Fatty acid composition of subcellular particles from sheep platelets and topological distribution of phosphatidylethanolamine fatty acids in the plasma membrane

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18∶1; 32–41%), linoleic (18∶2, 10–17%), stearic (18∶0; 13–15%), palmitic (16∶0; 11–15%) and arachidonic (20∶4; 8–12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22∶0), 18∶0 and 16∶0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18∶1n−9. Our findings suggest that fatty acids are asymmetrically distributed between thecholineversus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzenesulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18∶2 and significantly less 18∶1 and 20∶5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturatedversus saturated PE acyl chains.     

Membrane lipid profile of an edible basidiomycete <Emphasis Type="Italic">Lentinula edodes</Emphasis> during growth and cell differentiation

The basidiomycetous mushroom Lentinula edodes (Shiitake) exhibits a unique process of cell differentiation termed “fruiting-body formation”. To clarify the relationship between membrane lipids and fruiting-body formation in this fungus, we investigated variations in levels of phospholipids, cerebrosides, fatty acyl residues in the major phospholipids, and fatty acyl and sphingoid base residues in cerebrosides during vegetative growth and fruiting-body formation. PC, PE, and PS were the primary phospholipids in the cells of L. edodes. After a shift in growth temperature of L. edodes mycelia has been shifted from 25 to 18°C, the proportion of unsaturated FA (UFA), such as linoleic acid (18∶2) and oleic acid (18∶1), increased. In contrast, during fruiting-body formation induced by the temperature downshift to 18°C, 18∶2 of PC in the primordia and fruiting bodies decreased, and the UFA of PF and 18∶1 of PC increased compared with the proportions in mycelia growing at 18°C. These results showed that the proportions of fatty acyl residues in PC and PE differed during fruiting-body formation in L. edodes. Moreover, the amount of cerebrosides in primordia increased compared with those in mycelia and fruiting bodies and, in these differentiating tissues, the proportion of 2-hydroxypentadecanoic acid increased whereas that of 2-hydroxyoctadecanoic acid decreased compared with that in the mycelia. However, the proportion of sphingoid base residues in cerebrosides did not change during fruiting-body formation in L. edodes.     

On the phospholipids ofCulex pipiens fatigans

The lipids of different developmental stages ofCulex pipiens fatigans, vector of bancroftian filariasis, have been investigated. The phospholipid composition of the developmental stages and of the subcellular fractions of fourth instar larvae of the insects were analyzed. The composition of fatty acids and their positional distribution have also been examined in the major phospholipids of the larvae. The insect eggs contained higher amounts of lipids than larvae suggesting that they were utilized during embryogenesis. Phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) comprised over 75% of the insect phospholipids. Of these, PE was present in the greatest amounts during all stages of growth and in the subcellular fractions of larvae. An ethanolamine containing sphingolipid was found as a component of the phospholipids of the insects. About 50% of the lipids of the larvae were localized in the cell debris and nuclei fraction which also contained most of the lysolipids of the insects. As in other Diptera 16∶0, 16∶1 and 18∶1 were the major fatty acids present in the insect lipids of which the fatty acid found in greatest amounts was 16∶1. Similar to the phospholipids of animal species, saturated fatty acids were predominantly linked to the 1 position of the major phospholipids of the insects while the unsaturated fatty acids were in higher amounts at the 2 position.     

Dietary cod liver oil decreases arachidonic acid in rat gastric mucosa and increases stress-induced gastric erosions

The purpose of this study was to examine the influence of long-term feeding of dietary fat rich in either n−3 or n−6 fatty acids on the availability of arachidonic acid (20∶4n−6) in major phospholipids of gastric mucosa in rats. Three groups of male Wistar rats were fed either a standard diet, a cod liver oil-enriched diet (10% by weight), or a corn oil-enriched diet (10% by weight) for 8 mon. Dietary cod liver oil significantly reduced the level of 20∶4n−6 in phosphatidylcholine (PC) and in phosphatidylethanolamine (PE) of gastric mucosa. The loss of 20∶4n−6 was compensated for by eicosapentaenoic acid (20∶5n−3) in PC, whereas the decrease in 20∶4n−6 in PE corresponded to the increase in three n−3 fatty acids: 20∶5n−3, docosapentaenoic acid (22∶5n−3), and docosahexaenoic acid (22∶6n−3). The level of 20∶5n−3 was higher than the level of 22∶6n−3 both in PC and PE of mucosa in rats fed cod liver oil. Diets supplemented with corn oil increased the level of 18∶2n−6 but decreased the monoene fatty acids 16∶1 and 18∶1n−7 in PC but not in PE of gastric mucosa. The 20∶4n−6 levels of both PC and PE were markedly reduced by dietary cod liver oil, to about one-third of control levels. Similar changes were also observed in the stomach wall. Gastric erosions were observed in all rats exposed to restriction stress, but this form of stress induced twice the number of erosions in rats fed fish oil compared to control rats or rats fed corn oil. We conclude that a diet rich in fish oil altered the balance between n−6 and n−3 fatty acids in major gastric mucosal phospholipids, markedly reduced the availability of 20∶4n−6, and increased the incidence of gastric erosions induced by restriction or emotional stress.     

Phosphatidylethanolamine metabolism in rats fed a low methionine,choline-deficient diet

The metabolism of phosphatidylethanolamine (PE) was studied in male rats fed a low methionine diet for 7 days with or without supplemental choline. Groups of animals were injected with 2-¹⁴C-ethanolamine and killed at intervals thereafter up to 72 hr. Liver phospholipids were isolated, and PE and phosphatidylcholine (PC) were separated by argentation chromatography into diene (18∶2), tetraene (20∶4) and hexaene (22∶6) fractions. Fatty acid composition and the distribution of radioactivity and specific activity in the total phospholipids and in the fractions were determined. Choline deficiency did not affect total liver phospholipid, but it did increase the amount of PE and decreased that of PC. The major effect of the deficiency on phospholipid fatty acids was to decrease the proportion of PE arachidonate and to increase that of docosahexaenoate. Ethanolamine incorporation into liver PE of deficient rats was only slightly less than in the controls, but loss of the radioactivity from the PE was slower. Ethanolamine radioactivity appearing in the PC of deficient rats was about half that of the controls, even though specific activities of the PE precursors were similar to the control rats. Choline deficiency increased the biological half-lives of the total PE and its fractions. Although the proportion of PE tetraenoic fraction was reduced, the total amount of this liver PE fraction in deficient rats was not affected. However the amount of hexaenoic fraction was doubled, and it accounted for most of the increased quantity of liver PE seen in deficient animals. The results suggested that in choline deficiency PE synthesis was delayed but not appreciably suppressed, and that limited availability of methionine for methylating the PE fractions in their proper proportions affected the concentrations of the PE fractions and impaired their normal conversion to PC. Presented in part at the AOCS Meeting, Houston, May 1971.     

Molecular species composition of the major diacyl glycerophospholipids from muscle,liver, retina and brain of cod (Gadus morhua)

The molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from white muscle, liver, retina and brain of cod (Gadus morhua) were determined by high-performance liquid chromatography of the respective 1,2-diacylglycerol 3,5-dinitrobenzoyl derivatives. A minimum of 69 diacyl species was identified. In muscle and liver saturated fatty acid/polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid/PUFA molecular species were predominant, particularly 16∶0/20∶5 and 16∶0/22∶6 in PC, 16∶0/22∶6 and 18∶1/22∶6 in PE and 18∶0/22∶6 and 18∶1/22∶6 in PS. Didocosahexaenoyl species were major components of PC, PE and PS from retina, comprising 29.3, 71.8 and 59.7% of the respective totals. Didocosahexaenoyl species were also abundant in PE and PS from brain, accounting for 13.8 and 24.0% of the totals, respectively. DiPUFA species were important in muscle, totalling 21.2% in PC and 38.3% in PE. PC from all tissues had the largest amounts of species containing only saturated or monounsaturated fatty acids, accounting for 59.8% of PC from brain, including 12.8% of 18∶1/24∶1 plus 24∶1/18∶1.     

Influence of diet on conversion of14C1-linolenic acid to docosahexaenoic acid in the rat

¹⁴C₁-Linolenic acid was incorporated into lipids of hearts, livers, and carcasses of male rats. We studied the influence of diet composition on extent and distribution of radioactivity. A CHOW diet, a purified, essential fatty acid (EFA)-deficient diet, a purified control diet, and EFA-deficient diets with four fatty acid supplements were used. Supplements of 18∶2n−6, 20∶4n−6, 18∶3n−3, and 22∶6n−3 were given as single doses. Radioactivities in liver phosphatidyl ethanolamines (PE), phosphatidyl cholines, and neutral lipids were measured. The distribution of radioactivity among the fatty acids in liver phospholipids was determined. Rats on CHOW diet incorporated far less radioactivity than any other group into lipids of hearts and livers. Most of the activity in livers was recovered as 20∶5n−3 and 22∶6n−3 in all rats. In EFA-deficient rats, the radioactivity in 22∶6n−3 of liver PE was still increasing 36 hr after¹⁴C₁-linolenic acid had been administered. The n−6 supplements (18∶2n−6 and 20∶4n−6) seemed to reduce the conversion of 20∶4n−3 to 20∶5n−3 (desaturation), whereas the n−3 supplements (18∶3n−3 and 22∶6n−3) reduced the conversion of 20∶5n−3 to 22∶5n−3 (elongation). Formation of 22∶6n−3 may be controlled by 22∶6n−3 itself at the elongation of 20∶5n−3 to 22∶5n−3.     

Dietary linoleic acid and the fatty acid profiles in rats fed partially hydrogenated marine oils

The influence of the linoleic acid levels of diets containing partially hydrogenated marine, oils (HMO) rich in isomeric 16∶1, 18∶1, 20∶1 and 22∶1 fatty acids on the fatty acid profiles of lipids from rat liver, heart and adipose tissue was examined. Five groups of rats were fed diets containing 20 wt% fat−16% HMO+4% vegetable oils. In these diets, the linoleic acid contents varied between 1.9% and 14.5% of the dietary fatty acids, whereas the contents oftrans fatty acids were 33% in all groups. A sixth group was fed a partially hydrogenated soybean oil (HSOY) diet containing 8% linoleic acid plus 32%trans fatty acids, mainly 18∶1, and a seventh group, 20% palm oil (PALM), with 10% linoleic acid and notrans fatty acids. As the level of linoleic acid in the HMO diets increased from 1.9% to 8.2%, the contents of (n−6) polyunsaturated fatty acids (PUFA) in the phospholipids increased correspondingly. At this dietary level of linoleic acid, a plateau in (n−6) PUFA was reached that was not affected by further increase in dietary 18∶2(n−6) up to 14.5%. Compared with the HSOY- or PALM-fed rats, the plateau value of 20∶4(n−6) were considerably lower and the contents of 18∶2(n−6) higher in liver phosphatidylcholines (PC) and heart PC. Heart phosphatidylethanolamines (PE) on the contrary, had elevated contents of 20∶4(n−6), but decreased 22∶5(n−6) compared with the PALM group. All groups fed HMO had similar contents oftrans fatty acids, mainly 16∶1 and 18∶1, in their phospholipids, irrespective of the dietary 18∶2 levels, and these contents were lower than in the HSOY group. High levels of linoleic acid consistently found in triglycerides of liver, heart and adipose tissue of rats fed HMO indicated that feeding HMO resulted in a reduction of the conversion of linoleic acid into long chain PUFA that could not be overcome by increasing the dietary level of linoleic acid.     

Dietary fats containing concentrates ofcis ortrans octadecenoates and the patterns of polyunsaturated fatty acids of liver phosphatidylcholine and phosphatidylethanolamine

The effects of the mixedcis- 18∶1 isomers and mixedtrans-18∶1 isomers present in partially hydrogenated soybean oil (PHSO) upon the patterns of polyunsaturated fatty acids (PUFA) in liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in rats fed concentrates ofcis- 18∶1 ortrans- 18∶1 isomers isolated as triacylglycerides from PHSO. Thecis- 18∶1 andtrans- 18∶1 concentrates were fed at levels equal to those present in PHSO fed at 17.9% of the diet. All diets contained the required amounts of both linoleic and linolenic acids. Thetrans- 18∶1 concentrate was found to suppress the levels of 20∶4ω6 and 20∶3ω9, and to increase the levels of 18∶2ω6 and 20∶5ω3 in PC and PE. Thecis- 18∶1 concentrate suppressed 20∶4ω6 in PC, 20∶5ω3 in PC and PE, and 18∶2ω6 was more effective than thetrans concentrate in suppressing 22∶6ω3. Thetrans- 18∶1 concentrate was more effective in suppressing 20∶4ω6. Thetrans-18∶ isomers appear to modify PUFA metabolism by inhibition of PUFA synthesis, whereas thecis- 18∶1isomers appear to compete with 2-position fatty acyl transfer and to inhibit ω3 PUFA acylation.     

Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats

The effect of very low levels of dietary long-chain n−3 fatty acids on Δ6 desaturation of linoleic acid (18∶2n−6) and α-linolenic acid (18∶3n−3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20∶3n−6), in liver microsomes and its influence on tissue fatty acids were examined in obese and lean Zucker rats and in Wistar rats. Animals fed for 12 wk a balanced diet containing ca. 200 mg of long-chain polyunsaturated n−3 fatty acids per 100 g of diet were compared to those fed the same amount of α-linoleic acid. Low amounts of long-chain n−3 fatty acids greatly inhibited Δ6 desaturation of 18∶2n−6 and Δ5 desaturation of 20∶3n−6, while Δ6 desaturation of 18∶3n−3 was not inhibited in Zucker rats and was even stimulated in Wistar rats. Inhibition of the biosynthesis of long-chain n−6 fatty acids was reflected in a decrease in arachidonic acid (20∶4n−6) content of serum lipids when fasting, and also in the phospholipid fatty acids of liver microsomes. On the contrary, heart and kidney phospholipids did not develop any decrease in 20∶4n−6 during fish oil ingestion. Docosahexaenoic acid (22∶6n−3), present in the dietary fish oil, was increased in serum lipids and in liver microsome, heart, and kidney phospholipids.     

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

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

Phospholipid composition of the granular amebocyte from the horseshoe crab, Limulus polyphemus

The phospholipid composition was determined for the amebocyte of the primitive arthropod Limulus polyphemus. The total fatty acid composition of the cells' lipids was analyzed by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters (FAME). The FAME analysis revealed high levels of 20-carbon polyunsaturated fatty acids (PUFA), especially arachidonic (20∶4n-6) and eicosapentaenoic (20∶5n-3) acids. Almost 20% of the total lipid profile was comprised of dimethyl acetals of 16- to 20-carbon chain lengths, indicative of plasmalogens in the phospholipid pool. Phospholipids, analyzed by high-pressure liquid chromatography, included phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SPH), and cardiolipin (CL). PE and PC levels predominated at 42.2 and 36.3%, respectively. Smaller amounts of PS (9.0%) and PI (6.2%) were present, as well as low levels of SPH (4.6%), CL (1.6%), and trace amounts of lysophosphatidylcholine. The major phospholipid species, PE, PC, PS and PI, were collected and their molecular species were examined by electrospray-ionization mass spectrometry. The molecular species within the phospholipid classes reflected the high levels of PUFA seen in the total lipid profile. PI was mainly composed of 18∶0a/20∶4. Over half of the PS consisted of 18∶0a/18∶1 and 18∶0a/20∶4. The major PE species were 20∶1p/20∶5, 20∶1p/20∶4, 18∶0p/20∶5, and 18∶0p/20∶4. PC had the largest distribution of molecular species, and its most abundant species were 16∶0e/20∶5, 16∶0e/20∶4, and 16∶0p/20∶4. The presence of 16∶0e/20∶4 is the first documentation of a specific precursor to platelet-activating factor in an invertebrate hemocyte. Note: at the sn-1 position: [a=1=O-acyl, e=1-O-alkylether, and p=1-O-alk-1′-enyl (plasmalogen)].     

Selective deposition oftrans-8- andcis-9-octadecenoates in egg and tissue lipids of the laying hen

The deposition oftrans-8-octadecenoate-8(9)-³H (8t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids and in organ lipids from the laying hen.trans-8-Octadecenoate was preferentially incorporated into only the phosphatidylethanolamines (PE), whereas discrimination against 8t-18∶1-³H occurred in the phosphatidylcholines (PC), triglycerides (TG) and cholesteryl esters (CE). The 1-acyl position of both PE and PC contained three times more 8t-18∶1-³H than 9c-18∶1-¹⁴C. Almost total exclusion of the 8t-18∶1-³H from the 2-acyl position of these phospholipids was found. Preferential incorporation of 9c-18∶1-¹⁴C occurred at the combined 1- and 3-acyl positions and at the 2-acyl position of yolk TG. Tissue lipid analyses indicated that there was preferential deposition of 9c-18∶1-¹⁴C into all organs. Individual liver lipid classes displayed the same relative order of discrimination against 8t-18∶1-³H as did egg yolk lipids (CE>TG>PC>PE).     

Membrane lipid metabolism and phospholipase activity in insect Spodoptera frugiperda 9 ovarian cells

Although there is increasing use of insect ovarian Sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. In this study, analysis of membrane lipids of Sf9 cells indicated the presence of phosphatidylethanolamine (PE) (diacyltype) and phosphatidylcholine as major phospholipids, followed by phosphatidylserine and phosphatidylinositol (PI), and only trace amounts of ethanolamine plasmalogen. These phospholipids contain high proportions of monoenoic fatty acids, e.g., 16∶1 and 18∶1, which comprise more than 70% of the total fatty acids although small amounts of polyunsaturated fatty acids such as 18∶2 and 20∶4 are also present. When Sf9 cells were incubated in a culture medium containing [¹⁴C]oleic acid and [¹⁴C]arachidonic acid, a large portion of the labels were incorporated into membrane phospholipids. Using [¹⁴C]arachidonoyl-phospholipids as substrates for incubation with cell homogenate and subcellular fractions, results indicate the presence of a Ca2⁺-independent phospholipase A (PLA₂) in the Sf9 cell cytosol fraction. This PLA₂ shows a high preference for hydrolysis of PE and is active at a pH range of 7–9. Unlike the brain cells which contain active phospholipase C (PLC) specific for phosphatidylinositol, only limited amount of diacylglycerol (DAG) was released from [¹⁴C]arachidonoyl-PE in the Sf9 cells. Taken together, this study demonstrates active metabolism of membrane phospholipids in Sf9 cells, most likely mediated by acyltransferases and PLA₂. Furthermore, despite the absence of PLC for PI, limited amount of DAG could be generated through hydrolysis of PE.     

Acyl group distributions in tissue lipids of rats fed evening primrose oil (λ-linolenic plus linoleic acid) or soybean oil (α-linolenic plus linoleic acid)

Three groups of rats were fed diets with either 10 weight percent (wt%) of evening primrose oil, safflower oil or soybean oil for 11 weeks. Diets contained 7.1 wt% linoleic acid +0.8 wt% γ-linolenic acid, 7.6 wt% linoleic acid, or 5.3 wt% linoleic acid +0.7 wt% α-linolenic acid, respectively. In liver mitochondria as well as in heart, dietary γ-linolenic acid did not affect the fatty acid profiles of phosphatidylcholnes (PC), phosphatidylethanolamines (PE) or cardiolipins (CL), whereas dietary α-linolenic acid caused an increased formation of (n−3) polyunsaturated fatty acids (PUFA). The liver Δ6− and Δ5-desaturase activities determined in vitro were not affected by the dietary fats. In brain PE, which are rich in C22− and C20-(n−3) PUFA, as well as in testes PC and PE, which are rich in (n−6) PUFA, no effects were found from a partial replacement of dietary linoleic acid with γ-linolenic acid or α-linolenic acid. In kidney PC, PE, phosphatidylinositol (PI) and CL, 20∶3(n−6) was moderately elevated to ca. 1% following intake of γ-linolenic acid, whereas partial replacement of linoleic acid with α-linolenic acid was followed by increased deposition of 22∶6(n−3) in PC and PE of testes and kidney. Thus, no general effect of evening primrose oil on the content of (n−6) PUFA in rat tissue phospholipids was observed, wheras a significant incorporation of γ-linolenic acid into liver and adipose tissue triglycerides was found.     

Polyunsaturated fatty acids in tissues of rats fed trielaidin and high or low levels of linolenic acid

Male and female weanling rats that were born to dams fed a diet low in linolenic acid received diets of 15% lipids by weight containing 45% elaidic acid (as trielaidin) and 8.5% or 0.1% linolenic acid for 10 weeks. Four other groups, in which palmitic or oleic acid replaced elaidic acid in the diet, served as controls. The fatty acid profiles of several lipid classes were determined in adipose tissue, adrenals, testes, heart and brain. Elaidic acid was incorporated into tissue lipids in varying degrees, depending on the organ and on the lipid class. Feeding elaidic acid induced no changes in the polyunsaturated fatty acid (PUFA) profiles of testes lipids but resulted in definite modifications of the PUFA patterns of heart phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In linolenic acid-deprived rats, arachidonic acid was decreased in PC and linoleic acid was increased in both PC and PE; 22∶5n−6 was strongly depressed in both PC and PE. In linolenic acid-fed rats, 22∶6n−3 was decreased in PC and PE. These changes, on the whole, were more evident in females, and some also were observed in adrenal cholesteryl esters but only slightly in brain phospholipids. the apparent inhibition of the biosynthesis of PUFA induced by dietary elaidic acid appeared to be complex and of greater intensity in the n−6 fatty acid series than in their n−3 homologues.     

Nuclear magnetic resonance investigation of hydrocarbon chain packing in bilayers of polyunsaturated phospholipids

²H nuclear magnetic resonance (NMR) on chaindeuterated phospholipids has been used to study the influence of the degree of unsaturation on lipid chain packing and on area per molecule at the lipid water interface. Order and motions of deuterated stearic acid in position sn-1 of phosphatidylcholines (PC) containing 18∶0, 18∶1n-9, 18∶2n-6, 18∶3n-3, 20∶4n-6, 20∶5n-3, or 22∶6n-3 in position sn-2 were investigated in pure PC and in mixtures of PC in a phosphatidylethanolamine (PE) matrix. Results reveal that lipid packing in bilayers is mainly controlled by packing requirements at the lipid water interface. Increasing degrees of unsaturation lower chain order and increase area per PC molecule, whereas inclusion of PE in model membranes has the opposite effect. Chain order and motions in highly unsaturated lipid membranes are less sensitive to changes in temperature. Temperature sensitivity decreases further upon incorporation of PC into a PE matrix. Unsaturation induces chain disordering, which may be interpreted as an increase in area per molecule of lipids toward the center of the bilayer. This may result in a lower packing density of unsaturated lipids at the lipid water interface. We hypothesize that these differences in lipid packing and dynamics may influence activity of membrane proteins.     

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

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

Phospholipid fatty acid composition of various mouse tissues after feeding α-linolenate (18∶3n−3) or eicosatrienoate (20∶3n−3)

The selective incorporation of dietary α-linolenate (18∶3n−3) and its elongation product, eicosatrienoate (20∶3n−3), into various phospholipids (PL) of mouse liver, spleen, kidney, and heart, was examined in a two-week feeding trial by assessing mol % changes in associated fatty acids. Mice were fed fat-free AIN 76A diets modified with either 2 wt% safflower oil (control); 1% safflower and 1% linolenate; or 1% safflower and 1% eicosatrienoate. After linolenate or eicosatrienoate feeding, 20∶4n−6 was reduced by 36–50% in liver phosphatidylcholine (PC) and in liver and spleen phosphatidylethanolamine (PE). Linolenate was minimally incorporated into PL, but was desaturated and elongated to 20∶5n−3, 22∶5n−3, and 22∶6n−3, with notable differences in the quantity of these n−3 derivatives associated with different tissues and PL. Eicosatrientoate was uniquely incorporated into the cardiolipin (CL) pool of all organs. There was also considerable retroconversion of 20∶3n−3 to 18∶3n−3 (PC, PE). Dietary eicosatrienoate may therefore affect metabolism in diverse ways—20∶3n−3, which is retroconverted to 18∶3n−3, may provide substrate for 20∶5n−3 and 22∶6n−3 syntheses, whereas intact 20∶3n−3 may be incorporated into the CL pool. Acyl modifications of CL are known to affect the activity of key innermitochondrial enzymes, such as cytochrome c oxidase. This work was presented in part at the 73rd Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, LA, March 19–23, 1989.     

Turnover of label from [1-14C] linolenic acid in phospholipids of coho salmon,Oncorhynchus kisutch

Juvenile coho salmon were injected intraperitoneally with [1-¹⁴C] linolenic acid, and sampled at 24, 120, and 240 hr. Liver, heart, and gill lipids were extracted, analyzed, and halflives of individual liver glycerophospholipids and n-3 fatty acids determined from rates of loss of radioactivity. Incorporation of label into gill was much less than into either heart or liver. Total acyl halflife was shorter for the choline phospholipids than for the ethanolamine phospholipids, as were the halflives of all individual n-3 fatty acids. Eicosapentaenoic acid (20∶5n-3) had the shortest halflife in both phospholipids (50–60 hr), while docosapentaenoic acid (22∶5n-3) and docosahexaenoic acid (22∶6n-3) had much longer halflives. Specific activities of the shorter chain n-3 fatty acids were much greater than the longer, more unsaturated homologs at all times, suggesting possible differences in their mechanisms of incorporation into phospholipids. Diacylglycerol analysis indicated that de novo synthesis could be responsible for the incorporation of only a small portion of the labeled long chain fatty acids found in phospholipids. The fatty acid halflives reported here for salmon are in general agreement with those found previously in mammals. Technical Paper No. 5238, Oregon Agricultural Experiment Station, Oregon State University, Corvallis, Oregon 97331. This material is taken in part from a thesis submitted in partial fulfillment of the requirements for the MS degree in 1978.