods utilizing high performance liquid chromatography (HPLC) techniques are now available for the study of the molecular species of all three classes, namely diacyl, alkenylacyl (plasmalogen), and alkylacyl. Different molecular species may have different functions. This possibility was studied by examining the rates of incorporation of [3H]arachidonic acid into the three major molecular species of each of the three classes of ethanolamine glycerophospholipids. After the intracerebral injection of [3H]20:4 into rat brain, it was rapidly converted to 22:4(n-6). Of the total radioactivity, 10-20% was located in 22:4 in alkenylacyl and diacyl-GPE. In the alkylacyl-GPE, labeled 22:4 was preferentially incorporated and accounted for 50-60% of the total radioactivity. The primary arachidonoyl molecular species of alkenylacyl, alkylacyl, and diacyl-GPE were the 18:1-20:4, 16:0-20:4, and 18:0-20:4 species. The alkylacyl class contained almost equal proportions of these three molecular species. On the other hand, the 20:4 in alkenylacyl and diacyl classes was combined largely with 18:0 groups at the sn-1 position. In particular, the 18:0-20:4 species comprised about 80% of arachidonoyl molecular species of the diacyl class. In all three classes, the highest specific radioactivities were found in the 18:1-20:4 species, whereas the 18:0-20:4 species had the lowest specific radioactivity. Over the period 60 min-24 hr, the diacyl 18:0-20:4 and all three arachidonoyl molecular species of the alkenylacyl class increased in specific radioactivity more rapidly than the other arachidonoyl molecular species.     

Relative degradation of different arachidonoyl molecular species of choline glycerophospholipids in opsonized zymosan-stimulated rabbit alveolar macrophages

The relative degradation of arachidonoyl molecular species of glycerophospholipids prelabeled with [3H]20:4 caused by opsonized zymosan was studied in rabbit alveolar macrophages using a recently developed high-performance liquid chromatographic method. The opsonized zymosan caused the release of [3H]20:4 only from choline glycerophospholipids, no significant changes being observed in the radioactivities of other glycerophospholipids and triacylglycerol. Choline glycerophospholipids were resolved into seven arachidonoyl molecular species, which differed as to the alkyl ether or acyl residue bound at the 1-position, by high-performance liquid chromatography. Arachidonate was predominantly located in the alkyl type having 16:0 at the 1-position which comprised more than half of the total arachidonoyl molecular species of choline glycerophospholipids. The radioactivities of all arachidonoyl molecular species of choline glycerophospholipids, except for the 18:2-20:4 and 18:1-20:4 species of diacylglycerophosphocholine, decreased to 80-85% of the control values as a result of the challenge with opsonized zymosan for 1 h. However, 50% of the released 20:4 came from the 16:0-20:4 species of alkylacylglycerophospholipids, which were the most predominant species of choline glycerophospholipids. The present results indicate that the 16:0-20:4 species of alkylacylglycerophosphocholine is a significant source of arachidonate and 1-O-alkyl-2-lysoglycerophosphocholine, the precursor of the platelet-activating factor, relative to other arachidonoyl species in activated alveolar macrophages.     

Metabolism of unique diarachidonoyl and linoleoylarachidonoyl species of ethanolamine and choline phosphoglycerides in rat testes

Selected molecular species of rat testicular 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15-3H]arachidonic acid, the 20:4-20:4 and 18:2-20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4-20:4 and 18:2-20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1-20:4 and 18:0-20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0-20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonate. These data indicate that the 20:4-20:4, 18:2-20:4 and 18:1-20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [3H]arachidonic acid and that they appear to serve as the source of the [3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.     

Composition and incorporation of [3H]arachidonic acid into molecular species of phospholipid classes by cultured human endothelial cells

Based on quantitative high-performance liquid chromatographic analyses of molecular species in selected phospholipid subclasses from culture human umbilical vein endothelial cells, the relative degree of unsaturation was ethanolamine plasmalogens greater than phosphatidylethanolamine greater than phosphatidylcholine. A total of 36 different molecular species were identified in the phosphatidylcholine fraction. Interestingly, the phosphatidylcholine contained a significant amount (11.7%) of the dipalmitoyl species, a lipid normally associated with lung surfactant. The arachidonoyl-containing molecular species of phosphatidylserine/inositol were labeled to the highest extent and the ethanolamine plasmalogens contained the lowest specific radioactivity after incubating [3H]arachidonic acid with human endothelial cells for 4 h. Within each phospholipid subclass the arachidonoyl species where both acyl groups of the phospholipid are unsaturated (20:4-20:4, 18:2-20:4 + 16:1-20:4, and 18:1-20:4) had higher specific radioactivities, after labeling with [3H]arachidonic acid, than those that contained saturated aliphatic chains (16:0-20:4 and 18:0-20:4). This indicates that the unsaturated species have higher turnover rates.     

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.     

Highly unsaturated phospholipid molecular species of rat erythrocyte membranes: selective incorporation of arachidonic acid into phosphoglycerides containing polyunsaturation in both acyl chains

This study describes for the first time the complete molecular species composition and turnover of [3H]arachidonic acid in various glycerophospholipid classes of rat erythrocytes, a model system that has been extensively used to investigate numerous membrane phenomena. Quantitative analysis of the individual molecular species of the choline, ethanolamine, serine, and inositol glycerophospholipid classes was possible by preparing their diradylglycerobenzoate derivatives that can be quantitated by on-line uv detection in conjunction with high-performance liquid chromatography; turnover of the molecular species containing arachidonate was evaluated in erythrocytes labeled with [3H]arachidonic acid. A unique observation was the significant amounts of 22:6-20:4, 20:4-20:4, and 18:2-20:4 species observed in the diacyl fractions of phosphatidylethanolamine and phosphatidylserine. Moreover, the analysis of the specific radioactivities of individual phospholipid species from erythrocytes incubated with [3H]arachidonic acid demonstrated a selective incorporation of arachidonic acid into the most highly unsaturated molecular species in all of the phospholipid classes examined. Although the 22:6-20:4, 20:4-20:4, and 18:2-20:4 species represented only 4.5% of the total mass of the diacyl phosphoglycerides, these species accounted for a major portion (37%) of the arachidonic acid incorporated into the phospholipids. These results demonstrate the existence of unique populations of phospholipid molecules in rat erythrocytes with a high degree of unsaturation that exhibit a very rapid metabolic turnover rate.     

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.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).     

Release of arachidonic acid from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, a precursor of platelet-activating factor, in rat alveolar macrophages

Platelet activating factor and the bioactive metabolites of arachidonic acid are secreted by alveolar macrophages in response to stimulation by phagocytic agents or calcium ionophore. We have previously shown a deacylation-acetylation sequence in the formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) from alkylacyl-(long chain)-GPC (Albert, D.H. and Snyder, F. (1983) J. Biol. Chem. 258, 97-102). This sequence may be an important source of 20:4 during inflammatory reactions since, in alveolar macrophages, the ether lipid precursor of PAF represents 35% of the choline glycerophospholipids and has a much higher content (35%) of 20:4 in the sn-2 position than does diacyl-GPC (17%). Alveolar macrophages prelabeled with 14C-labeled fatty acids (16:0, 18:1, 18:2 and 20:4) and [1-3H]alkyllyso-GPC were used to study the release of fatty acids from ether-linked and diacyl phospholipids. Each of these fatty acids was incorporated primarily into the choline glycerophospholipids of alveolar macrophages. The release of 20:4 from macrophage phospholipids was increased by treatment of the labeled cells with the calcium ionophore A23187 (2 microM) or zymosan (1 mg/ml), whereas the release of 16:0, 18:1 and 18:2 was not increased above control levels by either stimuli. Although more of the labeled 20:4 is released from the diacyl-GPC (50% of the total released), substantial amounts (44%) of 20:4 are derived from alkylacyl-GPC after incubating the stimulated cells for 60 min. The loss of 20:4 continued from the diacyl species throughout the incubation period studied, whereas a slower net release of 20:4 lost from the alkylacyl-GPC fraction was evident after 2 h. We conclude that the deacylation-reacylation cycle is an important aspect of the metabolism of 20:4 and alkylacyl-GPC during inflammatory stimulation of alveolar macrophages and that the deacylation of this ether-linked phospholipid (which is the first step in the formation of PAF) is responsible for a significant amount of the 20:4 released.     

Composition of phospholipid molecular species from mammary tumors

The molecular phospholipid species of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in rats that were fed diets containing 20 or 3% sunflower-seed oil and different levels of calcium were analyzed by high-pressure liquid chromatography. Twenty-seven molecular species of phospholipids were identified. Phosphatidylcholine was predominantly composed of palmitoyl-arachidonoyl (16:0-20:4) (17-21%), palmitoyl-oleoyl (16:0-18:1) (19-21%), stearoyl-arachidonoyl (18:0-20:4) (12-13%), and 1,2-dipalmitoyl (16:0-16:0) (10-14%) species. The major molecular species of phosphatidylethanolamine were 18:0-20:4 (37-39%) and 16:0-20:4 (10-11%). The composition of diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species from rat mammary tumors was not greatly affected by the different diets.     

Selective changes of docosahexaenoic acid-containing phospholipid molecular species in monkey testis during puberty

Puberty has a profound effect upon the biochemical composition of the testis. We previously demonstrated that puberty was accompanied by great increases in the content of docosahexaenoic acid (DHA; 22:6 n-3) and dihomogamma-linoleic acid (20:3 n-6) and decreases in arachidonic acid (AA; 20:4 n-6) in the phospholipids of testis. In this report, we analyze the composition of the phospholipid molecular species of the ethanolamine and choline glycerophospholipids in the testis of prepubertal (2 years old) and young adult (7-8 years old) monkeys, There was an increase in the DHA species and a decrease in arachidonic species. Interestingly, with few exceptions, among the three molecules with DHA or AA at the sn-2 position, only 16:0-22:6 and 18:0-20:4 changed selectively in opposite directions for both ethanolamine and choline glycerophospholipids. In contrast, there was no such selectivity seen in molecular species containing dihomogamma-linoleic acid or linoleic acid at the sn-2 position. All three dihomogamma-linoleic acid species increased and all three linoleic acid species decreased during puberty. In summary, at puberty, i.e., the onset of spermatogenesis, there are selective changes in the phospholipid molecular species, particularly those containing DHA and AA. These changes suggest a specific functional role of DHA-containing molecular species in the lipid bilayer membranes of sperm cells. A possible link between the composition of DHA-phospholipid molecular species and cellular function is discussed.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Separation of dimethylphosphatidates of alkylacyl glycerophosphocholine and their molecular species by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for separation of the alkylacyl and diacyl analogs of choline glycerophospholipids (CGP) of guinea pig polymorphonuclear leukocytes and their molecular species is described. CGP were hydrolyzed with phospholipase D and then methylated with diazomethane to convert them to dimethylphosphatidates. The dimethylphosphatidates were then separated into the alkylacyl and diacyl subclasses by HPLC on a silica gel column within 15 min. The alkylacyl and diacyl analogs were then separated into individual molecular species by reverse-phase HPLC. Dimethylphosphatidates were resolved into 15 separate peaks, and 11-16 different molecular species of alkylacyl and diacyl glycerophosphocholines were identified on gas-liquid chromatography. The present results indicate that the CGP of polymorphonuclear leukocytes are composed of 27 major molecular species. In the alkylacyl subclass, the most predominant species was the 16:0-18:2 species (32%), followed by the 18:1-18:2 (18%), 16:0-16:0 (16%), and 16:0-18:1 (15%) species. The diacyl type consisted mainly of species with 18:2 at the 2-position, such as the 16:0-18:2, 18:0-18:2, and 18:1-18:2 species, the total percentage of which was 57%.     

Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil

In this study, the initial incorporation of arachidonic acid into human neutrophils has been examined. Neutrophils pulse labeled for 5 min with [3H]arachidonic acid rapidly incorporated this fatty acid into 1,2-diacylglycerophosphocholine. However, when neutrophils were pulse labeled with [3H]arachidonic acid for 5 min, washed, and allowed to incubate for an additional 120 min, the relative amount of [3H]arachidonic acid increased in alkylacylglycerophosphocholine molecular species. Similar, when neutrophils were pulse labeled, washed, and allowed to incubate in the presence of 30 microM unlabeled arachidonic acid for 120 min, [3H]arachidonic acid was also remodeled into alkylacylglycerophosphocholine. These results implied that the initial incorporation of [3H]arachidonic acid proceeded via a free fatty acid intermediate into 1,2-diacyl-GPC, while the subsequent remodeling of arachidonate-containing glycerophospholipids did not. This initial incorporation was further investigated in a number of cell-free systems. Disrupted neutrophils incubated with [14C]arachidonoyl-CoA incorporated [14C]arachidonic acid into 1,2-diacyl-GPC containing 16:0, 18:0, and 18:1 at their sn-1 position in a pattern similar to that seen when whole neutrophils were incubated with arachidonic acid for 5 min. A small percentage of [14C]arachidonate from [14C]arachidonoyl-CoA was incorporated into 1-alkyl-2-acyl-GPC. The enzymatic activity responsible was found predominately in the membrane fraction of the broken cell preparation. This selectivity of the CoA-dependent acyltransferase for 1-acyl-linked glycerophosphocholine was further examined by adding [14C]arachidonoyl-CoA and various 1-radyl-2-lyso-GPC to neutrophil membrane preparations. These studies provide evidence that the initial incorporation of arachidonic acid into sn-glycero-3-phosphocholine takes place by an arachidonoyl-CoA: lysophosphatidylcholine acyltransferase(s) which is selective for the 1-acyl-2-lyso-GPC.     

Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis

Glycosylphosphatidylinositol (GPI) anchor is a major lipidation in posttranslational modification. GPI anchor precursors are biosynthesized from endogenous phosphatidylinositols (PIs) and attached to proteins in the endoplasmic reticulum. Endogenous PIs are characterized by domination of diacyl species and the presence of polyunsaturated fatty acyl chain, such as 18:0-20:4, at the sn-2 position. In contrast, the features of mammalian glycosylphosphatidylinositol-anchored proteins (GPI-APs) are domination of alkyl/acyl PI species and the presence of saturated fatty acyl chains at the sn-2 position, the latter being consistent with association with lipid rafts. Recent studies showed that saturated fatty acyl chain at sn-2 is introduced by fatty acid remodeling that occurs in GPI-APs. To gain insight into the former feature, we analyzed the molecular species of several different GPI precursors derived from various mammalian mutant cell lines. Here, we show that the PI species profile greatly changed in the precursor glucosamine (GlcN)-acyl-PI and became very similar to that of GPI-APs before fatty acid remodeling. They had alkyl (or alkenyl)/acyl types with unsaturated acyl chain as the major PI species. Therefore, a specific feature of the PI moieties of mature GPI-APs, domination of alkyl (or alkenyl)/acyl type species over diacyl types, is established at the stage of GlcN-acyl-PI.     

Altered acyl chain compositions of alkylacyl, alkenylacyl, and diacyl subclasses of choline and ethanolamine glycerophospholipids in rat heart by dietary fish oil

The effects of dietary fish oil containing n - 3 polyunsaturated fatty acids on the fatty acid compositions of the alkylacyl and alkenylacyl species of choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) were studied in rat heart and compared with the corresponding diacylglycerophospholipids. After a 7 week feeding period, all phospholipid classes from the fish oil group exhibited much higher levels of the n - 3 polyunsaturated fatty acids including eicosapentaenoic acid (20:5(n - 30)), docosapentaenoic acid (22:5(n - 3)) and docosahexaenoic acid (22:6(n - 3)), as well as lower levels of the n - 6 series (18:2, 20:4, 22:4 and 22:5), relative to animals given sunflower seed oil-enriched in 18:2(n - 6). However, the docosahexaenoic acid rather than eicosapentaenoic acid provided a much greater contribution to the n - 3 accumulation (fish oil group) in the ether-containing CGP, as indicated by the 20:5(n - 3)/22:6(n - 3) molar ratios of 0.32, 0.26 and 0.56 in the alkylacyl, alkenylacyl and diacyl classes, respectively. In addition to accumulating very high levels of docosahexaenoic acid (e.g., 47.2 mol% of fatty acids in alkenylacylglycerophosphoethanolamine of fish oil group), both ether-linked classes of EGP exhibited significantly higher levels of docosapentaenoic acid than the diacylglycerophosphoethanolamine (GPE) and all classes of CGP. These findings may bear relevance to possible beneficial effects of dietary fish oil on pathophysiological states (including myocardial ischemia) in cardiac tissue and their mediation via platelet-activating factor, 1-alkyl-2-acetylglycerophosphocholine (PAF) and arachidonic acid (20:4(n - 6))-derived eicosanoids.     

Positional and species analysis of membrane phospholipids extracted from goldfish adapted to different environmental temperatures.

1. The proportion of ethanolamine phosphoglycerides in microsomal fractions of goldfish intestine increases at low environmental temperatures. The fatty acyl composition also changes, the proportion of C22:6 and C20:4 fatty acids increasing in positions 1 and 2 and position 2 respectively. The proportion of C16:0 and C18:0 fatty acids falls in position 1 and there is an apparent switch of C18:1 and C20:1 fatty acids from position 2 to position 1. 2. The proportion of choline phosphoglycerides does not depend on the previous environmental temperature of the fish. Temperature-dependent changes in fatty acyl composition in positions 1 and 2 take place in a way similar to that described for ethanolamine phosphoglycerides, but in this case C22:6 substitution is confined to position 2. 3. Choline phosphoglycerides have been further separated into 7 different molecular species. The amounts of species 3 to 7 increase and the amount of species 2 decreases at low adaptation temperature. These changes only account for part of total change in fatty acyl composition. The remaining changes occur by chain substitution within species. 4. Present results show temperature adaptation to be highly complex, involving both quantitative and qualitative changes in different phospholipids. The possible physiological significance of these changes are discussed together with the effects these changes might have on cholesterol-phospholipid interactions.     

DIFFERENTIAL DISTRIBUTION OF [U-14C]GLUCOSE AND [U-14C]GLYCEROL AMONG MOLECULAR SPECIES OF PHOSPHATIDYL CHOLINE,PHOSPHATIDYL ETHANOLAMINE AND 1,2-DIACYLGLYCEROL IN RABBIT BRAIN12

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-¹⁴C]glucose or [U-¹⁴C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of ¹⁴C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.     

On the metabolic relationships between monogalactosyldiacylglycerol and digalactosyldiacylglycerol molecular species in Dunaliella salina

Dunaliella salina cells were pulse-labeled for 2 min with [14C]palmitic acid, [14C]oleic acid, or [14C]lauric acid in order to trace the pathway of galactolipid biosynthesis and desaturation. Through the use of high performance liquid chromatography it was possible to follow the movement of radioactivity through many individual molecular species of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) for periods of 24 h and, in some cases, as much as 120 h. Analysis of the fatty acid fluxes permitted us to refine current views regarding biosynthesis of the predominantly "prokaryotic" galactolipids. The initial D. salina MGDG molecular species, containing paired oleate and palmitate (18:1/16:0), can follow two metabolic routes. If the palmitoyl chain is desaturated to 16:1, the resulting 18:1/16:1 MGDG is subject to rapid further desaturation to varying degrees, and a part of these products is subsequently galactosylated to DGDG. Contrary to widely held opinions, these DGDG molecular species can themselves be further desaturated toward a 18:3/16:4 final product. In a separate series of reactions, a smaller portion of the nascent 18:1/16:0 MGDG is directly galactosylated to 18:1/16:0 DGDG. This molecular species can then be sequentially desaturated to 18:2/16:0 DGDG and 18:3/16:0 DGDG. However, there is only very limited desaturation of the palmitoyl group attached to these molecular species.     

The molecular species composition of diacyl-, alkylacyl- and alkenylacylglycerophospholipids in rabbit alveolar macrophages. High amounts of 1-O-hexadecyl-2-arachidonyl molecular species in alkylacylglycerophosphocholine

The relative composition of molecular species of diacyl-, alkylacyl- and alkenylacylglycerophospholipids in rabbit alveolar macrophages was determined with reverse-phase high-performance liquid chromatography (HPLC). Diacylglycerophosphocholine (GPC) (22.3% of the total glycerophospholipids), alkylacylGPC (11.3%) and alkenylacylglycerophosphoethanolamine (GPE) (15.8%) were the predominant glycerophospholipids in rabbit alveolar macrophages. DiacylGPE (6.9%), diacylGPI (5.5%) and diacylGPS (3.6%) also occurred. 1,2-Diradyl-3-acetylglycerol derived from glycerophospholipids were each resolved into 19 separate peaks with reverse-phase HPLC. By gas-liquid chromatographic quantitation of each peak, 19-29 different molecular species were identified. DiacylGPC, GPE and GPS were mainly composed of saturate, monoene and diene species, such as the 16:0-16:0, 16:0-18:1, 18:0-18:1, and 18:0-18:2 species. The predominant molecular species composing diacylGPI was the 18:0-20:4 species, which represented 40% of this glycerophospholipid. Distinct differences were found in the distributions of arachidonyl molecular species between diacyl- and ether-containing GPC and GPE. Although diacylGPC and GPE included a small amount of arachidonyl molecular species, the 16:0-20:4 species was by far the most prevalent one which composed alkylacylGPC (39% of the total) and alkenylacylGPE (49% of the total). The 16:0-20:4 species of alkylacylGPC and alkenylacylGPE together comprised 60% of the total arachidonyl molecular species of glycerophospholipids. The high amounts of the 16:0-20:4 species in alkylacylGPC may serve as a good source of both the potent platelet-activating factor and the products of arachidonic cascade in the stimulated alveolar macrophages.